In order to provide production advice of high-quality upper tobacco leaves,field experiment was carried out to elucidate the effects of nitrogen application on the growth,development,carbon and nitrogen metabolism of upper leaves of different tobacco varieties.Three treatments,37.5(LN),75.0(MN)and 112.5 kg/ha nitrogen application rate(HN)were conducted with NC89 and Yunyan 87 as the test varieties.The effects of different treatments on the agronomic traits,photosynthetic characteristics,leaf tissue structure,key enzymes activity of carbon and nitrogen metabolism,and chemical composition of the upper leaves of were studied.The results showed that the leaf length,leaf width,leaf area,and leaf dry mass of the upper leaves of both varieties were significantly improved with the increased of nitrogen application rates.At 115 days after transplanting(DAT),the leaf area of NC89 and Yunyan 87 of HN were significantly increased by 63.10% and 68.43% compared to LN,respectively.Increasing nitrogen rate improved the leaf chlorophyll content of NC89,the leaf chlorophyll content of HN was 6.67%—37.50% higher than that of LN,respectively.The leaf net photosynthetic rate was notably improved by increasing nitrogen rate of Yunyan 87,especially at 70 DAT and 80 DAT.The leaf palisade tissue,leaf spongy tissue,and leaf thickness were markedly promoted with the increased nitrogen application rate on 85—115 DAT.The palisade tissue and spongy tissue thickness of LN and MN were almost stable from 95 to 115 DAT,but HN improved by 9.82%—14.08% and 10.72%—13.72% respectively.The leaf carbon content and leaf carbon and nitrogen ratio of both varieties were significantly decreased by reduced nitrogen rate,while leaf nitrogen content significantly increased.The activities of invertase,sucrose phosphate synthase,sucrose synthase,and glutamate synthase of both varieties were increased with the increased nitrogen application rate.HN decreased the amylase activity of Yunyan 87,but increased that of NC89.At 115 DAT,the amylase activity of HN of Yunyan 87 was 27.53% lower than MN,while HN increased amylase activity by 33.86% and 21.74% compared to LN and MN of NC89,respectively.The activity of glutamine synthetase of Yunyan 87 significantly increased with the increased nitrogen application rate,while there was no significant difference among different treatments of NC89.The content of flue-cured tobacco leaf reducing sugar and total sugar were reduced,but the content of nicotine and total nitrogen were improved with increased nitrogen application rate.Under the same nitrogen application rate,the content of flue-cured tobacco leaf nicotine,total nitrogen,and potassium of Yunyan 87 were higher than NC89,while the content of reducing sugar,content of total sugar(except LN),sugar and nicotine ratio,and nitrogen and nicotine ratio were lower than NC89.The effect of nitrogen application rate on upper leaves of tobacco varieties is difference.Increasing nitrogen application rate could promote the growth,development and carbon metabolism,reduce sugar and nicotine ratio and nitrogen and nicotine ratio,and improve the coordination of chemical components of NC89.However,increasing nitrogen application rate could lead to excessive nitrogen metabolism,delay the transformation from nitrogen metabolism to carbon accumulation metabolism,and cause late maturity of Yunyan 87.

To study the effects of ridge height on soil heat status,root growth,and tobacco maturity tolerance in tobacco planting,a 2-year field experiment was conducted using the flue-cured tobacco variety Yueyan 97 as the test material from 2022 to 2023.Three treatments were set up,including a ridge height of 30 cm(CK),a ridge height of 38 cm(T1),and a ridge height of 46 cm(T2),to analyze the changes in soil temperature and heat flux,root appearance and growth indicators,root vitality,and tobacco maturity related indicators under different ridge heights.The results indicated that the daily temperature difference in soil varies with the depth of the soil layer,and the daily temperature difference in the surface layer of the soil was the largest.Increasing ridge height can increase the average temperature of tobacco planting soil by 0.4-1.8 ℃ and increase soil heat flux by 4-56 W/m².In the 2 a experiment,the maximum increase in root length for a ridge height of 38 cm compared to a ridge height of 30 cm was 27.26%,the maximum increase in root dry mass was 26.21%,and the maximum increase in root vitality was 14.97%,with significant differences.Compared with a ridge height of 30 cm,the soluble protein content,peroxidase activity,and cell membrane stability index of a ridge height of 38 cm increased by 17.99%,27.82%,and 9.05 percentage points(2022),respectively and by 10.23%,12.44%,and 8.16 percentage points(2023),respectively.The maximum decrease in malondialdehyde content was 24.84% and 44.43%,respectively.Correlation analysis showed that root activity was significantly negatively correlated with malondialdehyde content,peroxidase activity was extremely significantly positively correlated with root growth indexes, and soluble protein content was significantly positively correlated with root length.In summary,increasing ridge height is beneficial for improving the thermal status of tobacco planting soil,promoting root growth,enhancing root vitality,enhancing leaf antioxidant capacity,and enhancing tobacco maturity tolerance.A ridge height of 38 cm is an appropriate ridge height for promoting root growth and improving tobacco maturity tolerance in southern tobacco regions.

The WRKY transcription factor family plays an important role in regulating abiotic stress.To systematically analyze the sequence characteristics and expression patterns of NtWRKY11 gene in tobacco,and explore the response mechanism of NtWRKY11 under abiotic stress such as low temperature and drought,the full-length cDNA sequence of NtWRKY11 gene was amplified by PCR using common tobacco cDNA as a template.The basic properties of NtWRKY11 protein were analyzed by bioinformatics software and the subcellular localization of NtWRKY11 protein was studied by constructing a plant expression vector.The expression of NtWRKY11 gene was detected by qRT-PCR technology in different tissues at the full flowering stage and under different abiotic stresses.The results showed that the full-length cDNA of NtWRKY11 gene in tobacco was 999 bp,encoding 332 amino acids,and it shared 54.23% similarity with ramie BnWRKY11.Its promoter region contained three kinds of cis-acting elements(one MBS,one MYB and three ARBE),which probably worked together to enhance the drought resistance of plants.It also contained three salicylic acid-responsive cis-acting elements(TCA-element),which could improve the low temperature tolerance of plants.Subcellular localization results indicated that the NtWRKY11 protein was located in the nucleus.The expression analysis of NtWRKY11 in different tissues at the full flowering stage showed that NtWRKY11 was highly expressed in old leaves,significantly higher than in roots.However,the expression in flowers was significantly lower than that in roots,and there was no significant difference between stems,new leaves and roots. The expression analysis under abiotic stress showed that the relative expression of the gene was significantly higher than that of normal (CK).The relative expression level under high salt stress was not significantly different from CK,while the relative expression level under high temperature stress was significantly lower than CK.All in all,NtWRKY11 is highly expressed in old leaves,and its expression level is enhanced under abiotic stresses such as drought stress and low temperature stress,indicating that this gene acts as a forward transcription factor to regulate drought and low temperature stress.

To investigate the effects of Piriformospora indica combined with Arbuscular mycorrhizal fungi (AMF) on drought resistance of tobacco,Yunyan 87 was inoculated with sterile water (CC),P.indica (CP),AMF (PC),P.indica and AMF (PP),and the contents of proline (Pro),superoxide dismutase (SOD),peroxidase (POD),malondialdehyde (MDA) and the expression of drought-related genes NTNAC1 and NAC4 in tobacco leaves were determined under stress in the form of natural drought.The results showed that P.indica and AMF could promote the growth of above-ground and underground parts of tobacco,and chlorosis of leaves and symptoms of their drought were slight.After 7 days of drought stress,the contents of Pro in tobacco leaves of CP,PC and PP groups were 1.39,1.59 and 1.78 times higher than those of CC in group,respectively.The activities of SOD and POD in tobacco leaves increased first and then decreased.The activities of SOD in CP,PC and PP groups were 1.15,1.22 and 1.33 times higher than those in CC group,POD activity was 1.33,1.46 and 1.85 times higher than those of CC group,respectively.The content of MDA in tobacco leaves was decreased by 21.98%,23.98% and 24.84% in CP,PC and PP groups respectively.The expression levels of NTNAC1 and NAC4 in tobacco leaves were up-regulated.The expression levels of NTNAC1 in CP,PC and PP groups were 3.37,3.88 and 5.07 times higher than those in CC group,the expression levels of NAC4 gene were 3.04,3.59 and 5.56 times higher than those of CC group,respectively.This study indicates that P.indica and AMF showed significant synergistic effects,which could significantly improve the drought resistance of tobacco.

Define the cultivation measures of long-leaf tobacco seedlings in tobacco-rice rotation area to promote the early growth and rapid development of paddy-tobacco.Yunyan 87 was used as the material.The effects of three factors on dry matter and nutrient accumulation and distribution,and fertilizer use efficiency of flue-cured tobacco were studied,namely,the aperture of nursery site(136,200 pores),microbial agent(substrate with Miaoqiangzhuang microbial agent,substrate without microbial agent),and transplanting leaf age(8-leaf transplanting,10-leaf transplanting).The results showed that increasing the aperture of nursery site and adding microbial agent in the substrate to cultivate long-leaf tobacco seedlings could increase dry matter accumulation,improve dry matter distribution,increase nitrogen,phosphorus and potassium accumulation,promote fertilizer absorption efficiency,and increase fertilizer utilization rate and production efficiency;the contribution rates of aperture of nursery site,microbial agent,transplanting leaf age and the interaction of three factors to dry matter accumulation were 22.87%,27.73%,37.41% and 11.99%,respectively.The contribution rates to nitrogen accumulation were 20.34%,23.45%,33.62% and 22.59%,respectively.The contribution rates of phosphorus accumulation were 24.41%,27.81%,32.85% and 14.93%,respectively.The contribution rates of potassium accumulation were 15.48%,26.05%,34.61% and 23.86%,respectively.The contribution rates to nitrogen efficiency were 20.81%,44.67%,23.11% and 11.41%,respectively.The contribution rates of phosphorus fertilizer efficiency were 32.15%,31.66%,14.91% and 21.28%,respectively.The contribution rates of potassium fertilizer efficiency were 25.31%,38.71%,31.67% and 4.31%,respectively.In Hunan paddy-tobacco growing area,increasing the aperture of nursery site and adding microbial agents to the substrate can make up for the defects of two-stage seedling raising,and jointly improve nutrient uptake and fertilizer utilization efficiency,which has a certain popularization value in flue-cured tobacco production.

