To study the expression of grape stress tolerance gene (CAN70200.1),a 1 354 bp promoter fragment (named as P_CAN) upstream of the gene CAN70200.1 was isolated by using PCR technology.Promoter sequence was analyzed by the database of PlantCARE and PLACE.The result showed that the P_CAN sequence contained basic elements CAAT-box,TATA-box and some cis-acting elements that response to abiotic stresses,light and plant hormones.To verify the expression pattern of the promoter,the P_CAN fragment was fused with GUS reporter gene located on pCAMBIA1391Z to construct a plant expression vector p1391Z-CAN,followed by transformation into tobacco by Agrobacterium-meditated method.The expression activity of P_CAN promoter reached highest at 120 min after drought stress treatment or at 30-60 min under 4℃ cold treatment condition,indicated that the P_CAN promoter could express under the condition of treatments with cold and drought.

Using homology cloning method,the full-length cDNA of galactinol synthase gene VvGolS2, VvGolS3 and VvGolS4 were cloned from Vitis vinifera cultivar Zuoyouhong tissue culture seedling.The analysis of VvGolS2, VvGolS3 and VvGolS4 sequencing indicated that three genes were 954,978 and 1 011 bp ranging in size,coded as 317,325,336 amino acids respectively with molecular weight were 36.65,36.97,38.12 kDa and the isoelectric point were 5.12,5.33,5.16 respectively.Three VvGolSs all possessed a conserved hydrophobic APXAA pentapeptide domain in the C-terminal region.Real-time PCR analysis showed that three VvGolSs were expressed different in all tested tissues,with the highest expression of VvGolS2 and VvGolS4 in leaves,and VvGolS3 was highly expressed in flowers and tendrils. Using tissue culture seedling,the relative expression levels of VvGolS2, VvGolS3 and VvGolS4 after different stresses or different stress singling factors treatment were checked.The VvGolS2 and VvGolS4 were highly induced by salt stress,while the VvGolS3 was only induced by low temperature.Moreover, VvGolS2 was significantly induced by stress-related signal molecules,such as abscisic acid (ABA),ethylene (ACC),and hydrogen peroxide (H₂O₂).The VvGolS4 was induced by ACC,H₂O₂ and H₂S,and the VvGolS3 was induced by ABA and ACC.Together,these results suggested that these three VvGolSs were involved in abiotic stresses resistance in grape with different functions.

To explore the potential role of Resveratrol synthase(Rs) in fruit development of Vitis vinifera cv. Kyoho, the structure and function of Rs sequence were analyzed, and the spatio-temporal expression specificity at different developmental stages of fruit was identified. The bioinformatics analysis and qRT-PCR method were used to analyze the sequence of Rs gene in Kyoho grape. The structure and subcellular localization of Rs gene were predicted, and its expression in different tissues of fruit at different developing stages was analyzed. The full-length cDNA of Rs gene was 1 539 bp, with an open reading frame(ORF) of 1 179 bp, encoding 392 amino acids. The predicted molecular weight was 42.88 ku, and the theoretical pI was 6.09. The gene contained chalcone, stilbene synthase activity sites and complete stilbene synthase family characteristic sites. The protein interaction predicted that Rs interacts with OMT2.1, which catalyzes resveratrol biosynthesis of Pterotilbene. The results of subcellular prediction showed that the Rs gene mainly expressed in the cytoplasm and chloroplast. The promoter analysis showed that the expression of Rs gene might be regulated by light, MYB, fungi and hormones, presenting a certain tissue specificity. qRT-PCR analysis showed that the Rs gene of grape expressed at different stages of the peel and flesh, but the expression level was highest in the 25th day after anthesis. Combined with the role of Rs gene accumulation in resveratrol, it could be inferred that there might be a high accumulation of resveratrol in the fruit peel during the early period of fruit development. The Rs gene of Kyoho grape had a high conservation during evolution, and its expression might be regulated by environment, fungi and hormone. It had the interaction with OMT2.1 and had a certain specificity in different tissues and development periods.

