In order to study the effects of continuous drought treatment in whole growth period on cotton growing development and final yield,in this experiment,pot-culture method was taken and Xinluzao 19hao,Xinluzao 27hao and Xinluzao 54hao as materials,the treatments were light and moderate drought stress,then measured morphological indexes,physiological indexes and photosynthetic parameters at seedling stage,bud stage,blooming stage and boll opening stage,and investigated yield components at harvest time.Results showed:cotton height and stem diameter decreased with the strengthen of drought stress; The content of reactive oxygen species and antioxidant enzyme activities were great difference at different growth stage,correlation analysis showed that there were significant or extremely significant correlation between active oxygen content and antioxidant enzyme activity,indicted that antioxidant enzymes activity changed regularly with the content of reactive oxygen species,so as to eliminate the excessive oxygen species in vivo and prevent the damage of reactive oxygen species; The photosynthesis of cotton at bud stage decreased with the strengthen of drought stress,but increased at boll opening stage,except Xinluzao 27hao which possibly due to persistent drought treatment caused the different developmental stage of cotton at later growth stage; Drought stress reduced the yield per plant and fiber length of Xinluzao 27hao and Xinluzao 54hao,light drought stress had little effect on single boll weight,even played a role in promoting; There were four indexes of cotton at blooming stage had significant correlation with yield per plant,indicted that cotton blooming stage might be the key period which easy to be affected by environment and determined the final yield.

MYB transcription factor proteins are ubiquitous in plants and play an important role in biological and abiotic stresses. To explore the function of cotton MYB gene, the paper cloned a MYB gene from cotton leaves using homologous cloning technology, and analyzed its bioinformatics and expression under different stresses. The results showed that a new MYB transcription factor gene GhMYBPA1 (gene entry site XM_016869420) was successfully cloned from Gossypium hirsutum cv. Zhongmian 35, the full of cDNA was 825 bp in length with a 630 bp ORF, which encoded a 210 amino acids peptide. Bioinformatics analysis results showed that the molecular weight of GhMYBPA1 was 20.183 ku, GhMYBPA1 contained two conserved DNA-binding domains at N-terminal, which belonged to R2R3-MYB transcription factors. Amino acid homology analysis showed that GhMYBPA1 had higher identification with GaMYB12-like from Gossypium arboreum. Based on qRT-PCR analysis, GhMYBPA1 was constitutively expressed in cotton roots, stems, leaves, and it was dominantly expressed in flowers and then was leaves. Moreover, the results of plant treated with various stresses showed that the expression of GhMYBPA1 gene changed under high salt, low temperature and drought stress, it was suggested that GhMYBPA1 might play an important regulatory role in the abiotic stress process of cotton. The results could lay a theoretical foundation for further researches on the function of GhMYBPA1 gene.

In order to study the function of GhAOP2-like in cotton,based on the previous proteomic data from the laboratory of Institute of Cotton, Hebei Academy of Agriculture and Forestry Sciences, this study obtained the gene sequence of GhAOP2-like using homologous cloning strategy.The physicochemical properties,structure and subcellular localization of GhAOP2-like protein were analyzed by bioinformatics method.The tissue-specific expression of GhAOP2-like and the change of expression level under drought,salt and hormone treatment were detected by quantitative real-time polymerase chain reaction(qRT-PCR).The results showed that the CDS sequence length of GhAOP2-like was 972 bp and encoded 323 amino acids.GhAOP2-like was located on chromosome D13.GhAOP2-like protein did not contain signal peptides and transmembrane domains,and was located in the cytoplasm with the molecular formula of C₁₆₅₄H₂₅₂₅N₄₂₉O₄₇₈S₁₉ and the theoretical isoelectric point was 5.20.The clustering analysis results showed that the AOP sequences in cotton and other species were clearly divided into two groups,but had the closest relationship with the AOP sequences of Hibiscus syriacus.qRT-PCR results showed that GhAOP2-like was expressed in roots,stems,leaves,and developing seeds,but the expression level of GhAOP2-like was the highest in roots.GhAOP2-like was up-regulated under drought,salt,GA₃,MeJA and ABA treatments,but the expression level changed most in MeJA treatment,indicating that GhAOP2-like might improve the resistance of upland cotton by participating in JA signaling pathway.

