Functional Analysis Of α-Expansin-like Gene GhEXLA1 In Cotton

Cotton fiber develops from a single cell in the ovule epidermis,which is a good material for studying plant cell elongation and cell wall development.Fiber cell elongation is accompanied by the synthesis of primary and secondary cell walls,and the properties of cell walls can determine the quality of mature fibers.Therefore,analyzing the functions of cell wall-related genes is of great significance to the research on cotton fiber quality improvement.In plants,Expansin is the first cell wall extensin to be discovered and plays an important role in plant development,mediating cell wall relaxation and promoting cell elongation under acidic conditions.In the early stage of this experiment,a preliminary functional and structural study of the proteins of the Expansin family was carried out.The function of a gene Gb EXPATR encoding α-type Expansin protein was verified,and the structure of an EXLA-type protein GhEXLA1 was also analyzed by structural biology,but the function of GhEXLA1 has not been studied.In this study.GhEXLA1 gene was cloned and transformed in cotton,and two overexpressing lines were obtained.Through the phenotypic investigation of the transgenic overexpression Lines,the analysis of the transcriptome and biochemical experiments,the biological function of GhEXLA1 gene and the biochemical basis of GhEXLA1 acting on the cell wall are enriched and perfected.The results of this study are as follows:1.GhEXLA1 is predominantly expressed in cotton roots,petals and ovulesUsing the Analyze function of Cotton FGD to find out the gene and tissue expression levels of all EXLAs by keyword of Expansin.It was found that GhEXLA1 was predominantly expressed in roots,petals and ovules.Using the BLAST function of NCBI to search for the homologous protein of GhEXLA1.GhEXLA1 has high homology with Arabidopsis At EXLA1,Cocoa Tc EXLA1 and rice Os EXLA1.2.GhEXLA1 protein glycosylation site identification and polysaccharide binding assayThe GhEXLA1 protein sequence has a typical conserved domain of Expansin protein,and its N-terminal has a signal peptide of 18 amino acids,19 to 137 constitute Domain 1,145 to 259 constitute Domain 2,and the two domains are connected by 7 amino acids.Since the protein expressed by insect cells was larger than the predicted size,it was speculated that GhEXLA1 had a post-translational modification.The website predicted that the 99 th Asparagine(N)of GhEXLA1 was a glycosylation modification site.Then the site was mutated to Glutamine(Q),and it was found that the mutated protein was more similar with the predicted size.The wild-type protein and the mutant protein were digested with PNGase A protease that specifically recognizes glycosylation modification sites.It was found that the wild-type protein could be cleaved,but the mutant protein did not change.The difference between the wild-type protein and the mutant protein was detected by mass spectrometry,and it was found that the molecular weight of the two proteins differed by1.37 k Da,which was most likely the size of the sugar chain on GhEXLA1.Furthermore,The binding ability of GhEXLA1 protein with 6 kinds of cell wall polysaccharides was tested,and it was found that GhEXLA1 protein can specifically bind to rhamnogalacturonic acid I.It shows that GhEXLA1 is very likely to contact the pectin of the cell wall to change the features of the cell wall and affect the Cells stretch.3.GhEXLA1 promotes root elongation in transgenic cottonGhEXLA1 overexpressed cotton lines was screened for copy number and expression level,and finally two stably inherited single-copy lines were obtained.The results of phenotypic investigation showed that overexpression of GhEXLA1 could significantly promote the root elongation of cotton,but reduced the size of mature seeds,and had a certain but not significant effect on fiber length.Through comparative transcriptome analysis of root tissues of different materials,it was found that the expression of reactive oxygen species-related genes and auxin regulation-related genes increased after overexpression of GhEXLA1.The up-regulated expression of these genes that positively regulate root development may promote cotton root elongation.