Expression Of Genes Related To Sugar Metabolism Under Low-temperature Acclimation Of Vitis Amurensis And Functional Identification Of VvAGPS1 Gene

Covering soil to overwinter has became a possibility to adapt the low temperature and dry climate of northern China cultivating in grape.The measure has dramatically aggravated the deterioration of the ecological environment and an increaseing in cost of planting.Therefore,it’s vital to seek grape germplasm resources with cold tolerance for our northern area.In this study,Vitis amurensis with significance cold resistance,which was used as materials.Under the natural low-temperature condition in the north,we set five different growth periods,the growth period（A,28±1°C）,early cold resistance（B,5±1°C）,mid-cold resistance（C,0±1°C）,late cold resistance（D,-5±1°C）and deep dormancy（E,-10±1°C）,respectively.Then we measure the change of sugar and the activity of its related enzymes in the mature branches.Through transcriptome sequencing of these five periods,a large number of differentially expressed genes were obtained,the function of VvAGPS1 gene were identified in Micro-Tom tomato.This study obtained the following results:（1）With the decline of temperature during exercise period of low temperature,the content of sucrose and fructose was gradually increasing in the phloem of branches of grape,while the glucose first increased and then decreased.The activity of sucrose synthase（SS）increased gradually,and the activity of sucrose phosphate synthase（SPS）was significantly decreased in the period of B,since that the activity of the enzyme was not significantly changed until the E period.The acid invertase（AI）had a peak of activity during the C period,and then there was a downtrend of its activity with the decrease of the temperature,the content of ABA was increased when temperature was decreased,while the content of IAA is significantly decreased.（2）Fifteen cDNA sequencing libraries were constructed for the phloem of the grape vines from five various low temperature exercise periods.A total of 69.34 Gb Clean reads were obtained,and the Clean reads of each sample up to 4.62 Gb.By setting differential express gene thresholds,8935 differentially expressed genes were screened,including 4064（45.48%）up-regulated genes and 4871（54.53%）down-regulated genes.The co-existing up-regulated genes were 567,and the co-existing down-regulated genes were 1224 in all low-temperature exercise periods compared with growth phase.The analysis of GO enrichment find that the differentially expressed genes were mainly enriched in molecular function during the low temperature exercise period compared with the growth phase,while except for the accumulation of DvsA and EvsA in the cellular components,but not in BvsA and CvsA.The analysis of KEGG show that the common pathway was not enriched in all four combinations,and the ether lipid metabolism was only enriched in DvsA,but not in the other combinations.BvsA and CvsA are together enriched in starch and sucrose metabolism,steroid biosynthesis and cysteine and methionine metabolism in the level of p-Value≤0.05.The common enrichment pathway of BvsA and EvsA was amino acid biosynthesis and Mannitol metabolism;the common pathways enriched are amino acid biosynthesis and arachidonic acid metabolism in CvsA and EvsA.Analysis of the starch and sucrose metabolic pathways of the main study revealed a total of 147 annotations,including 73 up-regulated genes and 74 down-regulated genes.There were commonly 29 difference up-regulated gene commonly among the three combinations of CvsA,DvsA and EvsA,and the total number of differentially expressed down-regulated genes was 31 in all the low-temperature treatment periods compared with the blank period.By analyzing the transcription factors,it was found that MYB transcription factors were the largest number of differential genes in enriching,followed by bHLH,and the transcription factors with the least differentially expressed genes were VOZ and LFY.（3）By the study of the metabolic pathways of starch and sucrose,we found that the VvAGPS1 gene was highly expressed when temperature gradually begain to decline during the low temperature exercise period.Therefore,the VvAGPS1 gene was cloned and transformed it into tomato.The full length of VvAGPS1 gene is 2703 bp,the length of CDS region is 1584 bp,the total amino acid number is 518,the molecular formula is C₂₆₀₈H₄₁₃₀N₇₁₆O₇₆₉S₂₃,the molecular weight is 58557.20,and the theoretical isoelectric point is 8.20.The VvAGPS1 was transformed into tomato,showing the height and leaf area of the transgenic lines were to be larger than the wildtype.Under the treatment of 6°C low temperature stress for 0h,12h and 24h,we found that the content of sucrose in transgenic tomato leaves increased significantly with the increase of stress time,and the glucose content decreased in wild type tomato with the increase of low temperature time,while the transgenic lines was up.the number of dead cells in the transgenic lines is less than that in the wild type.The activities of POD,SOD and CAT were different in both transgenic and wildtype tomato,and the activity of antioxidant enzymes in transgenic lines was significantly higher than that of wild-type as the low temperature time increased.The conductivity of transgenic lines is lower than the wild type.Under the condition of low temperature stress,the expression of VvAGPS1 gene was significantly up-regulated with time increasing.SlAGB1 and SlAGB3 were up-regulated in wild-type tomato leaves under low temperature treatment for 24h,while SlBAM1 and SlBAM8 were highly expressed at 12h（β-Amylase）.These results further demonstrate that VvAGPS1 plays an crucial role in cold tolerance in grape.