The purpose of this study was to find the relevant candidate genes for development of fiber or floral organ in cotton.Based on the available genome data of cotton,the WOX gene family was identified by bioinformatics analysis.The chromosome distribution,gene and protein structures,phylogenetic relationship and gene expression patterns of this family were comprehensively compared.The results showed that a total of 37 WOX genes in cotton genome were found and then designated as GhWOX1-GhWOX37,which were distributed in 20 subgenomes except for A04,A06,A09, D04,D06 and D09.A subgenome had one more GhWOX gene than D subgenme.The multiple sequence analysis of GhWOX gene family presented the highly conserved homeodomain.Phylogenetic tree analysis showed that 37 WOX genes could be divided into Ⅰ,Ⅱ and Ⅲ subfamily and the subfamily respectively contained 23,7 and 7 WOX family genes in cotton.A Blast search on the NCBI EST database showed that 11 GhWOX genes had no matching EST sequence,and other 26 GhWOX genes were expressed in various tissues of root,stem,leaf,flower,ovule,fiber and seed.The results would be helpful for further analysis of WOX family proteins in other plants.

Expansin can loosen the components of rigid plant cell walls and thereby allow cell expansion.To get insight into expansin genes in Gossypium raimondii,genome-wide exploration and comprehensive characterization of the G.raimondii expansin gene family members were conducted,and 39 expansin genes(including 26 EXPA,4 EXPB,3 EXLA and 6 EXLB,which was classified based on the phylogenetic tree) were identified.The results revealed that expansin family genes were located on 12 of 13 G.raimondii chromosomes.And the gene structures were relatively diverse(not conserved) in each subgroup.Evolutionary analysis of expansins revealed that chromosome segmental duplications contributed mainly to the three evolutionary expansions of expansin family,which had experienced negative selection pressure.The expression pattern of expansin genes under series of fiber development stages,in leaf and petal indicated that most expansin genes(showing diverse and specific expression pattern) might participate in fiber development processes including fiber initiation and elongation,and in morphogenesis of leaf and petal.This identification and characterization provided the complete profiles of Gossypium expansin family genes for future study on their functions related to the molecular mechanisms of fiber and other tissues development.

In order to further understand the structure and function of NF-YB gene family,we have systematically investigated the number,subcellular localization,chromosome distribution,evolutionary relationships,motif and tissue expression pattern of of family in the genome of Gossypium hirsutum L.acc.TM-1 by bioinformatics method.41 NF-YB genes were identified in TM-1 genome;the 41 NF-YB genes contain the same CBFD_NFYB_HMF domain,and most of them were located in nucleus; they distributed on 19 chromosomes,and there were 19 pairs of the genes on the A subgroup and D subgroup were orthologous genes; 41 NF-YB genes can be divided into groupⅠand groupⅡ,and there were similar motif type and arrangement in each group;.24 NF-YB genes were expressed,but the expression pattern varied among different tissues.

