Bioinformatics Analysis Of Invertase Family And Cloning And Functional Verification Of GbVIN1 Gene In Gossypium Barbadense L.

Cotton is an important economic crop that is the source of worldwide textiles,and the quality of fiber is an important indicator to weigh the value of cotton.It is known that the normal development of cotton fibers requires a large supply of sucrose,and sucrose invertase is one of the key enzymes involved in sucrose metabolism in organisms,in which vacuolar sucrose invertase can be irreversibly decomposed into glucose and fructose through the catalysis of sucrose,resulting in osmotic pressure will promote cell elongation.To investigate whether GbVIN1 gene has an effect on the elongation of cotton fibers,the present study mainly focused on overexpression of GbVIN1 gene in Arabidopsis thaliana.By observing the related phenotypes of transgenic Arabidopsis plants,the transgenic Arabidopsis plants were identified.Leaf epidermal hair density,root hair density and root length have all increased.The main results of this experiment are as follows:（1）In this study,in order to better understand the quantity and distribution of sucrose converting enzyme in cotton genome and so on,we used the 17-invertase family gene as reference sequence in Arabidopsis to determine the cotton genome database in G.barbadense,G.hirsutum,G.raimondii and G.arboreum.The results showed that the sucrose invertase gene family contained in the G.barbadense was significantly more than that of G.hirsutum,while G.raimondii was more one than that of G.arboreum,and the number of invertase gene family were 65,46,26 and 25respectively.By analyzing the gene structure of members of the invertase gene family,Theαsubgroup of neutral/alkaline invertases and acidic invertase gene mostly owned 6 or 7 exons andβsubgroup owned4 or 5 exons.In addition,in the genetic structure of some G.barbadense and G.raimondii existed a long UTR area,compared with G.hirsutum and G.arboreum;The acidic invertase basically contained three conserved motifs,namely DNPNG,FRDP,WECPD,indicating the evolutionary conservatism of the same gene family in different species;The clustering analysis infered that the VIN subfamily genes might be related to cotton fiber development.（2）According to the preliminary data and spectral analyses of fiber development transcriptome,we cloned GbVIN1 gene from cotton（Gossypium barbadense L.）cultivar“Xinhai21”using reverse transcription PCR method.The gene has an open reading frame of 1938 bp,encoding 645 amino acids;The multiple sequence alignment analysis showed that the protein is highly conserved and has a conserved domain of the GH32 family;The phylogenetic tree analysis showed that the GbVIN1and the Gh VIN1 are in the same branch of the evolutionary tree.Real time fluorescence quantitative PCR analysis showed that GbVIN1 gene was expressed in different periods of cotton fiber development,and the highest expression is found in 5 DPA and 10 DPA fibers.（3）Construction of the plant expression vector p CAMBIA1304-GbVIN1 into model Arabidopsis thaliana.Phenotypic observations of transgenic Arabidopsis thaliana plants in the T₃generation revealed that overexpression of the GbVIN1 gene resulted in an increase in the epidermal hair and root hair density and length of Arabidopsis plants.It is speculated that the gene has a certain effect on the elongation of cotton fibers.