To analyses the effects of water deficit in different stages on the variation tendency of soil biological characteristics (soil invertase,urease,soil microbial biomass carbon,soil organic carbon)and the fruit yield and quality of greenhouse grape under delayed cultivation,the experiment was carried out at Zhangye Irrigation Experiment Station in Gansu Province in 2013-2015.The results showed:water deficit treatment during germination and shoot growth stage could improve soil invertase activity.There was no difference in water deficit treatment in flowering period compared with CK treatment.The treatment of water deficit at the fruit expanding stage and the coloring maturity stage could inhibit the activity of soil invertase.The germination period was the most suitable period to improve the activity of soil invertase by water deficit.The treatment of water deficit at the germination stage and at the color maturity stage could inhibit the activity of soil urease.The water deficit treatment from the shoot growth stage to the fruit expanding stage could improve the soil urease activity in the corresponding growth stage.For the soil microbial entropy,the treatment of water deficit at the growth stage of new shoots had a significant effect on it.According to the results of correlation analysis and canonical correlation analysis,the soil invertase activity increased with the increased of fruit yield,while the content of soil microbial biomass carbon decreased significantly.The content of titratable acid increased significantly when soil urease activity increased.

To investigate the function of VvMSA, the full-length cDNA of VvMSA was cloned from Vitis vinifera cultivar Vidal Blanc tissue culture seedlings.Bioinformatic analysis and quantitative Real-time PCR was used to analyze its characteristics.The results showed that VvMSA amplified fragment size of 450 bp encoding 149 amino acids with molecular weight 16.703 kDa,isoelectric point 5.68.VvMSA was an unstable hydrophilic protein and contained a conserved domain ABA/WDS encoding by 65 amino acids.VvMSA and the known homolog LeASR1 in tomato belongs to different sub-families.Real-time PCR analysis showed that the VvMSA was expressed in all tested tissues with the highest expression in flower. VvMSA was induced by salt,drought and cold stress in different levels,and highest expression level was showed after 3 h salt treatment.In addition, VvMSA was also induced by nitric oxide(NO),hydrogen sulfide(H₂S),abscisicacid(ABA) gibberellin(GA₃),auxin(IAA)and ethylene(ET),while salicylic acid(SA)inhibited its expression.The expression pattern of VvMSA by salt treatment was similar with the expression pattern by ABA treatment.In total,these results suggested that the VvMSA was involved in salt resistance by ABA signaling.

In order to provide high efficient viticultural management in Beijing alluvial plain,we studied the water consumption regularity of grapevine growing in such areas. The experimental vineyard was located in Pinggu District,Beijing. Rain shelter cultivation,soil mulching and drip irrigation were used during the growing season,the row and plant spacing was 3 m×2 m. By using a soil moisture sensor(Beijing Insentek Corporation) in the whole year,the soil moisture data of different depth(10-30,0-50,50-70,70-90,90-110 cm) of six-year-old Ruiduxiangyu(Vitis vinifera) are collected. The decrement of soil moisture during day time was considered as water consumption while during night time was considered as infiltration. Followed by this logic,the water consumption of different soil layers in each phenological period were calculated;Combined with monthly precipitation during almost 30 years in Beijing,we found out the regularity of grapevine water consumption. The results indicated that:From January to June,the amount of water consumption was quite close to precipitation;While during July to September that contributed 78% to the whole year's precipitation,the rainfall during these three months was 300 mm more than the water consumption. The water consumption during each phenological period were as follows:budburst was 12 mm,shoot rapid growing was 24 mm,blooming was 30 mm,fruit development was 72 mm,verison was 43 mm,fruit maturation and nutrient back-flowing period were the same,about 40 mm.The daily water consumption was about 1 mm from budburst to shoot rapid growing;It increased to 2.1 mm per day during blooming and 2.4 mm during fruit development;Then it decreased to 1.34 m during verison and 1.0 mm from fruit maturation and nutrient back-flowing period. During the first half of growing season,the proportion of water consumption of 10-50 cm was almost 70%;The proportion of water consumption of 50-70 cm was 8%-10% during budburst and shoot rapid growing,while maintained around 13%-19% from blooming to nutrient back-flowing period;The proportion of water consumption of 70-90 cm was 10%-14% from budburst to verison while increased to 18%-20% from fruit maturation to nutrient back-flowing period;The proportion of water consumption of 90-110 cm was only 7% from budburst to verison,while increased to above 20% from maturation to nutrient back-flowing period. In conclusion,daily water consumption changes dramatically during the first half of growing season,it remained relatively stable during the latter period;The highest daily water consumption occurred at fruit development. It presented logarithm relationship that the water consumption decreased as the depth of the soil increased. Besides,water consumption of grapevine root system showed temporal and spatial variation,shallow root system consumed much more during the former period of growing season while deep root system showed the opposite.