To study the effects of cotton straw returning soil on soil microbes quantities and enzyme activities,experiments were conducted at the Experimental Station of Shandong Agricultural University with two treatments(4-year straw returning soil and no straw returning soil) to investigate soil microbes quantities and enzyme activities across 0-60 cm depths after cotton straw returning for four consecutive years.The main results indicated that cotton straw returning soil significantly increased the quantity of total microbe by on average 19.87%(0-20 cm),20.07%(20-40 cm) and 56.15%(40-60 cm),including soil bacteria and fungi quantity significantly increased by on average 20.91%,26.38%(0-20 cm),20.59%,31.18%(20-40 cm),56.85%,32.30%(40-60 cm),respectively,comparing to no straw returning soil treatment;soil actinomyces quantity increased by on average 4.29%(P>0.05),11.62%(P<0.05),54.00%(P<0.05)across 0-20 cm,20-40 cm,40-60 cm depths,respectively,comparing to no straw returning soil treatment.Cotton straw returning soil had no significant effect on soil urease activities at 0-20 cm depth,comparing to no straw returning soil treatment,while straw returning soil significantly in creased soil urease activities across all sampling times except September at 20-40 cm and all sampling times except July at 40-60 cm depths by on average 13.43% and 24.03%,respectively.Except May at 20-40 cm and August at 40-60 cm depths,soil sucrase activities after cotton straw returning soil significantly increased across 0-20 cm,20-40 cm and 40-60 cm depths by on average 27.08%,46.96% and 57.59%,comparing to no straw returning soil treatment.Except July and August at 0-20 cm depth,cotton straw returning soil significantly increased soil catalase activity at by on average 8.73%,comparing to no straw returning soil treatment,while straw returning soil had no significant effect on soil catalase activity across 20-40 cm and 40-60 cm depths except May and June at 20-40 cm depths.These results indicated that cotton straw returning soil could maintain and improved the biological characteristics of the soil.

Currently,two EPSPS genes from Gossypium hirsutum have been submitted to NCBI.It is more convinent to identify EPSPS genes in genome level of upland cotton comprehensively following the publication of Gossypium hirsutum genomic sequence.Four EPSPS homologous genes were identified from the genome sequence database of allotetraploid cotton (Gossypium hirsutum L.acc.TM-1),and they were found to be distributed on subgenomes of A07,A12,D07 and D12.Under the comperation of nucleotide sequences,amino acid sequences and gene structures,phylogenetic analysis of constructing phylogenetic tree and the study of transcription situation of these four genes,it was more clearly that the two genes located on A07 and D07 subgenomes belong to highly homologous co-linear gene,and the two genes located on A12 and D12 subgenomes as well.Sequence alignment results showed that the two EPSPS genes submitted to NCBI were the two genes located on A12 and D12 subgenomes respectively.The study results of this paper would provide an theoretical basis on cloning and functional research of EPSPS genes which located on A07 and D07 subgenomes.

GhTGA1 and GhTGA9.2 of transcription factor genes were isolated by RT-PCR technology from Zhongmiansuo 12 cultivar of resistance to Fusarium oxysporum. Sequence analysis showed that the ORF of GhTGA1 and GhTGA9.2 were 1 281 bp and 1 461 bp,respectively.They encoded 405 and 486 amino acids,respectively. GhTGA1 and GhTGA9.2 all contained two conserved bZIP and DOG1 domains. They belonged to the TGA transcription factor gene of bZIP subfamily. The GhTGA1 and GhTGA9.2 expression feature were detected by Real-time quantitative PCR(qRT-PCR),the results showed that GhTGA1 gene was up-regulation expressed with the duration of the treatment of Fov,ethylene (ET) and jasmonic acid (JA). In each treatment time point,the expression of GhTGA1 gene was lower than that of Mock with the treatment of salicylic acid (SA). We speculated that GhTGA1 gene might be involved in ethylene (ET) and jasmonic acid (JA) signaling pathway and regulated resistance to Fusarium oxysporum in cotton; After the treatment of Fusarium oxysporum,the expression of GhTGA9.2 gene was lower than Mock at different treatment time points,and was down-regulated expression. In other hand,it was up-regulation expressed with the duration of the treatment of hormone (ET,JA,SA).It suggested that GhTGA9.2 might participate in stress response through hormone (ET,JA and SA) pathways. The analysis of gene expression characteristics of GhTGA1 and GhTGA9.2 provides a basis for further study.