In order to understand the application effect of rice-straw application combined with decomposing agent on tobacco.The pot culture experiment was conducted to analyze the variation of the tobacco's growth,yield,chemical composition and coordination after the rice-straw application combined with chemical fertilizer and decaying agent containing different microbial dominant strains respectively,the research object was to explore the effects of rice-straw application combined with decomposing agent on the tobacco's growth,yield and quality.The results showed that the rice-straw application combined with decaying agent significantly increased the stem circumference,maximum leaf area,biomass,yields and the ratio of medium & superior grade of tobacco.The rice-straw application combined with decaying agent significantly lowered nicotine content of the upper tobacco,and increased its' total sugar content,reducing sugar content and total sugar-nicotine ratio,which resulted in reducing the irritability,bitterness and pungency of the upper tobacco and improving its' smoking quality;meanwhile,the rice-straw application combined with decaying agent significantly raised the content of total nitrogen,total sugar and reducing sugar in central tobacco,but there were no obvious variations for the ratio of total nitrogen to nicotine,reducing sugar total nitrogen and reducing sugar to nicotine,which meant the effect of rice-straw application combined with decaying agent on the central tobacco's chemical coordination was not significant;moreover,the rice-straw application combined with decaying agent increased the content of total sugar and reduced sugar in lower tobacco,and raised its' ratio of reducing sugar to total nitrogen and sugar to nicotine,which was beneficial to increase the aroma of lower tobacco and reduce its' impurity.According to the results of principal component regions between tobacco's agronomic characters and biomass,chemical composition and coordination indexes of different parts of cured tobacco,the rice-straw application combined with the decomposing agent containing dominant microorganism of bacteria+fungi or bacteria+fungi+actinomycetes had no significant difference in promoting tobacco's growth,yield and quality.Therefore,the rice-straw application combined with the decomposing agent containing different types of dominant microorganism could promote tobacco's growth,rationalize the chemical composition of upper and lower tobacco,and be beneficial to improve its sensory quality and increase tobacco's yield and quality.

To explore the effect of planting density on disease and quality of tobacco and the response characteristics of soil microorganisms in rainy regions,a field experiment was conducted in the Rainy Regions.The random block design was adopted to study the effects of different planting densities(CK was 16 650 and T₁ was 18 525 plants/ha) on yield of tobacco and soil microbial community structure during the experimental periods. Results suggested that:compared with the CK treatment,T1 treatment increased by 6.02% for the tobacco plant height,and 8.65% for yield,respectively. Compared with CK treatment,T1 treatment decreased by 15.38% for tobacco black shank,11.76% for tobacco black root rot,and 18.18% for tobacco anthracnose at the mature stage,respectively. T1 treatment increased by 1.62%for the species diversity and decreased by 0.99% for the richness of the rhizosphere soil bacterial community,increased by 3.76% for the species richness and decreased by 4.71% for the diversity of the fungal community when compared with CK treatment. The bacterial and fungal community structure of the T1 treatment was similar to that of the CK treatment,but the microbial dominant taxa in the soil samples was inconsistent. In the horizontal structure of phylum,T1 treatment relative abundance of bacteria in the soil Proteobacter,Acidobacteria and Actinobacteria,and fungal in the soil Ascomycota was higher. In the horizontal structure of genera,T1 treatment relative abundance of bacteria Sphingomonas and fungi Penicillium was significantly improved. Redundancy analysis (RDA)showed that environmental factors (sunshine duration,rainfall,and temperature)significantly affected the bacterial and fungal community structure,and the influence of soil bacterial community was rainfall>temperature>sunshine duration,the influence of soil fungal community was sunshine duration>rainfall>temperature,respectively. This study indicated that properly increasing the planting density of flue-cured tobacco in rainy regions could improve the soil microenvironment,and reduce tobacco diseases,which could improve the quality of flue-cured tobacco and increase the income of tobacco farmers.

In order to study the biological function of NbEHD1 in Nicotiana benthamiana,its gene structure,conserved domains,phosphorylation sites,subcellular localization and evolutionary relationship were predicted by bioinformatics methods.The encoding sequence of NbEHD1 was 1 638 bp,and its genomic sequence contained 16 exons and 15 introns.NbEHD1 protein was predicted located in the cytoplasm,which had 42 phosphorylation sites and had no signal peptide and transmembrane region.NbEHD1 belonged to the P-loop_NTPase superfamily and had a conserved domain specific to the EHD family.Phylogenetic relationship showed that NbEHD1 was closely related to EHD sequences of Solanum tuberosum and Solanum lycopersicum.Blast analysis against N.benthamiana sub-data base in SNG was performed to obtain the predicted full length of NbEHD1,then its full nucleic acid sequence was amplified by gene-specific primers.After obtaining NbEHD1 CRISPR/Cas9 gene editing construct,the vector was successfully transformed into the leaves of N.benthamiana by Agrobacterium-mediated genetic transformation.Eventually 16 T₀-generation plantlets were successfully identified,which provided materials for further research to determine the biological function of NbEHD1.

In order to investigate the growth, dry matter accumulation and nutrient use efficiency of rich flavor style flue-cured tobacco in Hunan paddy-tobacco, case study methods were carried out in two typical paddy-tobacco fields, suitable yield level(2 250-2 700 kg/ha) and high yield level(2 700-3 150 kg/ha), in Chenzhou Guiyang County and Hengyang Leiyang of Hunan Province in 2019. The differences in agronomic traits, leaf area index, root morphology, dry matter accumulation and distribution, nutrient accumulation and distribution, and nutrient utilization efficiency of flue-cured tobacco in different periods were analyzed. The results showed that:The above-ground part of flue-cured tobacco grown well at high yield level, the root system was well developed, the dry matter and N, P, K nutrients accumulated much, the utilization efficiency of N, P, K fertilizer was high, but the proportion of N, P, K nutrients in flue-cured tobacco leaves at suitable yield level was large, N, P, K nutrient harvest index was high. Flue-cured tobacco plants in Leiyang were tall, thick stems, thick roots, the dry matter and nutrient accumulation in roots and stems were higher;The leaf area of flue-cured tobacco in Guiyang County was large, many lateral roots, and the dry matter and nutrition of flue-cured tobacco were more accumulated. In the production of flue-cured tobacco of rice-tobacco rotation, in view of large soil masses and low temperature and rainy weather, it is necessary to mature the soil, cultivate strong seedlings, improve the quality of transplanting, create a good soil environment for the root growth of flue-cured tobacco, and promote the early growth and rapid development of flue-cured tobacco.

In order to improve the soil environment and soil quality of tobacco planting in rice growing area of Changsha. Through field experiments, 1 deep vertical rotary tillage(T1) treatment and 2 conventional controls were set up, including the ploughed furrow(T2) and direct rotary ploughing(T3), to explore the effects of different tillage methods on soil physical characteristics and dry and fresh weight of flue-cured tobacco roots in the rice-tobacco zone area in Changsha. The results showed that deep vertical rotary tillage improved the soil surface physical structure. Within the overall range of 0-30 cm surface layer, T1 deep vertical rotary tillage treatment showed an average reduction in bulk density and compactness of 8.8%, 29.7%, and an average increase in porosity of 10.67 percentage points in 2018, compared with T3 conventional treatment(local tillage method). In 2019, T1's bulk density and compactness decreased by 11.25%, 21.45% on average compared with T3, and porosity increased by 12.8 percentage on average. Within 3-5 months of flue-cured tobacco transplanting, the average moisture content of the soil at T1 was 0.62 percentage points higher than that at T2 in 0-10 cm, 3.23, 4.92 percentage points higher than that at T2 and T3 in 10-20 cm, 9.43, 12.3 percentage points higher at 20-30 cm, 9.56, 13.88 percentage points higher at 30-40 cm, respectively. At the same time, compared with conventional treatment, the D value was increased and the stability of soil aggregates was improved. In addition, the dry mass of flue-cured tobacco roots under T1 treatment was 69.75%, 28.93% and 28.33% higher at maturity than that under T3 treatment, and the volume of roots was 20.07%, 55.61% and 36.34% higher than that under T3 treatment. To sum up, deep vertical rotary tillage bed improved soil physical properties such as soil bulk density, compactness, porosity, soil moisture, soil aggregate stability and so on, improved soil environment, increased the biomass and volume of flue-cured tobacco roots and laid a foundation for improving the yield and output value of tobacco leaves.

To determine the genetic diversity and genetic structure of Phytophthora nicotianae from Henan Province,34 P. nicotianae isolates were analyzed by randomly amplified polymorphic DNA (RAPD) marker. The results showed that 30 bands were amplified using 6 RAPD primers and 90% of them were polymorphic. Nei's gene diversity index was 0.271 0,and Shannon's information index was 0.402 8. The genetic similarity coefficient of 34 isolates ranged from 0.59 to 1.00. Based on UPGMA dendrogram,34 isolates were divided into 6 groups with 0.80 genetic similarity. Group Ⅰ was the dominant group including 26 isolates. The result of principle coordinate analysis was basically similar to that of UPGMA dendrogram. The result of genetic structure analysis revealed that P.nicotianae population in Henan originated from 2 ancestral subpopulations. The subpopulation Ⅰ and subpopulation Ⅱ accounted for 72.73%,27.27%,respectively. The subpopulation Ⅰ was the dominant subpopulation. Genetic component of 55.88% of isolates almost consisted of only one subpopulation. Therefore,the genetic diversity of P.nicotianae from Henan was low and the genetic structure of them was also simple.