Toll-like receptor (TLR) family plays a key role in innate immunity and various inflammatory responses,for recognizing the bacterial pathogen and mediated the signal path of inflammation.To investigate the expressions of TLR2 in mammary glands of Small-tail han sheep were infected with Staphylococcus aureu, and to explore the mechanism of TLR2 in mastitis,the qRT-PCR and IHC technology was used to detect the expression of TLR2 mRNA and TLR2 protein in the mammary gland of control and experimental groups.The results showed that the TLR2 mRNAs and protein were expressed in all mammary glands of Small-tail han sheep in control and experimental groups.The levels of TLR2 mRNA and protein was lowest in control group,was highest at 48 h after Staphylococcus aureu infusion,significantly higher than that of control and 96 h group(P<0.05).After infected 96 h,the levels of TLR2 mRNA and protein was significantly reduced than that of 48 h group(P<0.05).The protein of TLR2 were located in the epithelial cell of mammary gland acinus,were expressed in epithelial cell of mammary gland acinus dropped and inflammatory cells as lymphocytes after effected Staphylococcus aureu.The results implied that the expression of TLR2 in the epithelial cell of mammary gland was up-regulated to recognize pathogenic bacteria,arouse the innate immunity.

The purpose of this study was to identify and analyze HXK gene family in grape. Based on the HXK(hexokinase) gene registration sequence of Arabidopsis thaliana and maize, four HXK genes were identified from the whole genome of Vitis vinifera, namd as VvHXK1, VvHXK2, VvHXK3 and VvHXK4. The bioinformatics analysis were performed on these four genes. The results of protein physicochemical properties showed that the number of amino acids encoded by the four genes was between 412 aa and 524 aa, of which VvHXK4 was a basic amino acid, and the other three were acidic amino acids. In addition, subcellular localization analysis indicated that all four VvHXK genes were localized in the cytoplasm. VvHXK4 was mainly localized in chloroplasts, and VvHXK2 might appear in mitochondria and nucleus. Meanwhile, the secondary structure prediction of the proteins found that the proteins encoded by the four VvHXK genes were mainly α-helix. The structural analysis of exons indicated that VvHXK1 contained 10 exons, and others contained 9 exons, showing that the VvHXK genes were relatively conserved; The initiation of cis-acting element analysis indicated that four VvHXK genes contained ABA response elements, MYB binding region and WRKY transcription factors, but no low temperature response component were found in VvHXK4. Phylogenetic tree analysis showed that the homology relationship between VvHXK1 and Arabidopsis AtHXK1 was the closest, indicating that it not only had the functions of regulating growth and development and affecting the growth of roots, but also had the effect of accelerating aging. The homology relationship between VvHXK2 and potato StHXK1 was recent, showing that it could increase the glucose phosphorylation activity in leaf extract or chloroplast. The homology relationship between VvHXK3 and potato StHXKRP1 was the closest, suggesting that VvHXK2 might be able to induce the expression of sugar in leaves. The homology relationship between VvHXK4 and spinach SoHXK1 was recent, which was important for studying the glucose metabolism of grapes. Fluorescence quantitative analysis showed that VvHXK2, VvHXK3 and VvHXK4 had the highest relative expression under the treatment of 20 g/L glucose, which was the most significant for glucose phosphorylation, and low expression under the treatment of fructose and sucrose. This is important for studying the sugar metabolism of grapes.