In order to provide references for nitrogen fertilization in field production, the effects of different NH₄⁺/NO₃^- ratio of nitrogen nutrition (NH₄⁺-N/NO₃^--N were 0/100, 25/75, 50/50, 75/25, 100/0)on morphological characteristics and dry matter accumulation, hydroponic experiments were conducted using Nongda 601 as material. The results showed that the plant height, stem diameter, leaf area and main root length of cotton increased gradually with the growth stages changed. In addition to the main root length showed a continuous elongation, the plant height, stem diameter, leaf area and dry weights of cotton increased firstly and then decreased with the increasing of NH₄⁺ concentration in the ammonium nutrient solution. The plant height of cotton seedlings was the highest at the NH₄⁺/NO₃^- ratio of 50/50 and 25/75 respectively 7, 14, 21 d after enhanced ammonium nutrient solution treatment;both stem diameter and leaf area were the largest at the NH₄⁺/NO₃^- ratio of 50/50 after enhanced ammonium nutrition treatment 7, 14, 21 d, indicating that the concentration of NH₄⁺ more than 50% could inhibit significantly the growth of cotton seedlings. In terms of dry matter, total dry weight, stem dry weight and leaf dry weight were the highest at the NH₄⁺/NO₃^- ratio of 75/25, while there was no significant difference in root dry weight and root to shoot ratio, indicating that the effect of enhanced ammonium nutrition on the dry matter weight was positive mainly in the aboveground, while the effect in the underground was not obvious. Moreover, the ratio of root to shoot was the highest, the main root length was the longest when supplied with only NH₄⁺, indicating that single NH₄⁺ could promote root growth. All results showed that ammonium and nitrate nitrogen had different mechanisms for regulating growth and development in cotton underground and aboveground organs, mixed nitrogen was more favorable to the aboveground growth of cotton seedlings.

In order to analyze the structure and function of the promoter of cotton NAC transcription factor gene GhSNAC3,the promoter fragment was cloned and identified from Lumianyan 32 in Gossypium hirsutum L..The cis-acting elements in the promoter sequence was predicted and analyzed by using the online database of Plant CARE,PLACER and BDGP.A plant expression vector consisting of GhSNAC3 and its promoter was constructed and transformed into Nicotiana tabacum L.to investigate the transcription activities of the promoter under different stress treatments.The results showed that besides the basic cis-acting elements such as CAAT-box,there existed several MeJA,GA and ABA-responsive elements as well as multiple light-responsive elements and stress-induced controlling elements in the promoter.Under salt stress GhSNAC3 had a high level of expression in the roots of transgenic tobacco plants,while the expression level was low in the stems and leaves,suggesting that the gene GhSNAC3 might played an important role in stress responses in cotton,and its promoter might be tissue-specific and salt-inducible.The results provide the basis for the further study of cotton NAC signal pathway as well as salt tolerance improvement through genetic engineering in the future.

To further study the mechanism of cotton JAZ genes in low temperature response,a cold response gene GhJAZ1 was cloned from the upland cotton Zhongmiansuo 36 by RT-PCR (GenBank accession number KJ562212).The CDS sequence of GhJAZ1 was 810 bp in length and encoded 270 amino acids.The predicted molecular weight of this protein was 29.808 ku,and the theoretical isoelectric point was 8.33.It was not a transmembrane protein.The encoded protein contained a N-terminal NT domain,a TIFY domain,and a C-terminal Jas domain.Phylogenetic tree analysis showed that GhJAZ1 was much closer to that in Theobroma cacao and Corchorus olitorius.Quantitative real-time PCR analysis showed that GhJAZ1 had significantly high expression in hypocotyls and roots.In addition,the expression of GhJAZ1 was also induced by abscisic acid(ABA) and methyl jasmonate (MeJA).The trend of expression was first raised and then decreased. Under MeJA treatment, the gene expression varied more obviously. The expression pattern of GhJAZ1 varied in different cold resistant cotton materials under cold stress. The up-regulated expression of GhJAZ1 gene in cold-resistant cotton variety-Zhongmiansuo 36 was more significant, which suggesting that GhJAZ1 gene may be involved in cold response in cotton. Subcellular localization showed that GhJAZ1 protein was localized in the nucleus.This study lay foundation for further researches on the molecular mechanism of GhJAZ1 in cold response.

