Identification And Genetic Transformation Of GmXTH18 Gene Related To Cell Wall Development In Soybean

XTH is an important cell wall modification enzyme.It is involved in the regulation of a series of complex physiological and biochemical processes during plant growth and development.In order to understand comprehensively the characteristics of the xyloglucan endotransglucosylase/hydrolase（XTH）gene in the soybean genome,bioinformatics method was conducted to identify all the family members of the XTH gene in soybean.The gene structure,chromosome location,phylogenetic relationship and conserved domain were predicted.According to the differently expression data of transcriptome collected from the key stage of plant height development,The Gm XTH18 was filtered as a key gene and its vector of the overexpression and knockout was successfully constructed,and its bioinformatics analysis and expression pattern analysis were carried out.The main results of current study were as follows:1.Gm XTH18 was identified as a key gene for plant height development through the regulation of secondary cell wall in soybean.2.The Soybean Gm XTH gene family consists of 59 gene members,which are unevenly distributed on 19 chromosomes except chromosome 4.The amino acid length of 59 soybean XTH protein ranges from 229-345 aa,and the amino acid length of Gm XTH37 encoded the smallest number of amino acids,229 aa,Gm XTH41 encoded the largest number of amino acids,345 aa.The molecular weight is 26.23-40.46 k Da,the isoelectric point is 4.73-9.60,and 31 members are uns Tab.proteins.From the perspective of gene distribution on chromosomes,except for chromosome 4,Gm XTH gene family are distributed on the remaining 19 chromosomes,and the most genes are located on chromosome 13 with 8 members,followed by chromosome 17 with 7 1,9,10,11,16,18,19 members,respectively.The same numbers of genes were located on chromosome 20,with 3 genes,and chromosome 3,6,7,14,and 15 had only 1 member.Plant CARE was used to analyze the promoter of Gm XTH18 gene.The results showed that the promoter had light response element,stress response element and hormone response element.The promoter should be binded by LRR receptor-like kinase and ubiquitin using the BLI method.3.In order to further reveal the diversity of soybean XTH protein structure,MEME online analysis software was used to analyze the conserved domains of 59 soybean XTH proteins.All members contain important conserved domain DEIDFEFLG（E was the conserved site）.In the evolutionary tree,the XTH members in the same group have similar conserved structure.There were 42 members of soybean XTH protein,which contained 9 motifs.The sequence of the 9 motifs was consistent,starting with Motif7 and ending with Motif9.Similar conserved domains may have similar gene functions.Only Glyma.01G013900 and Glyma.09g208300 contain only 5 motifs,and only 8 members have motif10,located at the 5’end.4.The expression pattern of Gm XTH18 gene under different biological stress and exogenous ABA stress was analyzed by real-time quantitative method.The results showed that high salt,drought and ABA could induce the expression of Gm XTH18.However,the expression pattern of Gm XTH18 gene was different under different abiotic stresses,and Gm XTH18 gene was different in root and leaf.5.Gm XTH18 gene was cloned and its protein was analyzed using bioinformatics method.The results showed that the ORF of Gm XTH18 was 801 bp in length and encoded 266 amino acids.The relative molecular weight of the protein was 29.90 k Da,the theoretical isoelectric point was 7.74,the protein was hydrophilic with one transmembrane helix,and a signal peptide.6.Plant expression vector,PTF101-Gm XTH18,was constructed,and five T₁plants were obtained by agrobacterium tumefaciens mediated cotyledon node-mediated infection of soybean.7.The knockout vector,Cas9-Gm XTH18,was constructed and infected by Agrobacterium tumefaciens mediated cotyledon node-mediated method and obtained 18 T₁plants.