Sugar Accumulation Mechanism And Key Gene Mining In Grape Fruit

Carbohydrate accumulation is the key component of grape fruit quality,which is not only an important index to measure its quality,but also the focus of fruit quality production research.In this study,qualitative and quantitative targeted metabolome detection and analysis of the components and contents of carbohydrates in the fruits of 18 grape cultivars was carried out;through field and physicochemical analysis,HPLC,transcriptome,metabolomics,bioinformatics and multi-omics analysis,q RT-PCR and other techniques,we explored the mechanism of carbohydrate accumulation and related key genes in the fruit of two cultivars‘Flame seedless'and‘Victoria'with large difference in sugar content.It will provide scientific basis for the regulation and breeding of key traits and quality of grape.The main research results obtained are as follows:(1)Based on GC-MS technology,the saccharides in the fruits of 18 grape cultivars were accurately detected,and 13 kinds of saccharides were identified.In the sugar accumulation of grape fruits,fructose and glucose were the main components,followed by sucrose.Followed by sucrose The content of 9 kinds of sugars,including D-arabinose,lactose,maltose,and trehalose were very small,with an average content ranging from 0.01 to 1.04 mg/g.Comprehensive analysis of these,we screened out the‘Flame seedless'and‘Victoria'which have a large difference in the source of the population and the sugar content close to the maturity period as high-sugar types and representatives of low-sugar cultivars to carry out further research.(2)Analysis of the leaf characteristics,anatomical structure and photosynthetic characteristics of‘Flame seedless'and‘Victoria'showed that the chlorophyll content of‘Victoria'was 13.90%higher than that of‘Flame seedless',the difference was significant,and the leaf thickness was higher than that of‘Flame seedless'71.65%,the thickness of the upper epidermis was 50.88%higher than that of‘Flame seedless',the thickness of fence tissue was 86.89%higher than that of‘Flame seedless',and the thickness of sponge tissue was higher than that of‘Flame seedless'82.17%.The analysis of light response characteristics showed that compared with‘Victoria',‘Flame seedless'had higher maximum net photosynthetic rate,apparent quantum efficiency and intrinsic quantum efficiency,stronger photosynthetic capacity and more organic matter accumulation.(3)Observation of the fruit anatomical structure during the sugar accumulation process of‘Flame seedless'and‘Victoria'fruits showed that there was a big difference in the thickness of the stratum corneum of the two varieties.Victoria's was 134.24%larger.The‘Victoria'epidermal cell space was tighter.The sub-epithelial cell thickness and cell transverse diameter of‘Flame seedless'were 114.24%and82.71%larger than those of‘Victoria'at the fruit ripening stage,and the difference was extremely significant.At the mature stage,the longitudinal diameter and intercellular space of the vascular bundle cells of‘Victoria'were 67.84%and 39.44%larger than those of‘Flame seedless',respectively.(4)The dynamic changes of hormone content during the sugar accumulation process of‘Flame seedless'and‘Victoria'fruits showed that the change trend of IAA content in the early stage of sugar accumulation was different,and the change was more consistent at the later stage of maturity.At 100 d after flowering,‘Flame seedless'the content of IAA in the fruit is 26.60%higher than that of‘Victoria'.The GA₃ content was at a low level,and the GA₃content reached 15.95 ng·g^-1 and 16.62 ng·g^-1 respectively at100 d after flowering.The ABA content was higher,and the ABA content in‘Flame seedless'was 26.39%higher than that in‘Victoria'at 100 d after flowering.90-100 d after flowering,the ZR content of‘Victoria'showed a downward trend,while the ZR of‘Flame seedless'ZR showed an upward trend,eventually46.46%higher than that of‘Victoria'.At maturity,IAA,ABA and ZR contents of‘Flame seedless'fruit were significantly higher than those of‘Victoria'fruit.(5)Glucose and fructose were mainly accumulated during the development of‘Flame seedless'and‘Victoria'fruits,supplemented by sucrose.Glucose content was significantly higher than fructose content in the early stage,and slightly lower than fructose at maturity.The content of glucose and fructose in‘Flame seedless'during the maturation period was significantly higher than that of‘Victoria'.The difference mainly occurred from 40 days after anthesis to maturity.The soluble solid content of‘Flame seedless'80 d after flowering was 61.8%higher than that of‘Victoria'.During fruit development,the activities of AI and NI showed first increasing and then decreasing;while SS-s and SS-c showed a gradual increase trend,SPS showed a trend of first increasing,then decreasing and then increasing,AI activity and SS-c activity has always been in a high state.AI,SPS and SS-s were positively correlated with the accumulation of various sugars in fruits,namely,the accumulation of carbohydrates in grape berries was coordinately regulated by the three enzyme activities of SPS,AI and SS-s,among which SS-s was a key regulatory factor related to sugar accumulation.(6)A total of 9820 differentially expressed genes were identified by transcriptome analysis at four stages of fruit development,namely‘Victoria'and‘Flame seedless'Functional enrichment analysis was performed on them.Genes related to carbohydrate metabolism,photosynthesis,hormones,cell wall material metabolism and cell wall metabolism were found.Transcription factors were not only differentially expressed in different developmental stages of the same cultivar of fruits,but also differentially expressed between varieties.From 40 days to 80 days after flowering,4,889 and 2,802differentially expressed genes were screened for‘Victoria'and‘Flame seedless',respectively.Through q RT-PCR verification,it was found that sugar accumulation during berry development Vv SWEET15,Vv HXK and Vv MYB44 genes were significantly up-regulated after flowering,while Vvb HLH104 was significantly down-regulated.These genes were key genes associated with sugar accumulation in grape fruits.(7)Through the target-like metabolomics study of the fruits of‘Victoria'and‘Flame seedless',it was found that there were 47 different metabolites with significant changes in the fruits of the two varieties.Compared with‘Flame seedless',the content of metabolites such as organic acids,amino acids and flavonoids in‘Victoria'is higher during fruit development.The‘Flame seedless'fruit had significantly higher levels of 15 sugars during fruit development than‘Victoria'.(8)The transcriptome and metabolome association analysis showed that the gene transcription expression levels and metabolite expression levels of‘Victoria'and‘Flame seedless'could be clearly distinguished.Among them,the metabolism and transcription of carbohydrates,amino acids,organic acids and phenols were significantly different between the two cultivars.