In order to study the effect of different rootstocks on the disease resistance of tobacco grafted seedlings, and to clarify the mechanism of Yunyan 87 (Y87) as rootstock to improve the resistance of scion Hongdajinyuan (HD) to bacterial wilt, this study used split grafting and raw material band winding method to construct HD/HD grafted and HD/Y87 flue-cured tobacco seedlings respectively, using HD as the scion and Y87 as the rootstock. The PAL activity of HD/HD and HD/Y87 protective enzymes was measured. Finally, mRNA of 5 HD/HD or HD/Y87 were extracted and reverse transcribed to construct a total cDNA library by high-throughput sequencing technology and Illumina HiSeqTM 4000 sequencing platform. After sequencing and filtering low-quality Reads, the reference genome was compared with TopHat2, and the genes expression amount and differentially expressed genes were obtained by Cufflinks and DESeq software Heterogene expression. The results showed that the PAL enzyme activity of HD/Y87 was significantly higher than that of HD/HD at different sampling times, 48 414 474-48 697 874 Clean Reads and 38 272-40 938 genes were obtained from four samples, respectively, and the number of selective shear events of HD/Y87 was higher than HD/HD. After comparing the gene expression between HD/Y87 and HD/HD, 3 904 differential genes were obtained, 3 096 genes were up-regulated, 808 genes were down regulated; among the up-regulated genes, there were 3 genes ecoding phenylalanine ammonia lyases, 5 genes ecoding Myb family transcription factors, and 5 genes ecoding polyphenol oxidase involved in lignin synthesis, it also had 2 disease-related proteins and 3 genes ecoding ERF transcription factors. These results showed that Y87 as rootstock could improve the activity of HD PAL and gene expression of various resistance genes (including PAL, PPO and disease related protein) in scion, so as to improve the resistance of HD to bacterial wilt and other diseases.

A plant expression vector pBI121 -StPR1 was constructed to clarify the role of potato StPR1 gene in disease resistance, and introduce StPR1 gene into tobacco by Agrobacterium transformation method. The disease resistance and physiological characteristics of transgenic tobacco were investigated using different pathogen treatment, including bacterial disease soft rot (E. carotovora subsp. Carotovora Borgey, Ecc; E. chrysanthemi Burkholder. Atroseptica Dye, Ech; E. carotovora subsp. Mc Fadden et Dimock, Eca), bacterial wilt (Ralstonia solanacearum, RS) and fungal disease dry rot (F. sambucinum, F. avenaceum). The results showed that the diameter of lesions leaves increased with the prolongation of stress time in transgenic tobacco and wild type (WT) under the pathogen stress, and the diameter of transgenic tobacco lesions were significantly smaller than WT. In the physiological characteristics analysis, the physiological indexes of leaves increased with the prolongation of stress time in WT and transgenic tobacco, but the activities of SOD, POD and CAT in transgenic tobacco were all increased to different degrees compared WT. The results of disease resistance and physiological characteristics showed that transgenic tobacco had stronger resistance to pathogenic bacteria and fungi, indicating that the StPR1 gene played an important role in the disease resistance of potato, which could provide a theoretical basis for PR1 gene resistance in plants.

In order to explore the function of BdDREB1-like gene in transgenic tobacco, it was cloned and analyzed through bioinformatics technology. The open reading frame of this gene was 684 bp, encoded 228 amino acids, and the molecular weight of its encoding protein was 24.178 ku. It contained only one typical AP2 domain and belonged to the DREB family of AP2/EREBP superfamily. Multiple sequence alignment and evolutionary analysis showed that it was closely related to Rye ScCBFI. Subcellular localization analysis showed that BdDREB1-like was located in the nucleus. It was then constructed into plant expression vector and transformed into tobacco by Agrobacterium-mediated method to further confirm its function. PCR and RT-PCR screening showed that BdDREB1-like gene was not only integrated into tobacco genome, but also expressed stably. Transgenic tobacco seedlings with BdDREB1-like gene had higher germination rate under hydrogen peroxide stress. In order to further determine the relationship between BdDREB1-like gene and oxidative stress, we treated the one-month-old transgenic tobacco plants under oxidative stress with MV (Methyl viologen). The results showed that there were no significant differences in the contents of hydrogen peroxide, catalase and other physiological indicators except for SOD between wild type and T3 transgenic line, while there were only two physiological indexes of POD and chloroplast content of T1 transgenic line, which were significantly different from those of wild type under normal growth condition. Under oxidative stress, transgenic tobacco plants with BdDREB1-like gene had lower malondialdehyde and hydrogen peroxide content, higher peroxidase activity, catalase, superoxide dismutase and chlorophyll content compared with wild type. These results suggested that overexpression of BdDREB1-like gene enhanced the tolerance of plant to oxidative stress.

In order to explore the effects of tillage depth on the growth, development, yield and quality of tobacco plants in the Zhaoyang tobacco-growing area, there was experimental research on the depth of the tillage to the treatments 20 cm (T1), 25 cm (T2), 30 cm (T3) and CK (15 cm) by the randomized block design in the field. The results showed that there were significantly different at 0.05 of volume-weight and soil porosity between T2 and T1 in the long-term, and there was significantly different at 0.05 level of volume-weight between T2 and T3. The T2 dry matter accumulation of tobacco plants roots, stems and leaves was comparatively large at different times, followed by T3 and T1, and CK was the worst. The root-knot nematode disease of T3 was the lightest, the incidence rate was 25.23%, and there was significantly different at 0.05 level with CK; the disease index T3 (4.04) < T1 (5.27) < T2 (5.39), significantly lower than CK (18.64) at 0.01 level. In the agronomic traits, the plant height of T2 was 112.85 cm, the leaf area coefficient of T2 was 3.09, and the stem circumference of T3 was 12.07 cm, which was significantly higher than CK at 0.05 level. In the economic traits, T2 yield, upper grade tobacco ratio (α=0.01), medium grade tobacco ratio (α=0.05) were significantly higher than CK; T3 yield (α=0.05) was significantly higher than CK; T1 medium grade tobacco ratio (α=0.01) was significantly higher than CK. On the whole, the economic traits of T2 flue-cured tobacco was the best, followed by T3 and T1, and CK was the worst. The study found that the continuous ploughing depth of 25-30 cm can improve soil physical properties, reduce the occurrence of root-knot nematode disease, promote the growth and development of tobacco plants, and improve the yield and quality of tobacco.

The aims of this study were to reveal the appropriate mode for water-potassium coupling of flue-cured tobacco in Qiandongnan Prefecture, Guizhou province. Three irrigation rates: 0.50 L/plant(W₁), 1.00 L/plant(W₁) and 2.00 L/plant(W₃), and three potassium fertilizer rates:135 kg/ha(K₁), 270 kg/ha(K₂) and 405 kg/ha(K₃) were set in this field plot experiment, resulting in a total of nine treatments. The effects of different potassium application rate and water irrigation rate coupling treatments on the growth of flue-cured tobacco, yield and quality of tobacco leaf were studied. Different water and fertilizer amounts significantly or extremely significant influenced the plant height, maximum leaf area, stem girth and root volume, and interaction effect existed between water and fertilizer amounts. Water and fertilizer amounts significantly influenced total sugar, reduced sugar, alkaloid, total nitrogen, potassium and chloride contents in tobacco leaves(C3F grade), the smoking quality and appearance quality of flue-cured tobacco leaf increased with the increase of irrigation rate and K rate, and interaction effect existed between them.Water and fertilizer rate significantly influenced tobacco yield, and interaction effect existed between them. Considering all above, when irrigation water quantity was 1.00-2.00 L/plant, K application rate was 270 kg/ha (W₂K₂ and W₃K₂), the growth of tobacco plant, yield and quality of tobacco leaves were improved. Linear fitting revealed a positive relationship between plant height, root volume and yield of tobacco leaves within a certain range. When irrigation rate was 1.00-2.00 L per plant, K application rate was 270 kg/ha, water-potassium coupling treatment had a good effect in promoting tobacco plant growth, increasing yield and improving quality of tobacco leaves in tobacco-planting in Qiandongnan Prefecture, Guizhou province. From the view of obtaining high yield and saving water and fertilizer, W₂K₂(1.00 L/plant, 270 kg/ha)and W₃K₂(2.00 L/plant, 270 kg/ha)treatment can be used as an appropriate combination under the present experimental conditions.

In order to clarify the decomposing characteristics of different rates of tobacco straw and their effects on rice yield. In this study, one field experiment was conducted. Then, the decomposition rates of tobacco straw and rice yield among different treatments were analyzed. Meanwhile, the quantitative relationship between the external input of carbon and nitrogen ratio and rice yield was discussed. The results showed that： the decomposing characteristics of the tobacco straw in all treatments were fast in the early stage and slow in the late stage. At the 5th day, the amount of decomposed tobacco and the average daily decomposing amount were reached the maximum, which were 0.87 g and 0.29 g/d, respectively. The cumulative decomposing rate of tobacco straw also showed a similar trend, and the cumulative decomposing rate of tobacco straw in the whole rice season was 42.31%. However, there was not significant different for the total and daily decomposed amount of tobacco straw among all treatments, but the cumulative decomposing rate of the semi-returning treatment was higher than the equivalent and 1.5 times returning. The correlation between the input of carbon-nitrogen ratio and rice yield could be fitted by the quadratic curve equation. Moreover, it indicated that the reasonable input of carbon-nitrogen ratio was 10.1, and the corresponding maximum yield of rice was 10.4 t/ha. Therefore, in the rotation of tobacco and rice, the reasonable carbon-nitrogen ratio input was one key pattern to improve rice yield, although the characteristics of decomposing under different amounts of tobacco straw was similar.

