Function Of Alkaline/Neutral Invertase SlCIN7 Gene In The Development Of Tomato Fruit

Plants use invertase to decompose sucrose into glucose and fructose to provide plants with energy materials for growth and development,as well as signal factors that respond to the external environment to regulate plant growth and development cycles.Invertases are divided into acid invertases and alkaline / neutral invertases,cytoplasmic invertases(CINs).At present,the function of acid invertase and its mechanism of action have been intensively studied,but CINs are not stable due to the non-glycosylation characteristics of this family of proteins,and little is known about their functions.Recent studies have shown that it plays an important role in organ development and product quality formation of idopsis,rice and other model plants.However,its function in tomato,a hortiural crop with fruits as product organs,is unclear.In this study,Micro-Tom tomato was used as a test material to study the invertase activity,tissue cell localization,gene silencing and overexpression of SlCIN7 gene with high expression in fruits,and its function was preliminary explored.The main findings are as follows:1)Bioinformatics analysis of SlCIN7,we found that the open reading frame is 1996 bp long,it encodes a protein with an isoelectric point of 5.84,655 amino acid residues,and a molecular weight of 73.51 k Da.By constructing a phylogenetic tree,SlCIN7 was classified as an alpha subfamily.Subcellular localization prediction showed that it was localized in mitochondria.2)The pET-SlCIN7 prokaryotic expression vector was constructed and induced to express in E.coli,and the purified protein SlCIN7 was obtained.In vitro enzyme activity analysis showed that SlCIN7 has invertase activity and can catalyze the decomposition of sucrose in vitro,and its optimum enzyme activity p H is 7.0.Sucrose is the only catalytic substrate of SlCIN7.3)The full length of the promoter of the SlCIN7 gene was obtained by amplification.A GUS expression vector containing the SlCIN7 promoter was constructed using the Gateway technology.After transforming Agrobacterium GV3101,it was transiently expressed in tomato fruits.Expressive activity in tube bundles and glia.A SlCIN7-GFP fused plant expression vector was further constructed and transiently expressed in tobacco mesophyll cells.The results showed that the SlCIN7 protein was localized in mitochondria.4)Constructing the RNA interference vector and overexpression vector of SlCIN7,using Agrobacterium-mediated transformation of tomato Micro-Tom,through PCR identification of selectable markers and expression analysis of target genes,50 overexpression lines and 40 silent plants were obtained.5)There was no significant change in the phenotype of transgenic lines overexpressing the SlCIN7 gene compared with non-transgenic plants.However,after silencing the SlCIN7 gene,there was almost no seed in the transgenic tomato fruit.Furthermore,the pollen viability test results showed that the vitality of pollen was significantly reduced,and the reactive oxygen species staining results showed that the content of reactive oxygen species in transgenic pollen grain 9 was significantly increased.In this study,we have preliminarily regulated the expression of SlCIN7 gene in tomato,laying a foundation for further research on the function of SlCIN7 gene in tomato fruit maturation.