In order to excavate the role of exogenous silicon(Si) in salt stress response and explore the regulation mechanism of salt stress response, the cotton variety Xinluzao 45 was used as test material, and was treated with 3 levels of salt(NaCl:0, 100, 200 mmol/L) and 2 levels of silicon(K₂SiO₃:0, 262.3 mg/L). The changes of seedling growth, osmotic regulation, reactive oxygen, malondialdehyde(MDA) content and electrolyte leakage rate were measured after the treatment of exogenous Si, and the effect of silicon in relieving the inhibitory effect of NaCl on cotton seedlings were studied. The results showed that, compared to CK, the fresh leaf weight, dry leaf weight, stem fresh weight, stem dry weight, root dry weight, root cap ratio, root activity, malic acid and citric acid of cotton seedlings all showed a downward trend along with the increase of NaCl levels, while the contents of free proline(Pro), hydrogen peroxide(H₂O₂) and malondialdehyde, the rate of electrolyte leakage and the oxygen free radical(O₂^·) production presented a upward trend, and SS and free amino acid contents increased first and then decreased. The biomass and osmotic regulator of cotton seedlings increased obviously in the same salt treatment after application of exogenous silicon, while the rate of O₂^· production, electrolyte leakage rate, the contents of hydrogen peroxide and malondialdehyde in cotton seedlings decreased obviously. Comprehensive analysis showed that the salt stress could inhibit the growth of cotton seedlings, and with the increase of salt concentration, the inhibition effect and osmotic stress degree aggravated. The exogenous silicon increased the accumulation of osmotic regulation substances and reduced the accumulation of active oxygen in cotton seedlings, so it could alleviate the inhibiting effect of salt stress on cotton seedling growth and improve the salt resistance of cotton seedlings.

In order to study the function of cotton CBF2 gene in plant resistance,the complementary expression vector pCAMBIA1300-AtCBF2P-GbCBF2-AtCBF2T was constructed,and transformed to Arabidopsis cbf2 mutant by genetic engineering.The four CBF2 genes were cloned from cDNA of the cotton Xinhai 16 and Arabidopsis CBF2 gene promoter AtCBF2P and termination AtCBF2T were also cloned by PCR method.Firstly,these genes were connected with pBluescript SK,and obtained the intermediate transition vector pBluescript SK-AtCBF2P-GbCBF2-AtCBF2T,then the AtCBF2P-GbCBF2-AtCBF2T fragment was obtained by cutting the intermediate transition vector with Sac Ⅰ and BamH Ⅰ,after the fragment inserted into pCAMBIA1300,the complementary expression vector pCAMBIA1300-AtCBF2P-GbCBF2-AtCBF2T was constructed.The cotton CBF2 gene was transformated into Arabidopsis cbf2 mutant by Agrobacterium mediated method and regenerative seedlings were obtained.These results will lay the foundation for screening cotton negative regulator CBF2 gene and stress-resistance molecular breeding.

It is of great significance to investigate the response of arginase gene to abiotic stress and its physiological function in cotton. Bioinformatics analysis showed that the ORF sequence of GhARG1 was obtained by In silico cloning and RT-PCR technique. Salt stress, PEG6000 stress and ABA, SA and MeJA treatment cotton seedlings, and GhARG1 was expressed in E. coli prokaryotic. The ORF sequence contained 1 023 bp, and encoded 340 amino acids with typical conserved domains of arginase. Quantitative RT-PCR analysis showed that the expression of GhARG1 in leaves was up-regulated under stress condition of NaCl, PEG6000, and signal molecule of ABA, SA, respectively, but down-regulated by MeJA. The coding sequence of GhARG1 was fused to prokaroytic expression vector pCold-TF, and the combinant vectors were induced by IPTG and detected by SDS-PAGE. The molecule weight of protein GhARG1 was detected to be the expected 37 ku. The activity of arginase in the recombinant strain was 6 times that of the control. The results suggested that GhARG1, which coding product has arginase activity, might be involved in the response to salinity and drought stress through ABA and SA signaling pathway in cotton, and could lay foundation for further physiological functional identification.