In order to study the effect of 6-BA on grape fruit organic acid accumulation,and the possible molecular mechanism of 6-BA regulating organic acid synthesis,a grape cultivar,Rizamat was selected to study the effect of 6-BA on fruit organic acid accumulation and related genes expression. The inflorescences and fruit clusters were treated with 6-BA at 5 days before blooming,3 and 10 days after anthesis respectively.The results showed that organic acid content of the mature grape fruit was closely related to treatment concentration of 6-BA. Tartaric acid and total organic acid content of the mature fruits that treated by 30 mg/L 6-BA was significantly lower than control. But it didn't play important roles in the content of malic acid,citric acid and oxalic acid. For 10,20 mg/L 6-BA treatment,all kinds of organic acid didn't show significant difference between treated and control mature fruits. Gene expression result showed that the expression of IDH, MDH and PEPC were mainly suppressed by 6-BA treatment,but it also promoted the expression of ME. Therefore,we speculated that the reason for inhibiting organic acid synthesis was that parts of the organic synthesis related genes expression were affected by 6-BA treatment.

To explore the effects of different light qualities on the expression of CO4 gene in grapevine plantlet in vitro. The full-length cDNA sequence of CO4 was cloned from Vitis riparia×V.labrusca Beta in vitro by RT-PCR. Bioinformatics analysis of the second level structure, and phylogenetic tree of protein. The result showed that the size of CO4 gene fragment was 663 bp, the open reading frame (ORF)of CO4 gene was 747 bp, including 248 amino acid coding regions. By use bioinformation analysis, CO4 of Beta was a hydrophobic, unstable protein with good lipid solubility. CO4 had a signal peptide, it can be inferred that it was a transmembrane protein. Phylogenetic tree indicated that CO4 of Beta (Vitis riparia×V.labrusca)had the highest evolutionary relationship with Eucalyptus grandis (Eucalyptus grandis Hill ex Maiden).qRT-PCR analysis showed that the CO4 expressed specifically and strongly in the treatment of red to blue light (control), which increased 5.39 fold in comparison to the control. The difference was not significant when exposed to the treatment of white to red light and red to white light. However the expression of CO4 was the lowest in the white to blue light treatment, which was 54% of the control. Fluorescence parameter analysis showed that the maximum photochemical efficiency (Fv/Fm), potential activity (Fv/Fo)and photochemical quenching coefficient (qP)were significantly higher than those of the control under red to blue light. The non photochemical quenching coefficient (NPQ)was the lowest under that treatment, but was the highest under red light treatment, it was the lowest under this treatment. The CO4 of Beta was more sensitive to light. The treatments of red to blue light could not only promote the expression of CO4, but also could improve the Beta Grapevine in vitro photosynthetic capacity. Thus, red to blue light played an important role in the process of morphogenesis of Beta Grapevine in vitro.

This paper aimed to study the effects of nitric oxide(NO) and low temperature treatment on storage quality of grape fruits. Using Jufeng grapes as the experiment material,the grape fruits were soaked with 5,15 and 30 μmol/L NO solution and then stored at 0,25℃. The physiological index including fruits firmness,vitamin C,soluble protein,soluble sugar,reactive oxygen(ROS),malonaldehyde(MDA)and proline were investigated. Compared with 5 and 30 μmol/L NO treatment,15 μmol/L NO kept the grape fruits firmness effectively and delayed the concentration of soluble protein,the content of soluble sugar and vitamin C,also inhibited the increase of reactive oxygen and MDA content and increased the content of proline. 15 μmol/L NO made the grape fruits flavour kept long time during postharvest storage. However low temperature can keep grape fruits hardness effectively,reduce the content of MDA and reactive oxygen,slower the process of fruits rot greatly.The experimental results showed that 15 μmol/L NO and 0℃ storage could keep grape nutritional quality and delay the lower of fruits rot effectively,and then maintain the fruits flavour and enhance the fruits resistance.