In order to discover the function of Blind gene in Nicotiana tabacum, we cloned one Blind-like gene, NtBL1, from K326 by homologue cloning method. The protein domain and structure was predicted by PROSITE(ExPASy), Sopma and Swissmodel, the Phylogeny tree construction and Motif prediction of NtBL1 and its homologues were analyzed separately by Mega 7.0 and MEME, the expression pattern of NtBL1 in different tissues was analyzed by semi-quantitative PCR, and the expression changes of NtBL1 under abiotic stress treatment was detected by qRT-PCR. The results showed that the coding sequence of NtBL1 was 1 014 bp, encoding 337 aa. The expression pattern of NtBL1 in transcription level was generally detected in several organs and growing stages, while it had the highest expression level in root. Bioinformation analysis showed that NtBL1 was a typical R2R3 Myb protein, which contained 2 Myb-type HTH domains. The phylogenetic tree analysis showed that NtBL1 gene was shared lots of motifs with tomato Blind. The results of qRT-PCR showed that the expression level of NtBL1 gene was up-regulated by ABA treatment and the highest expression level appeared in 24 h treatment; The expression level of NtBL1 gene was down-regulated by NaCl treatment and the lowest expression level presented in 24 h treatment. In conclusion, NtBL1 gene might participate in regulation of axillary mersitem development by ABA and NaCl.

In order to further clarify the mechanism of over expression TaZAT8, a wheat C2H2 type zinc finger gene, to enhance the resistance of tobacco plants under P-starvation, the classical proteomics approach was applied to reveal the changes of protein expression between WT and transgenic tobacco plants. The results showed that there were 22 protein spots differentially expressed on 2-DE images, and among them, twenty protein spots were up-regulated and two were down-regulated respectively. Subsequently, these protein spots were identified by LC-MS/MS, which were classified into six functional groups, including primary metabolism, energy generation, stress response and defense, transcription and translation, protein turnover and unclear functions. Subcellular localization analysis indicated that these proteins were mainly located in five organelles, including cytoplasm, mitochondria, peroxisome, nucleus and chloroplast. Go analysis showed that the differentially expressed proteins participated in various metabolism processes or act as enzymes of different categories. Above results showed that over expression TaZAT8 laid the foundation for improving the plant tolerance to stress via regulating proteins involved in biological processes such as substance metabolism, energy production, stress response, etc.

In order to set up the basic theory for rational fertilization technology of tobacco in Southern Anhui Province,we analyzed the changes of agronomic characters,dry matter accumulation,nutrient absorption and fertilizer use efficiency of flue-cured tobacco under different nitrogen and potassium fertilizer application rates. The result showed that high nitrogen fertilizer rate caused the increment of plant height,leaf number,leaf length and leaf width of tobacco at different positions. The dry matter accumulation in different organs and the ratio of shoot to root also increased with the increase of nitrogen application rate. The increase of nitrogen fertilizer rate also increased the nitrogen and potassium accumulation in roots,stems and leaves. The amount of nitrogen fertilizer was positively correlated with the whole plant nitrogen concentration,and had significantly positive correlation with the total nitrogen and potassium accumulation. With the increase of nitrogen fertilizer amount,the utilization rate of nitrogen fertilizer did not change significantly,but the agronomic efficiency and partial productivity of nitrogen fertilizer increased significantly. The increase of potassium fertilizer had no significant effect on the agronomic traits of flue-cured tobacco. High potassium fertilizer rate caused the increment of nitrogen concentration in roots,and potassium concentration in stems and leaves,but the dry matter accumulation and nutrition accumulation in different organs were not influenced by potassium fertilizer rate. The amount of potassium fertilizer was only positively correlated with the whole plant potassium concentration. The utilization rate of potassium fertilizer was relatively low among the treatments. Among the treatments with the lowest application rate of potassium fertilizer,the utilization rate of potassium fertilizer,the agronomic efficiency and partial productivity of potassium fertilizer were the highest. In the experiment,the dry matter accumulation and the nutrition accumulation in whole plant,and the nitrogen fertilizer use efficiency increased significantly with the increment of nitrogen application rate. Applying nitrogen fertilizer had obvious effect on the increase of yield. With the increase of potassium fertilizer,there was no significant change in dry matter accumulation and nutrient accumulation. According to the results of leaf potassium concentration and the potassium fertilizer use efficiency,the potassium application rate should be reduced in Southern Anhui Province.

The aim of this study was to reveal the effects of application rates of chemical nitrogen(N) fertilizer combined with organic fertilizer(rapeseed cake,M)on the quality,N uptake and use efficiency of tobacco Yun yan 87,and to find the best organic and inorganic N application amount of tobacco in Qiandongnan prefecture of Guizhou Province,which could provide the technical reference for reducing the production cost,and increasing the yield and quality of tobacco. Six treatments,i.e. CK₁,CK₂ and MN_iPK(i=0,1,2 or 3)were set in a field plot experiment. The effects of each treatment on the leaf quality,N uptake and use efficiency of tobacco were analyzed. The results showed that,compared with CK₂ treatment,total sugar contents in tobacco leaves of MN₁PK,MN₂PK and MN₃PK treatments increased by 8.57%,14.18% and 13.03%,respectively,the reduced-sugar contents increased by 4.67%,9.66% and 8.26%,respectively,the potassium contents increased by 8.34%,19.08% and 17.12%,respectively,while the chlorine content decreased by 0.36%,8.19% and 3.56%,respectively. The ratio of potassium to chloride and the difference between total sugar and reduced-sugar were improved. Compared with CK₂ treatment,the stimulation and foreign smoke of tobacco leaves decreased and the fragrance quality and fragrance amount enhanced in combination of chemical N fertilizer with organic fertilizer. The combustibility enhanced,and the lasting smoke was more comfortable and longer. Compared with MN₁PK and MN₃PK treatments,MN₂PK treatment increased the N use efficiency by 2.99,4.54 percentage points,respectively. The chemical N fertilizer combined with organic fertilizer(organic N accounting for 36.00%-45.77%)coordinated the ratio of different chemical component of tobacco leaves,improved the smoking quality,increased N the nitrogen uptake of tobacco plant and enhanced N use efficiency. After comprehensively analyzing the effects of each treatment on chemical component,smoking quality and N use efficiency of tobacco leaves,the best treatment was that the organic N accounted for 45.77%,and the recommended ratio of organic N was 36.00%-45.77% of total N in tobacco land of Qiandongnan prefecture,which was suggested to be extended in the local areas,and could provide a reference for the neighboring areas.

It is known that the threonine at position 1 084 of the third coiled-coil (CC3) of the new member of the tobacco-driven family NtTkr is crucial for the binding of the target protein. Multiple candidate proteins interacting with NtTkr were obtained by yeast two-hybrid screening of NtTkr tail. In order to determine the importance of T₁₀₈₄ deletion or replacement mutation between NtTkr and target protein vitro, first the pBI121-NtTkr plasmid was take as a template to obtain the tobacco NtTkr tail T₁₀₈₄ deletion and replacement tail by overlap extension PCR, and cloned T_1084d,T1084A into pUC19, by the blue white spot screening, Sma Ⅰ-BamH Ⅰ double enzyme cuting the identification and gene sequencing, geting the right T₁₀₈₄ deletion and replacement NtTkr tail; Then restructured pUC19-T_1084d, pUC19-T1084A and pMXB10 with Not Ⅰ-Nde Ⅰ double enzyme, the target fragment and the carrier fragment are recovered and connected, and the ligation product transforms DH5α. The recombinant was screened to Not Ⅰ-Nde Ⅰ double enzyme identification, managed to build the required pMXB10-NtTkr-T1084A and pMXB10-NtTkr-T_1084d prokaryotic expression vector; Finally, pMXB10-NtTkr-T1084A and pMXB10 -NtTkr-T_1084dd were transferred into BL21 (DE3), and the protein expression was detected by 12% SDS-PAGE after the induction of 0.05 and 0.06 mmol/L IPTG concentrations, respectively NtTkr-T1084A-1320 of about 77 ku and NtTkr -T_1084d-1317 of 76.2 ku were highly expressed.

Under the optimization of C/N with organic fertilizer and N fertilizer, a field experiment was carried out to study the effect of potassium and the ratio of basic/dressing fertilizer on biomass, potassium uptake, soil available potassium content and aroma quality of tobacco. Split-plot experiment design was adopted. The main treatment was 3 K level:K1 (120.0 kg/ha),K2 (240.0 kg/ha) and K3 (360.0 kg/ha);and the subsidiary treatment was the ratio of basic/dressing K fertilizer:S1(7:3),S2(5:5) and S3(3:7). The results showed that the biomass of tobacco leaves increased with the increase of potassium application, while it decreased with the increase of potassium dressing ratio in the same K level in the fast growth and mature period. Under the K2 and K3 level, the leaf potassium content was lower in the S1 treatment than in S2 and S3 treatments, indicating that the increase of K dressing percentage was conducive to the transfer of potassium ions from lower leaves to middle and upper leaves. With the increase of potassium application, the content of available and slowly available potassium in rhizosphere soil increased. Under K2 level, the available potassium of soil reached to maximum at fast growth and bud stages, and was lower in S3 treatment than in S1 and S2 treatments at mature stage. Under K3 level, the available potassium in rhizosphere soil had no significant difference among the four growth stages, in which that of S3 treatment > S1 treatment > S2 treatment. Compared with other treatments, the contents of total nitrogen, total nicotine and protein in tobacco leaves were significantly higher in S3 treatment under K2 level, and the ratio of sugar/alkali and ratio of nitrogen/alkali were improved. The neutral aroma components had no significant difference in upper leaves under K1 level, but it improved significantly in S3 treatment under K2 and K3 levels, especially for S3 treatment under the K2 level, in which the quality, quantity and concentration of aroma in tobacco leaves were the best. It was concluded that under the optimization conditions of organic fertilizer C/N, 240.0 kg/ha potassium fertilizer was recommended in the sand mud soil of Southern China, and the basic fertilizer was 72.0 kg/ha and the dressing one was 168.0 kg/ha (the ratio of base to dressing was 3:7), which could effectively improve the potassium content and quality of tobacco leaves.

