Cloning And Functional Analysis Of Dwarf Soybean Expansin Gmexpa41Gene

Soybean is the world’s fifth largest crop. It’s not only a very important kind of vegetable protein and edible oil resource, but also an important quality of industrial raw material. Soybean comprehensive utilization and development are emphasized all over the world. However, in recent years, soybean production in our country grow slowly, this situation already cannot satisfy the demand of domestic soybean. In order to solve this problem, we must increase the yield of soybean, optimize soybean cultivation, and dwarf soybean studies favor the advantage of soybean cultivation, increasing soybean yield, which will meet the growing demand of Chinese soybean. While many literature suggests, expansin gene may be associated with plant height, so the dwarf soybean expansin research is helpful for us to study the reason of soybean phenotype at the molecular level, to lay the foundation for soybean molecular breeding.In this paper, I cloned the full-length sequences dwarf soybean expansin GmEXPA41gene and analysis GmEXPA41gene at the molecular level, then constructed dwarf soybean expansin gene into Escherichia coli expression vector pET32a, transformed into E. coli BL21(DE3) to carry out prokaryotic expression, after SDS-PAGE analysis, I screened high yield strain, with the aid of fermentation tank for fermentation, we laid the foundation for dwarf soybean expansin separation and purification, structural analysis and functional verification.The main results are as follows:1. I designed dwarf soybean expansin gene conserved primer, obtained expansin gene CDS about765bp by PCR technology. Through the5’RACE and3’RACE obtained804bp and828bp gene by PCR amplification, finally I got dwarf soybean GmEXPA41mRNA full-length sequence by splicing the three genes. By NCBI blast in comparison, the sequence is new series which submitted to the gene bank, get the landing No. JF694991.2. Expansin GmEXPA41gene analysis showed, gene CDS is765bp, encoding254amino acids. Normal soybean amino acid sequence homology is96.47%. With secondary structure prediction and comparison, I found dwarf soybean expansin has one more alpha spiral than normal soybean expansin, and beta folding slightly less than normal ones. By computer homology models, I obtained three dimensional structure of dwarf soybean and normal soybean expansin. In addition, I use normal soybean expansin protein receptor for molecular docking simulation, find10compounds which binding better with normal soybean expansin protein, and simulate their binding mode, preliminary studyed interactions between some compounds and expansin protein.3. Constructed recombinant Escherichia coli bacteria [pET32a-expansin BL21(DE3)], and get the efficient expression of the No.2strain.4. Using high efficient expression of the No.2strain as starting strain, cultured strain in test tubes and Erlenmeyer flask, and then switch the10%inoculum to3L fermentation tank,37℃, initial speed100rpm, ventilation150L· h-1. After3hours culture, correlation the speed and dissolved oxygen content, when A600is15, adding the final concentration of1mM IPTG to induce, after4h’s culture, collecting strain, finally fermentate mycelium weight94.66g.