Cloning And Expression Analysis Of Expansin Gene In Dioscorea Zingiberensis

Dioscorea zingiberensis is endemic to China and has important medicinal value.Its diosgenin is the first in the genus Dioscorea.Diosgenin is a raw material for a variety of hormonal drugs,and its global demand is large.Diosgenin contained in different tissues and different developmental stages of Dioscorea zingiberensis had the highest content in rhizomes.At present,the research on diosgenin mostly focuses on the excavation of key genes in the metabolic pathway,and pays less attention to its growth and development process.Expansin is an active protein that can relax the cell wall of plants.It plays a key role in plant growth and development by participating in cell elongation and cell wall modification.However,its role in the process of expansion and elongation of different tissues of Dioscorea zingiberensis has rarely been studied.In this paper,the full length of Dz EXP gene was obtained by RACE cloning technology.The nucleic acid,protein sequence and protein structure were analyzed by online website and software.The Dz EXP gene was inserted into p CXUN-YFP vector and transformed into rice protoplast.The subcellular localization of Dz EXP protein was observed by laser confocal microscopy.The Dz EXP gene was inserted into the vector p ET-28b-MBP-BST and transformed into Escherichia coli BL21(DE3).Prokaryotic expression was induced by IPTG.The solubility was observed by SDS-PAGE electrophoresis.The protein was purified by affinity chromatography and detected by Western blot.The differential expression of Dz EXP gene was analyzed by real-time quantitative PCR(q RT-PCR).The experimental results are as follows:1.The core sequence of Dz EXP gene was amplified by PCR using degenerate primers.The 5' and 3' ends were obtained by RACE kit.The full length was 1216 bp,and the ORF length was 630 bp,encoding 209 amino acids.Phylogenetic tree analysisfound that it is closely related to the pineapple(Ananas comosus).Structural analysis indicated that Dz EXP protein is a non-transmembrane protein with DPBB-1(Rare lipoprotein A(Rlp A)-like double-psi beta-barrel and Pollen-allerg-1 domains,belonging to the ?-expansin(EXPA)subfamily.Subcellular localization revealed that the Dz EXP protein was localized on the cell membrane.2.The p ET28b-MBP-Dz EXP expression vector was obtained and transferred into E.coli BL21(DE3)to prokaryotic expression.The SDS-PAGE electrophoresis showed that the product was a soluble protein.3.The differential expression analysis showed that the Dz EXP gene was expressed in different tissues(leaves,stems,rhizomes et al.)of Dioscorea zingiberensis,and the expression level in young leaves and young rhizomes was higher.The leaves were sprayed with 10 ?M Me JA,and the expression was highest at 8 h,and then began to decline.