The effects of different phosphorus application rate on growth and yield of tobacco Yunyan 87 were studied to explore the suitable phosphorus application rate in Qiandongnan prefecture tobacco region, Guizhou province, and to provide scientific basis for phosphorus fertilizer application in tobacco production. A field plot experiment was conducted, which was established six fertilizer treatments, i.e. CK, M, P₀,P₁, P₂ and P₃. In the growth period of tobacco, main agronomic characters, diseases situation and yield of flue-cured tobacco were recorded and investigated. The results showed that P₃ treatment was the best treatment, which had good field growth, good agronomic characters, strong disease-resistant ability and high yield of the all fertilizer treatments. At mature stage, as compared with CK treatment, tobacco plant of P₃ treatment increased plant height, maximum leaf area, leaf numbers, stem girth, internodal length and root volume by 63.19%, 72.67%,78.93%,37.93%,13.65% and 161.79%, respectively; As compared with P₀ treatment, tobacco plant of P₃ treatment increased plant height, maximum leaf area, leaf numbers, stem girth and root volume by 15.90%,21.46%,23.61%,11.25% and 28.75%, respectively. As compared with CK and P₀ treatment, tobacco plant of P₃ treatment decreased incidence of Red star disease, Mosaic and Black shank by 7.51, 7.92, 6.68 and 3.66, 6.52, 4.72 percentage point, decreased disease index of Red star disease, Mosaic and Black shank by 56.12%, 45.53%, 51.33% and 33.72%, 31.97%,43.53%, respectively. As compared with P₂ and P₀ treatment, P₃ treatment increased tobacco leaf yield by120.62% and 28.51%, respectively. The suitable chemical phosphorus fertilizer application rate was 112.5 kg/ha in Qiandongnan prefecture tobacco land, Guizhou province. Therefore, it is suitable to be extended in this tobacco region and can provide a reference in its neighbouring areas.

The current agricultural cultivation is highly dependent on chemical fertilizers,resulting in the deterioration of the micro-ecological environment of the soil. In order to improve the biological characteristics of the soil and fertility, The effects of green manure (an annual) as green manure or chemical fertilizer on the activities of invertase,urease,phosphatase and catalase in flue-cured tobacco soil were studied. The results showed that the activity of four enzymes could be significantly increased by overturning of grass rhinoceros,and the soil enzyme activity increased with the increase of the amount of green manure. Compared with the blank control (CK2),the third batch of grass rhinoceros (overturned on September 18 with a biomass of 32 180 kg/ha) had the largest increase in enzyme activity,with the maximum increase being:sucrase June 48.4%,91.5% for urease in April,72.4% for phosphatase in August,and 51.6% for catalase in October. In the treatment of green manure and chemical fertilizers,the treatment of green manure + chemical fertilizer was higher than that of the control (CK1,only chemical fertilizer), the effect of the third batch of overturning green manure plus conventional fertilization on soil enzyme activity was better than that of green manure plus 30% nitrogen reduction, The first batch (turnover on August 3,biomass was 19 350 kg/ha) and the second batch (biomass,22 930 kg/ha on August 18) were overturned.The different of soil enzyme activity between green manure+conventional fertilization and green manure+nitrogen reduction 30% was not significant,and urease and phosphatase were particularly prominent. The characteristics of the dynamic changes of enzymes in different growth stages of tobacco plants were as follows:soil sucrase,urease,and phosphatase in all treatments with overturn green manure green manure,and chemical fertilizers all peaked in the long-term (June) peak. Catalase A peak appeared in the early stage (August) of tobacco leaf harvest. The above results indicated that the soil metabolism was very active after the rhizome was pressed,which was conducive to the nutrient transformation of the soil and provides the necessary nutrition for the normal growth of the flue-cured tobacco.

In order to study the effect of garlic root decomposition on tobacco black shank in tobacco growing soil,the greenhouse pot experiment was carried out to study inhibition effects on tobacco black shank through indoor cultivation by garlic root decomposition with different decomposing time.The experimental result indicated that garlic root decomposition with different decomposing time could inhibit the growth of tobacco black shank fungus,and garlic root decomposition that decomposed 15 days had the best inhibition effect.Treated by garlic root decomposition that decomposed 15 days,myceliun appeared hyphae deformation,hyphae branch and protoplasm leak.Pot experiments showed that the control effect of garlic decomposition solutions decomposed in different times on tobacco black shank was to 4.30%-41.38% in ninth days after the transplanting of flue-cured tobacco.From the findings it is inferred that garlic root decomposition had a good inhibitory effect on tobacco black shank,and had highly activity on tobacco black shank.

The HKT protein family is mainly involved in controlling the absorption of K⁺ and the selective transport of K⁺/Na⁺, which plays an important role in improving the stress resistance of plants.In order to study the relationship between the tissue expression level of AtHKT1 and salt tolerance of plants,the homology of HKT proteins,the expression of AtHKT1 gene and the cis-acting elements of AtHKT1 promoter were analyzed and predicted using bioinformatics techniques. On the basis,the AtHKT1 promoter was introduced into Nicotiana benthamiana. GUS staining results were used to analyze the tissue expression level of AtHKT1 gene in Nicotiana benthamiana. The results of bioinformatics analysis showed that Arabidopsis thaliana and wheat were located in different evolutionary branches and their genetic relationship was far, suggesting that the function of AtHKT1 might be different from the corresponding protein in wheat. AtHKT1 gene was abundantly expressed in many organs and tissues of Arabidopsis thaliana, especially in leaves, roots and flowers, which confirmed that AtHKT1 gene was likely to have important physiological functions. The AtHKT1 promoter might be a stress-responsive promoter containing a number of important elements that responded to environmental stress. Therefore, the expression of AtHKT1 gene was likely to be regulated by environmental stress. Results of GUS staining showed that the coloration of leaves,vascular system,roots and flowers of the seedlings with pHKT1-gus was deeper,further confirming that the expression level of AtHKT1 in these regions was high. The above results indicated that the regulation of AtHKT1 gene expression was conducive to the translocation of Na⁺,thereby regulating the salt tolerance of plants.In addition, there might be other unknown features that further increase the complexity of AtHKT1 functionality. At present, the mechanism of K⁺ and Na⁺ transport of HKT proteins is not clear. The expression level of AtHKT1 gene in Nicotiana benthamiana was analyzed to provide reference for further understanding the action mechanism of AtHKT1 gene.

In order to study the function of CIPK3 in abiotic stress response,a CIPK family gene NtCIPK3 was cloned from common tobacco K326 by homologous cloning. The gene contained a 1 272 bp ORF encoding 423 amino acid. Protein sequence analysis indicated that the protein contained a transmembrane domain and belonged to a hydrophilic membrane protein. Protein alignment analysis revealed that NtCIPK3 contained a highly conserved N-terminal kinase region,a junction region,and a C-terminal regulatory region,with the highest homology to wild tobacco CIPK3,reaching 99%.Subcellular localization predictions showed that the protein was mainly located in the nucleus and had a bipartite typing nuclear localization signal sequence. The expression characteristics of this gene were analyzed by qRT-PCR. Analysis of tissue expression revealed that the gene was expressed in the roots,stems,leaves and flowers of tobacco and had tissue-specific expression and the expression level in leaves was significantly higher than that in other tissues,and it was speculated that it might function mainly in leaves.Meanwhile,NtCIPK3 gene was induced by low potassium,high salt,drought,ABA,H₂O₂,and low temperature treatment. It is speculated that NtCIPK3 gene played an important role in tobacco abiotic stress response. And successfully constructed the pBI121-NtCIPK3 overexpression vector,which laid the foundation for the future research of the gene under abiotic stress.

To explore the relationship between tobacco NtCIPK family gene expression and the potassium content of tobacco, and to provide reference for the molecular mechanism of potassium absorption in tobacco. For common tobacco varieties K326, Guiyan 5 and Yun 87 three different potassium concentrations (6.0, 1.0,0.2 mmol/L) were cultured in solution and their dry weight and potassium content were measured. At the same time, 11 genes of the NtCIPK family were analyzed for different potassium concentrations using qRT-PCR. Induced expression pattern. With the increased of potassium concentration in the culture medium, the dry weight and potassium content of the tested plants showed an increasing trend, and there were significant differences between treatments. Among them, Yun 87 is treated with different potassium concentrations (1.0, 0.2 mmol/L). Dry weight and potassium content were the highest. The expression of 11 genes in the NtCIPK family was induced by the change of potassium concentration, and showed up-regulated expression with the decreased of K⁺ concentration; only the expression of NtCIPK9 in K326 and NtCIPK24 in Guiyan 5 showed a downward trend.The results of path analysis showed that the expression of NtCIPK3 and NtCIPK5 was significantly positively correlated with the potassium content of K326 and Guiyan 5, respectively, at the concentration of 0.2 mmol/L K⁺, while NtCIPK23 was treated at the concentration of 6.0 mmol/L K⁺. The expression had a significant negative correlation with the potassium content per plant of Yun 87. The potassium content of tobacco was closely related to the expression of NtCIPK gene, and the enhanced expression of NtCIPK gene contributes to the absorption and accumulation of potassium by tobacco.