To investigate the expression of key enzymes affecting the composition of anthocyanins in the transformation stage of grape peel of Vitis amurensis. With the young leaves of Vitis amurensis as the test material, the genomic DNA of Vitis vinifera was extracted and the full length sequence of PAL (GenBank accession number:MH045991) was obtained by homologous cloning, and conducted a biological information analysis. The Q-PCR method was used to analyze the expression levels of 8 grapevine skins at different pigmentation stages. The results showed that the DNA sequence of the PAL gene was 1 763 bp in length and had a 1 671 bp open reading frame (ORF) encoding 556 amino acids. The molecular weight was 61.07 ku and the isoelectric point was 5.76, which was a stable protein. The whole polypeptide chain had a transmembrane helix structure with no signal peptide. Molecular phylogenetic tree analysis showed that the homology was higher with European and Asian grape species; The expression pattern of PAL gene in the 8 stages of the grape rind of P. villosa. The PAL gene was analyzed in the last 4 periods. With irregular expression levels, the PAL gene was hardly expressed during both the 50% color shift and 100% color shift. The regulation of PAL gene expression was regulated by many factors. It changed regularly in the early stage of growth and development of Vitis vinifera. In the last 4 periods, the expression level changes irregularly. PAL gene was in 50% color rotation and 100% color rotation. Almost no expression, which may be affected by some kind of inhibition of PAL enzyme activity factors.

In order to improve the effect of grape fruit coloring,the optimum concentration of ALA in solar greenhouse was discussed. In the enlargement and coloring period of the Flame seedless fruit,the use of 5-aminolevulinic acid 50,100,150 mg/L(ALA) spraying fruit,to investigate the effects of ALA treatment on color,quality and anthocyanin synthesis related enzyme activity of grape fruit appearance. The results showed that during the fruit ripening,the treatment of exogenous ALA could induce the accumulation of anthocyanin,promote the colouring of peel,and alleviate the degradation rate of chlorophyll and carotenoids; The content of soluble sugar in fruit increased significantly,and the content of titration decreased obviously. After treatment with ALA,the anthocyanin content of the peel was significantly correlated with PAL activity,there was no significant correlation with DFR activity,CHI activity was significantly related only after the treatment and the content of anthocyanin,but after treatment of ALA,PAL,CHI,DFR activity in pericarp than control had different degrees of improvement,by hypothesizing that the expression of ALA by upregulating the related regulation the factor to induce synthesis of anthocyanin accumulation,thereby improving fruit appearance and internal quality.

The aim of this study is prokaryotic expression analysis of BcKMO gene from Botrytis cinerea and obtain the purified BcKMO protein.The BcKMO gene was amplified by RT-PCR technology using the cDNA of the Botrytis cinerea wild type BC22,cloned into the pMD19-T vector and sequenced.The results of sequencing showed that the BcKMO gene sequence was right.The pMD19-T-BcKMO and pGEX4T-1 plasmids were digested using restriction enzyme.The BcKMO gene segments were collected and cloned into the pGEX4T-1 vector.The results of restriction enzyme digestion and sequencing showed that the vector pGEX4T-1-BcKMO-GST was successfully constructed.The vector pGEX4T-1-BcKMO-GST was transformed into E.coli BL21 strain.The results of IPTG inducement indicated that the pGEX4T-1- BcKMO -GST was successfully expressed in E.coli BL21 strain,with the molecular weight 71 kDa.The optimal conditions of the prokaryotic expression of BcKMO were determined as 0.2 mmol/L IPTG treatment 12 h.Western Blot results showed that the GST antibody could specifically bound to purified pGEX4T-1-BcKMO-GST fusion protein,suggesting that the expression of BcKMO gene was successfully in vitro.