BES1 gene is a major transcription factor responsible for brassinosteroid-regulated gene expression and plant growth.In order to know the family of GhBES1 gene in upland cotton,21 GhBES1 genes were identified from the genome sequence database of upland cotton (Gossypium hirsutum L.acc.TM-1).These GhBES1 genes were found to be distributed on different subgenomes.There were 9 pairs of GhBES1 genes in the A and D subgenomes with one-to-one correspondence relationship,and the gene sequence identity of the correspondence genes were higher than 95%.Based on the results of phylogenetic tree,the 21 GhBES1 genes could be divided into four subfamilies,and there were homologous genes of Arabidopsis thaliana L.in each subfamily,except for subfamily Ⅲ; GhBES1 genes had two exons, except for genes in subfamily Ⅳ and GhBES1-1 in subfamily Ⅱ.The result of subcellular localization indicated that 21 GhBES1 proteins were primarily located in nuclear and cytoplasmic,and 7 pairs of GhBES1 genes had the same location.The tissue expression analysis showed that GhBES1 genes were expressed in the leaf,root,stem and shoot apical meristem,but the expression level of GhBES1 genes in different subfamily were different.These results provide a theoretical basis on cloning and functional research of GhBES1 genes.

In order to explore the genetically modified effects of excessive expression of superoxide dismutase in the upland cotton.Cotton cytoplasmic superoxide dismutase gene sequences of nucleic acids was found in the Genbank database,a pair of specific primers were designed,the cotton cytoplasmic superoxide dismutase gene was cloned and the plant binary expression vector was built,the excessive expression of transgenic cotton plants were obtained by used a new technology of cotyledon axillary bud of Agrobacterium mediated genetic transformation.PCR and Southern Blot analysis indicated that the new copy Cu/Zn-SOD gene had been integrated into the genome of cotton.Germination experiment of genetically modified materials T₁ generation seeds showed that under the stress of NaCl,the average length of taproot,bud average long and average number of lateral root of genetically modified materials were greater than the negative control,the SOD activity in transgenic plants leaves were significantly improved than that in the control,so were the chlorophyll contents,the MDA content was dramatically declined,but praline contents was no changed.If excessive cytoplasmic SOD genes were expressed in the cotton,the SOD activity of plantlet can be increased,this can reduce salt stress caused by the cell membrane peroxidation,and enhance the ability of plants to resist salt stress.

Effects of optimized fertilization on plant growth and soil nutrients supply in saline fields of Dongying City were studied under filed condition for two years.Four treatments consisting of no fertilization, farmers traditional fertilization,optimized fertilization,optimized fertilization+organic fertilizer.The results showed that cotton seed yield,fertilizer agronomic efficiency and partial factor productivity was effectively enhanced by optimized fertilization and optimized fertilization+organic fertilizer,compared to farmers traditional fertilization.Composition of saline ions was changed by fertilizer application which was remarkably higher than no fertilization in various degree in the contents of K⁺,Ca²⁺,Mg²⁺and SO₄^2-,meanwhile the percentage of Na⁺and Cl^- was reduced 2.29%,3.45%and 6.15% by farmers traditional fertilization,optimized fertilization and optimized fertilization+organic fertilizer. Microorganism and enzyme activity of soil was increased by optimized fertilization+organic fertilizer significantly, that the activity of urase and alkaline phosphatase was enhanced 7.8%-17.0% and 5.0%-13.3% respectively, compared to farmers traditional fertilization and optimized fertilization. The available nutrients of soil were increased by fertilizer application, which showed that the available P and K contents of optimized fertilization+organic fertilizer treatment were in higher levels. Meanwhile, the NO₃^--N and NH₄⁺-N contents were farmers traditional fertilization > optimized fertilization+organic fertilizer > optimized fertilization > no fertilization, that it indicated that the contents of NO₃^-N and NH₄⁺-N were reduced by optimized fertilization,then the risk of running off would be re duced.In the condition of reducing chemical fertilizer, useing the optimized treatment of nitrogen and potassium basal application and dressing at bud and flowering stage could not only ensure the purpose of cotton yield, but also reduce the dosage of fertilizer, meanwhile the effect of cotton yield while cooperating with organic fertilizer was better.