Potassium channel is the main protein of plant uptake and translocation K⁺,and SKOR (Stelar K⁺ outwaed rectifier) belongs to Shaker channel family exogenous rectifier,which plays a key role in response to low potassium stress response in plants.In order to study the function and role of tobacco NtSKOR gene in abiotic stress response,one NtSKOR gene was cloned from Nicotiana tabacum K326 with homologous cloning strategy,gene expression patterns were analyzed by Real-time quantitative PCR,and their physicochemical properties,domains,phosphorylation sites and evolutionary relationships of proteins were predicted by bioinformatic analysis. Bioinformatics analysis showed that this gene contained 2 484 bp and encoded 827 amino acid. The predicted molecular weight was 94.75 ku and the isoelectric point (pI) was 6.52. The largest secondary structure element of this protein was α-helix,and the smallest was β-turn. NtSKOR contained six transmembrane domains(S1-S6),with three different kinase phosphorylation sites for serine,threonine and tyrosine. Phylogenetic analysis showed that the protein had 99% homology with Nicotiana sylvestris and 96% homology with Nicotiana tompentosiformis,thus it was named as NtSKOR. Expression patterns showed that the gene was expressed in roots,stems,leaves and flowers in mature stage,which had the highest expression in roots and the lowest expression level in flowers. Expression patterns under abiotic stress indicated the gene could respond rapidly to the adverse conditions of low potassium,high salt,drought,H₂O₂,ABA and 4℃. This suggests that NtSKOR gene plays an important regulatory role in tobacco abiotic stress,and the results provide a theoretical basis for further in-depth study of the NtSKOR function.

In order to study the function of tobacco NtRAP2-7 gene in abiotic stress response, NtRAP2-7 was cloned from K326 by homologous cloning. And the bioinformatics software was used to analyze the physicochemical properties, spatial structure and phylogenetic analysis of the encoded protein. Sequence analysis results showed that the gene contained 1 398 bp in length which encoded 465 amino acid residues and the predicted molecular weight of this protein was 51.16 ku. Based on 3-D modeling and secondary structure analysis, the protein consisted of three β-sheet and one α-helix and contained 58 phosphorylation sites. The subcellular localization prediction showed that the protein was mainly located in the nucleus and contained a monopartite nuclear location signal sequence. Phylogenetic analysis showed that NtRAP2-7 protein had the highest homology with Nicotiana sylvestris RAP2-7 protein sequence, which was 98%.The expression pattern analysis of the gene was carried out by qRT-PCR. Tissue expression analysis showed that the gene was expressed in the roots, stems, leaves and flowers, but had the highest expression level in roots. The expression levels of NtRAP2-7 was affected by abiotic stress treatments, indicating that the NtRAP2-7 was involved in the tobacco abiotic stress response. The pBI121-NtRAP2-7 overexpression vector was successfully constructed by double enzyme digestion, which laid the foundation for further study on the function of this gene in abiotic stress.

This study laid the theoretical foundation for the study of the function and structure of peroxidase genes in tobacco and cloned the cDNA of peroxidase gene NtPOD1 from tobacco cultivar K326 by homologous cloning and analyzed the bioinformatics. At the same time,qRT-PCR method was used to analyze the expression pattern of tissue and organ of this gene and its stress response. The results showed that the total length of cDNA was 981 bp,encoding 326 amino acid residues,the predicted molecular weight was 37.19 ku,and the isoelectric point was 8.89. Bioinformatics analysis showed that the protein was a hydrophilic protein containing four conserved disulfide bonds and two conserved calcium binding sites in the domain,which may be located in extracellular(including cell wall).The class Ⅲ secretion oxidase of the dependent peroxidase superfamily had high homology with Nicotuana attenuata POD42, Nicotiana sylvestris POD42,Solanum tuberosum POD42 and so on. The gene was expressed in tobacco roots,stems,leaves and flowers,with the highest expression in leaves and the lowest expression in flowers. Meanwhile, NtPOD1 expression was induced by high salt,drought,hypokalemia,ABA and H₂O₂. The results indicate that NtPOD1 belongs to tobacco peroxidase and may play a role in the response of tobacco to abiotic stress.

In order to study the influence of different application amounts of charcoal on the microbial diversity in tobacco rhizosphere soil, with the rhizosphere soil of flue-cured tobacco variety Yunyan 87 as the research object, we compared the situation by applying different amounts of charcoal, and conducted high-throughput sequencing of rhizosphere soil ITS2 region and 16S rDNA-V4 region with progressive treatment with 3 application amounts of charcoal; through bioinformatics analysis of data, we obtained the OTU abundance, distribution,α diversity, community species composition and abundance information of microorganisms in rhizosphere soil when different treatment methods were used, and conducted PCA clustering analysis and UPGMA clustering analysis of the community composition and abundance. The results showed that within the application scope of 50-150 g/tree, by increasing the application amounts of charcoal, it could increase the variety diversity and distribution uniformity of bacteria in rhizosphere soil, while it would reduce the variety diversity and distribution uniformity of fungus; compared to the control group where no charcoal was applied, after applying the charcoal with the amounts of 50, 100,150 g/tree, the abundances of Proteobacteria bacteria had declined by 4.1%, 2.7% and 0.7% respectively; the abundances of Acidobacteria bacteria increased by 10.4%, 8.1% and 7.7% respectively; the abundances of Actinobacteria bacteria were all lower than the abundance of control group; the difference in abundance of Gemmatimonadetes bacteria between various groups was very small. After applying the charcoal with the amount of 50 g/tree, the abundance of Zygomycota fungus had declined by 12.38% compared to the control group(50.92%), and then, with the increased of the application amount of charcoal, the abundance of Zygomycota fungus gradually increased to 53.68%; on the contrary, after applying the charcoal with the amount of 50 g/tree, the abundance of Ascomycota fungus had increased by 10.15% compared to the control group(30.63%), and then, with the increased of the application amount of charcoal, the abundance of Ascomycota fungus gradually declined to 29.11%; after applying the charcoal with the amount of 50 g/tree, the abundances of Basidiomycota and Chytridiomycota funguses almost had no change, and then, with the increased of the application amount of charcoal, the abundances of Basidiomycota and Chytridiomycota funguses presented the trend of increased first, and then declined; various treatment methods did not have significant influence on the abundance of Glomeromycota fungus. By studying the influencing method and action rules of charcoal application on the micro-ecology of tobacco rhizosphere, it can provide theoretical basis to the application of charcoal in tobacco field.

In order to study the effect of drought stress on physiological characteristics of grafting flue-cured tobacco,using KRK26 as scion, KRK26,K326 and Anyan 2 as rootstock,analysing and discussing root morphology, plant morphology and physiological indexes of resistance in pot culture under different treatment. The results indicated that under drought stress conditions at early period, the relative content of chlorophyll (SPAD readings) decreased, while the content of proline, superoxide dismutase (SOD) activities, catalase (CAT) activities and peroxidase (POD) activities in flue-cured tobacco demonstrated an increasing trend. The root length,surface area,plant height,stem girth, node spacing and LAI of the grafted tobacco plants showed a decreasing trend,the content of proline continued to increase in both grafted seedlings and non-grafted seedlings with the aggravation of drought stress when tobacco leaves were turning to mature, whereas, the SPAD readings, SOD activities, CAT activities and POD activities all demonstrated a decreasing trend. The speed of decreasing in different root grafted was slower than that in own root grafted. Compared with KRK26/K326 and KRK26/KRK26, KRK26/Anyan 2 had greater parameters under drought stress, but the SPAD readings did not have significant difference. All the results showed that less damage was made to KRK26/Anyan 2 than KRK26/K326 and KRK26/KRK26 under drought stress, and KRK26/KRK26 suffered the most serious damage. According to correlation analysis, there was a certain correlation between morphological indexes and resistance indexes of grafting flue-cured tobacco. In conclusion, there were obvious differences among grafted tobacco in physiological characteristics, and there response to soil drought. The order of drought resistance was KRK26/Anyan 2, KRK26/K326 and KRK26/KRK26 in turn from strong to weak, based on the main root morphology,plant morphology,physiological characteristics under drought stress.

2C protein phosphatase (PP2C) is a key component in the ABA signal transduction pathway and plays a pivotal role in plant growth and development,cell cycle regulation and adaptation to adversity stresses.To explore the function of the PP2C gene in tobacco adaptation to abiotic stress,a PP2C homologous gene was cloned from the tobacco cultivar K326,which contained a 1 617 bp ORF encoding 538 amino acid. The homology analysis showed that the gene had high homology with PP2C16 of Nicotiana tomentosiformis. So it was named NtPP2C16. Bioinformatics analysis showed that the NtPP2C16 catalytic region had 11 structural sub-regions which were relatively conservative in the PP2C family evolution. qRT-PCR analysis revealed that the expression of this gene was significantly induced by ABA and H₂O₂ signal molecules,and responded to drought,high salt,low temperature and low potassium stresses. NtPP2C16-pBI121 overexpression vector was constructed successfully,the results provided some basis for analysis of NtPP2C16 responsing to abiotic stresses in tobacco.

The expression of the lectin gene (NTL1) from Narcissus tazzetta var. chinensis in Nicotiana alata was studied by qPCR method in order to determine the aphid-resistant function of NTL1 and cultivate N. alata with aphid-resistant. By means of susceptible to aphid,inoculation in vivo and in vitro,the resistance to aphid in different transgenic tobacco was studied. The result showed that NTL1 gene had been integrated into the tobacco,but the expression of different tobacco was different,the expression of NTL1 in tobacco by descending order was 3,4,1,2. The bioassay of aphid indicated that transgenic tobacco had a certain resistance to aphid,but the capacity of aphid-resistance in different tobacco plant was slightly different;The average density inhibition rate of aphid in inoculation in vivo and in vitro was 8.22%-76.61% and 4.62%-65.65%. The transgenic tobacco could speed up the aphid's death. The relative expression of NTL1 gene in different transgenic tobacco and the result of resistance to aphid were compared,showed that two results came with a high degree of consistency.