For the further promotion of vegetative growth and effective fertilization in grape seedlings,taking the one-year-old potted Jumeigui grape seedlings as tested materials,using ¹⁵N tracer technique to study the effects of different fertilization methods (foliar fertilization and soil fertilization) on ¹⁵N-urea absorption,distribution and utilization.The whole plants were dissected 3 times to leaves,shoots,trunk and roots in mature stage of new shoots.The results showed that fertilizer was beneficial to shoot and leaf growth,and foliar fertilization had remarkable influences on total nitrogen of grape leaves on September 30.The Ndff values of different organs under different fertilization methods were different at the mature stage of new shoots.Ndff values in all organs under foliar fertilization treatment were significantly higher than those under soil fertilization treatment on September 10.On September 30,Ndff values of shoots reached to 10.03% when daubing on the leaves,which was higher than that when applying in soil (5.61%).The distribution ratios of ¹⁵N in leaves under foliar fertilization treatment were higher than those under soil fertilization treatment in three different period,which showed that foliar fertilization was beneficial to the absorption of nitrogen in leaves.¹⁵N utilization rate of daubing on the leaves were significantly higher than those of applying in soil on August 20 and September 10.On September 30,there was no significant difference of distribution ratios of ¹⁵N in different organs between two fertilizer manners,as well as the ¹⁵N utilization rate,and the difference of Ndff values in different organs reduced between two fertilizer manners.The foliar fertilization is beneficial to raise the ¹⁵N utilization rate in grape seedlings,and promote the vegetable growth.

To solve the problem of grape root uplift and soil acidification,the influences in root growth and soil properties were studied by planting Lolium perenne L.and Medicago sativa L.in the inline of vineyard located at Xingcheng in Liaoning Province.The results showed that both Lp and Ms could significantly increase the root length and surface area in different stages and root levels (P< 0.05), and the amplification of which from high to low was Lp,Ms and control.The soil in vineyard by control management had an acidification trend,the pH distributed from 6.14 to 6.39.The pH ranged between 6.22 and 7.04 in Lp treatment,while that in Ms treatment was lower than Lp treatment.Compared with the control,the content of organic matter in Lp treatment significantly increased by 24.71%,48.07% and 44.44% (P< 0.05),while the Ms treatment increased by 7.87%,29.88% and 34.07%,respectively.In fruiting period,veraison and harvest time,the content of alkaline nitrogen in soil by Lp treatment increased by 40.40%,51.46% and 22.15% (P< 0.05) to the control,respectively. In fruiting period,veraison and harvest time,while those in the Ms treatment increased by 29.88%,28.03% and 5.42%,respectively.Compared with the control,both Lp and Ms treatments significantly increased the content of available phosphorus in soil (P< 0.05),which in Lp treatment was the highest,followed by the Ms treatment and the control.Both Lp and Ms treatments could cause the content of each mineral elements significantly increased at a certain time (P< 0.05).The overall effect of Lp treatment on the root length,root surface area of grape,and the content of organic matter,essential nutrients in soil was better than that of Ms treatment and control.

In order to define the characteristic aromatic components of each grape flavor type，we detected and analyzed the aromatic components of the berries from fifteen grape varieties (include 2 non-aromatic, 5 strawberry aromatic,8 muscat aromatic) using solid phase micro-extraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS).Results showed that the primary components of non-aromatic grape Moldova, Victoria is aldehydes. The relative content of 2-hexenal was the highest among aroma substances;The aroma substances of strawberry aromatic grape Shenfeng, Kyoho, Hupei No.2, Liaofeng, Shuofeng were mainly aldehydes, alcohols and esters; The aromatic substances of muscat aromatic grape were mainly aldehydes and terpenes. The main aldehydes are 2-hexenal and 1-hexanal.Terpenes were mainly linalool, dihydrolinalool, alpha-terpineol, nerol and geraniol. There were differences in aroma substances and substance contents among different grape varieties. This study can provide a theoretical basis for the research of grape aroma mechanism, breeding and promotion in the later stage.