Characterization And Functional Validation Of Expansin Gene In Upland Cotton(Gossypium Hirsutum)

As an important cash crop,cotton provides important raw materials for the textile industry.At present,people’s quality of life and textile level are improving,so the requirements for cotton fiber quality are becoming increasingly strict.As an important model for studying cell elongation and cell wall structure,cotton fiber is a single-cell epidermal hair developed from epidermal cells outside the ovule.It has the characteristics of high elongation,no branching and thickening.Expansin is a cell wall-associated protein that has a positive effect on the initiation and elongation of fiber cells;in addition,the characteristics of the cell wall play a key role in the quality of mature cotton fibers.This study mainly identified the Expansin gene family of upland cotton through bioinformatics and analyzed its evolutionary characteristics,using transcriptome sequencing(RNA-seq)data to study the spatial and temporal characteristics of its expression,and using qRT-PCR to verify the expression profile.Based on the expression profile data,four genes were selected for cloning and functional validation: overexpression of the Expansin gene in Arabidopsis thaliana,VIGS(gene silencing)in cotton,and finally the overexpression vector was transferred into the seed solution by agrobacterium in cotton.The main results are as follows:1、Based on the amino acid sequence of Arabidopsis and rice expansin(as a query),BLAST alignment was performed,and 98 members of the expansin gene family were identified in upland cotton,of which 71 belong to the EXPA subfamily and 8 belong to the EXPB of the families,6 belong to the EXLA subfamily and 13 belong to the EXLB subfamily.There are 3-5 expansin genes on most chromosomes of upland cotton,and some genes appear tandem duplication.2、Collinearity analysis shows that most expansin genes are located in the collinear chromosomal segment,indicating that the expansion of the expansin family(up to 98)is mainly due to segmental duplications of chromosomes during evolution.Therefore,there is a certain paralogous relationship between the expansin genes in the subgenome;AA and DD species hybridized and then doubled,so as to evolve to At At Dt Dt upland cotton,facilitated the doubling of expansin gene members(although a few genes have been lost).In the phylogenetic tree,the terminal branch is composed of four genes of diploid cotton and tetraploid(upland)cotton.It indicates that the evolutionary relationship between the amino acid sequences of the two subgenomes(At and Dt)of tetraploid upland cotton and the Expansin members of the diploid cotton genome(A and D).Collinearity analysis,gene structure analysis and motif structure analysis further confirmed its evolutionary relationship.3、The average length of the acid sequence of 98 expansins of upland cotton is 248 aa,the average molecular weight is 27.00 k D,and the p I is 8.48(most of which are partial basic proteins).The instability index range from 20.03 to 46.80,and most of the proteins have good stability;the aliphatic index is 70.83,and the GRAVY range from-0.392 to 0.140,which is an amphiphilic protein.Among the 72 expansins,there are signal peptides of different AA lengths,all of which are located outside the cell.The expansin gene has a certain codon bias.4、RNA-seq data shows that the expansin gene has its own specific spatiotemporal expression pattern.The expression patterns of homeologous gene-pairs with partial homology are also different,indicating the diversification and complementarity of gene functions among family members in the evolutionary process after polyploidization.The RNA-seq data was verified by quantitative real-time PCR and the results showed that the expression profile data of the two methods had a high degree of agreement;each of the expansin genes expressed different abundances under different spatial and temporal conditions,and performed their own duties.5、Based on the expression profile data of RNA-seq and qRT-PCR,GhEXLA3 A,Gh EXPA03 A,Gh EXPA24 D,and Gh EXPA27 D,were selected for cloning and transformed into A.thaliana by the "trimming method".The results showed that overexpressing the gene Gh EXPA27 D would promote the root development of A.thaliana,bolting in advance,the number of branches increased,the number of pods and seed yield also increased.Using the VIGS technique to interfere with the expression of the gene Gh EXLA3 A on cotton plants,the results showed that the roots and plants of the seedlings after two weeks were smaller(compared to the control);qRT-PCR was performed on the plants treated with VIGS and the Gh EXLA3 A gene.The amount of expression did not change much.The seeds of Agrobacterium tumefaciens were soaked,and the overexpression vector of gene Gh EXPA27 D was transferred into cotton.The results showed that the leaves of T0 generation plants were partially curled,the plants were thin,and the cotton peach was small during the mature stage.