To explore the effect of different leaf age on the yield and quality of flue-cured tobacco,the main cultivars K236 in Luoping tobacco-growing area of Yunnan were used as test materials to construct different flue-cured tobacco varieties with different leaf age. Photosynthetic potential in growth period and economic characters of post-baked tobacco leaf were analyzed. The results showed that with the increase of transplanting age,the survival rate of tobacco seedlings increased first and then decreased,while the field growth period increased; With the advance of fertility in the field of flue-cured tobacco,the photosynthetic potential (LAD) of the treatments decreased first and then increased,and the total LAD of the treatments was the highest in 5 leaves transplanted tobacco seedlings,which was 20.14% higher than that of 6 and 8 leaves respectively,14.82%. The mean leaf angle (MLIA) of the population increased first and then decreased with the increase of leaf age,while the extinction coefficient (K)was an increasing trend.The leaf tobacco leaves with 5 leaves were transplanted with appropriate chemical components and the quality was the best,followed by 4 leaves;6,7,8 leaves,because of its reducing sugar,starch,potassium content does not meet the requirements of high-quality tobacco chemical indicators,poor quality;With the increase of transplanting age,economic traits showed a trend of increasing first and then decreasing. Therefore,the optimum tobacco leaf transplanting age of tobacco leaves in Luoping tobacco-producing areas in Yunnan is 5-leaf-stage,which can shorten the period of nursery and shorten the time of cutting and reduce the time when the high yield value can be maintained. Such as smoke and the proportion of medium and high smoke,improve the grade structure and site structure.

In order to explore the effect of different forms of nitrogen supply on flue-cured tobacco's curing characteristics. A field experiment,using K326(Nicotiana tabacum L.)as experimental material,with five different forms of nitrogen supply that N1(100% nitrate nitrogen),N2(30% ammonia nitrogen + 70% nitrate nitrogen),N3(50% ammonia nitrogen + 50% nitrate nitrogen),N4(70% ammonia nitrogen + 30% nitrate nitrogen)and N5(100% ammonia nitrogen)were conducted to study the effects of different treatments water,chlorophyll,polyphenol oxidase and malondialdehyde contents in leaves of tobacco leaves were compared,and the economic characters of flue-cured tobacco leaves were compared.The results showed that different nitrogen supply had different effects on the baking characteristics of flue-cured tobacco under 165 kg/ha nitrogen application rate.The water loss rate of tobacco leaves treated with 30% ammonia nitrogen+70% nitrate nitrogen during the curing process was close to the mean value of the water loss rate in 72 hours,and the chlorophyll degradation rate and the water loss rate was coordinated and synchronized.The activity of polyphenol oxidase in tobacco leaves was the lowest,the accumulation of malondialdehyde was slow and the membrane lipid peroxidation level was the lowest.In addition,the treatment of 30% ammonia nitrogen+70% nitrate nitrogen had the proportion of high-grade tobacco and the highest value.It could be seen that the tobacco leaf curing characteristics and leaf quality could be obtained by using 30% ammonia nitrogen+70% nitrate nitrogen in the northern part of Guangdong.

A field experiment was carried out in tobacco,to study the effect of K application on K content and aroma quality of leaves of flue-cured tobacco under the optimization of C/N with organic fertilizer.The results showed that:In the C3 or C1 level,the biomass of central leaf increased along with the increase of K application;The biomass of upper leaf in high K application was higher than that in low potassium;K application had no significant effect on the biomass of central and upper leaf in C3 level,and increased the biomass of leaf in C2 level;In the same C level,the content of K in leaf had the lower trend along with the growth period,and increased with the increase of K application;Under the same K application,C2 and C1 had no significant effect on K content of leaf;At the budding and mature period,compared with C1,in the medium and high K application,C levels increased K content of leaf,and the lower content of K in C3 level.K content of different parts of leaf was C2 > C3 > C1 level;The number of K bacteria showed an increasing trend from the rosette stage to vigorous growing period and a decreasing trend from bud period to mature period;The number of K bacteria in medium and high K application was higher than that in low K application;In the C2 level,the number of K bacteria in medium and high K application showed an increasing trend with the growth process,and those was significantly higher than other treatment;There was no significant difference in C3 and C1 between medium and high K application,but it was significantly higher than that in low K application;The chemical components of leaves treated with C2 level was higher between medium and high K application,which the total sugar content of the leaves was 25.39%-25.27%,reducing sugar was 22.75%-25.73%, total nitrogen was 1.67%-1.72%, nitrogen base ratio was of 0.79, sugar nicotine ratio was of 10.78-11.80, nicotine content was of 2.11%-2.18%,which showed that the application of K increased the content of total nitrogen,nic otine and protein,improved the ratio of sugar to nicotine,nitrogen base ratio,and increased the potassium content of tobacco leaf;The neutral aroma substances of 28 species in tobacco leaves was found in all treatments,and the total amount of low K application was lower than that of medium and high K application,and the difference was significant between the position of leaves;The upper leaf was lower than that in the middle leaves in the total amount;and the application of medium and high K increased the total amount of aroma substances,and the maximum amplitude in the upper leaves was found with C2 level.It can be seen,under the optimization of medium carbon(C/N ratio),the increased of K application was the best on the effect of neutral aroma substances in tobacco leaves,especially for the upper leaves.

In order to reveal mechanism of internal injury on leaves under transplanting stage for flue-cured tobacco seedling. The changes of chlorophyll fluorescence kinetics curve of young and mature leaves were studied under high light and low temperature environment transferred from the weak light and mildness greenhouse through the methods of artificial simulation.The results showed that photosynthetic performance index (PI_ABS) in young leaves was lower significantly than that of mature leaves and the maximum photochemical efficiency of PS Ⅱ (Fv/Fm) was lower than that of mature leaves, but the difference between them was not significant.Especially, under the environment of high light and low temperature, the parameters of Fv/Fm and PI_ABS of young leaves reduction were significantly higher than the mature leaves, which indicated that young leaves were more likely to occuring inhibition phenomenon under high light and low temperature. The relative variable fluorescence (V_J and V_I) in young leaves were significantly higher than that of mature leaves, which suggested that electron transfer rate in PS Ⅱ receptor side of young leaves were significantly lower than the mature leaves. Under high lights and low-temperature, the increasing extend of V_J were significantly greater than mature leaves, the inhibition of electrons transfer from Q_A to Q_B in PS Ⅱreceptor under high light and low temperature.V_I increased amplitude of young leaves were relatively smaller than mature leaves under high light and low temperature, which indicated that the effects of electrons transfer from Q_B to PQ in young leaves were fewer under high light and low temperature, the electron transfer blocked sites in young leaves were still Q_A to Q_B transfer process. The relative variable fluorescence (V_K and V_L) of young leaves were significantly higher than that of mature leaves, under high light and low temperature, the values of V_K and V_L of young leaves raise were significantly higher than the mature leaves, which indicated that damaged degree in young leaves were more serious than mature leaves, which showed that high light and low temperature led to more damaged for oxygen evolving complex and thylakoid membrane structure in young leaves. Conclusion, damaged degree of young leaves PS Ⅱ function under high light and low temperature stress in transplanting stage were bigger than the mature leaf, which were relevant with photochemical activity, electron transfer rate and OEC in young seedlings leaves were sensitive to high light and low temperature.

In order to explore the physiologic race type and the parasitism on tobacco of Jimo and Juxian populations of Heterodera glycines (SCN),the cup trial with quantitative inoculation was adopted,and the reproduction and infection performances were detected for the two populations on five soybean cultivars (including four host differentials and one susceptible standard cultivar Lee) and six tobacco ones.The results showed that the reproduction factor (Rf=P_f/P_i) of SCN Jimo population was 1.88(0.05-9.03) on the five soybean cultivars,with the Rf of 9.03 on the susceptible standard cultivar Lee,and the Rf was 0.01(0.00-0.04) on the six tobacco cultivars.The Rf of SCN Juxian population was 1.23(0.05-5.28) on the five soybean cultivars,with the Rf of 5.28 on the susceptible standard cultivar Lee,and the Rf was 0.16(0.00-0.65) on the six tobacco cultivars.The results indicated that the Jimo and Juxian populations of SCN were the physiologic race No.3 and No.6,respectively,both of which were found for the first time in Shandong Province.The SCN Jimo population had almost no parasitism on the six tobacco cultivars,and the Juxian population had certain parasitism on some tobacco cultivars such as Yunyan 85 and NC95.

In order to reveal the mechanism of tobacco leaf light energy utilization and photoprotective systems in aluminum (Al)-induced reactive oxygen species metabolism, two different Al-tolerant tobacco cultivars Yunyan 100 (Al-tolerant) and Yunyan 105 (Al-sensitive) were used to study effects of four Al concentrations (0, 50, 100, 200 μmol/L) on plant growth, reactive oxygen content, photosynthesis, chlorophyll fluorescence characteristics, photorespiration, activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in leaves of tobacco cultivar with solution culture method. The results showed that increasing Al concentrations significantly decreased the leaf chlorophyll content, net photosynthetic rate (Pn), maximal photochemical efficiency (Fv/Fm),quantum yield of electron transport at PS Ⅱ (Φ_{PS Ⅱ}) and coefficient of photochemical quenching (qP), increased excess energy of PS Ⅱ photochemistry leading to higher accumulation of leaf O₂^· and H₂O₂, and lower plant biomass, especially in Yunyan 105. With the Al concentrations increased from 0 to 200 μmol/L, the coefficient of non-photochemical quenching (NPQ), photorespiration rate, and activities of SOD and APX were promoted firstly and then reduced again. Compared with Al-sensitive cv. Yunyan 105, Al-tolerant cv. Yunyan 100 remained higher leaf Φ_{PS Ⅱ}, qP, NPQ, photorespiration rate, and activities of SOD and APX under different Al concentrations. These results indicated that tobacco could enhance the tolerance to Al stress by increasing photochemical reaction capacity, heat dissipation, photorespiration and activities of antioxidative enzymes in order to decrease excess energy of PS Ⅱ photochemistry, which prevents or eliminates reactive oxygen accumulation under the low Al concentration.

To seek new ways to improve the quality of tobacco, a field experiment was conducted to study the effect of vermicompost and vinasse on improving the growth and chemical properties of flue-cured tobacco using K326 as materials. Six treatments were set up, which were CK (conventional fertilization);T1(vermicompost 2 400 kg/ha);T2(vermicompost 1 800 kg/ha and vinasse 600 kg/ha);T3(vermicompost 1 200 kg/ha+vinasse 1 200 kg/ha);T4(vermicompost 600 kg/ha+vinasse 1 800 kg/ha);T5(vinasse 2 400 kg/ha),respectively. The results showed that vermicompost and vinasse could improve the plant growth, enhance the agronomic traits and chemical component of flue-cured tobacco, especially for the T2 (vermicompost 1 800 kg/ha and vinasse 600 kg/ha). The content of total nitrogen, total potassium and sugar content at topping stage were increased by 60.60%, 46.37% and 7.80%, while those at maturing stage were increased by 14.86%, 34.59%, 32.50%, as compared with CK, respectively. It also exhibited appropriate chemical property under T2 treatment. In conclusion, applying with vermicompost 1 800 kg/ha and vinasse 600 kg/ha can be suggested as a suitable fertilization models in tobacco-growing areas in Southern Shaanxi Province.

Black shank disease is one of the main diseases of tobacco production.The screening,identification of the disease resistance and genetic analysis of tobacco germplasm resources are the base of breeding for disease resistance. In this study,we used Physiological race 1 black shank to identify the resistance to black shank of 49 tobacco germplasm resources and used SSR markers to analysis genetic relationship. The results showed:Among them 24 were resistant,including dayeyongyan,G80,E9,MS212-8;7 were moderate resistant;6 were moderate susceptible;12 were susceptible;6 were high susceptible.The average effective number of alleles of 49 tobacco germplasm resources was 3.06(1.31),and the average Shannon's information index was 1.18(0.36);The 49 germplasm materials were classed into 4 groups at the position of coefficient of 0.66 by the 23 SSR markers. The first group was resistance variety K326;the second group included 6 germplasm resources;the third group included 4 susceptible germplasm resources;the fourth group included 38 germplasm resources. The most of resistance to black shank germplasm was clustered in the fourth group,suggesting the genetic basis of the resistance to black shank germplasm resources was narrow.

For further study of the flue-cured tobacco aroma content and smoking quality and TSNAs content which under control humidity baking. Experimental designed four difference dry bulb temperature under the same type of wet-bulb temperature.The content of neutral aroma substances,the smoking quality,and the content of TSNAs were analyzed.The results indicated that with the dry bulb temperature increased neutral aroma substances showed a trend of gradual decline,smoking quality first after rising downward trend,TSNAs content gradually reduced.

In order to measure the effect and action mechanism of cadmium(Cd) on the photosynthetic characteristics in the leaves of flue-cured tobacco,flue-cured tobacco was cultured in a solution containing different concentrations of Cd. The effects of Cd stress on the chlorophyll content,photosynthetic gas exchange and chlorophyll fluorescence parameters in the flue-cured tobacco leaves were investigated. The results showed that the chlorophyll content in the flue-cured tobacco leaves declined as the Cd concentration increased. The chlorophyll a content,in particular,was reduced to a much greater extent than chlorophyll b content. The photosynthetic capacity in the leaves of flue-cured tobacco was reduced mainly associated with the stomatal conductance until the Cd concentration reached 75 μmol/L. But,when Cd concentration reached 100 mol/L,the cause of flue-cured tobacco leaf photosynthetic capacity decreased to were the stomatal and non stomatal factors common restrictions. The non-photochemical queching(NPQ) and the quantum yield of light-dependent and ΔpH-and xanthophyll-mediated regulated thermal dissipation(Y_NPQ) was increased below the 50 μmol/L Cd,that means the flue-cured tobacco leaf was mainly by increasing non-radiative energy dissipation mechanisms by xanthophyll cycle to reduce the flue cured tobacco in excess excitation energy,but with the further increase of Cd concentration,NPQ and Y_NPQ was reduced,which leads to the excess light energy(1-q_P)/NPQ,the quantum yield or flux of thermal dissipation in non-functional PSⅡ and the degree of photoinhibition of PSⅡ reaction center increased dramatically,at this point,the heat dissipation and the reaction center inactivation was the major photoprotective mechanisms in leaves of flue-cured tobacco to defense the Cd poisoning. This study would provide some basic data for seeking Cd-resistant mechanism of tobacco.

To select the antagonistic bacterial strain that suited for high quality tobacco area of Anhui,the study on the antagonistically antibacterial effects of three kinds of antagonistic fermentation and the factors by the broth optical density and plate count.The results were that:three antagonistic strains had the better antibacterial effects;5,10,20,40,80 times concentration gradients had high optical density(OD) than the control(CK),and over 57%,reached to a significant level;The antagonistic order was AKJK-2013-02 > AKJK-2013-13 > AKJK-2013-11. The antibacterial effects were positively correlated with the fermentation time,and were weakened increasing storage time,and over 83.5% after storage 28 d.Three antagonistic strains had broth certain development values and application prospects.

To study the regulatory function of ethylene in plant stress tolerance,over 40 ethylene insensitive tobacco mutants were obtained from a genetic screen based on the typical seedling phenotype to ethylene in darkness,named "triple response",and most of the mutants displayed chilling resistance.The present study chose 4 mutant strains for further analyses in their chilling tolerance,physiological basis,and cold-regulated gene expressions.Under low temperature (0℃) stress,the electrolyte permeability of the tobacco ethylene insensitive mutants was significantly lower than that of the wild type,while the activities of SOD,POD and catalase were higher than those of the wild type.Further study indicated that the cold-induced genes showed highly transcription level under low temperature (0℃) stress. And,the transcripts levels of the downstream ERFs in the ethylene signal transduction pathway in the mutants were altered in the tobacco mutants.These data provide a direct molecular evidence for the strong resistance of the tobacco ethylene insensitive mutants to chilling stress.This study not only provides a new evidence for the relationship between ethylene signaling and chilling stress,but also lays a foundation for the use of biotechnology to improve crop stress resistance.

In order to realize the application of PRPs genes in plant genetic engineering,the GmPRP and SbPRP from soybean were cloned into plant expression vector of pRI101,respectively,and then introduced into tobacco by Agrobacterium-mediated transformation.Two positive transgenic tobacco plants of GmPRP and four positive transgenic tobacco plants of SbPRP were obtained.The transgenic tobacco plants were treated with salt,drought and cold stresses.The results showed that the contents of proline and soluble sugar in SbPRP transgenic plants were significant higher than wild-type plants and the contents of malondialdehyde in SbPRP transgenic plants were significant lower than wild-type plants under salt stress.While the contents of proline,soluble sugar,and malondialdehyde in GmPRP and SbPRP transgenic plants had no significant differences compared to wild-type plants under drought stress.The contents of proline in GmPRP and SbPRP transgenic plants were both significant higher than wild-type plants and the contents of malondialdehyde in GmPRP and SbPRP transgenic plants were both lower than wild-type plants under cold stress.These data indicated that the transgenic tobacco plants constitutively expressing SbPRP showed an increased tolerance to salt and cold stresses and the transgenic tobacco plants constitutively expressing GmPRP showed an increased tolerance to cold stress,which may provide a way toward the application of them.

In order to study the effects of different amount of biochar application combined with nitrogen fertilizer on the growth of flue-cured tobacco and the main chemical composition of tobacco leaves.Yunyan 87 was selected as testing material and a field experiment was designed to study the effects of nine different amount of biochar application combined with nitrogen fertilizer on the growth of flue-cured tobacco and the main chemical composition of tobacco leaves.The results showed that biochar application combined with nitrogen fertilizer could regulate and promote the growth of flue-cured tobacco,accelerate the speed of the leaves,but amount of biochar application was not positively correlated with the role of promotion tobacco growth,when applied to the biochar more than 3 750 kg/ha,and nitrogen fertilizer at 112.5 kg/ha,the role in promoting tobacco growth gradually decreased.Biochar application combined with nitrogen fertilizer could promote root growth,and improve the amount of dry matter accumulation of flue-cured tobacco.Coordinate the content of total sugar and reducing sugar,decline the content of starch,and improve the content of potassium in tobacco leaves and improve the combustibility of tobacco leaves.At the same time,it could also improve the ratio between sugar and nicotine,the ratio between sugar and nitrogen and the ratio between sugar and nitrogen of tobacco leaves.It was harmful to improve the ratio between sugar and nitrogen and the ratio between potassium and chlorine of tobacco leaves that a lower proportion of biochar application combined with nitrogen fertilizer that was 3 000 kg/ha biochar and 75.0 kg/ha nitrogen fertilizer.It was concluded that 3 750 kg/ha biochar and 112.5 kg/ha nitrogen fertilizer was the optimal proportion which had faster growth of tobacco plant and the appropriate content of main chemical composition of tobacco leaves.