银杏GbSAD基因对非生物胁迫的响应及原核表达  

Expression Analysis of Gb SAD from Ginkgo biloba in Response to Abiotic Stress and Its Prokaryotic Expression

作　　者：刘新亮[1] 蔡金峰[1] 王欢利[1] 曹福亮[1] 

机构地区：[1]南方现代林业协同创新中心(南京林业大学),南京210037

出　　处：《东北林业大学学报》2015年第12期1-6,共6页Journal of Northeast Forestry University

基　　金：国家"十二五"林业科技支撑项目(2012BAD21B04)

摘　　要：以悬浮培养的银杏愈伤组织为材料,采用荧光定量RT-PCR研究了Gb SAD基因在高盐和不同外源激素处理条件下的表达模式。结果表明:外施100μmol·L^(-1)水杨酸(SA)后,Gb SAD表达量发生显著变化;外施100μmol·L^(-1)脱落酸(ABA)、40μmol·L^(-1)乙烯利(ETH)、100μmol·L^(-1)茉莉酸甲酯(Me JA),Gb SAD表达量未呈现显著变化;外施200 mmol·L^(-1)氯化钠(Na Cl),Gb SAD表达量小幅度降低。Gb SAD组织表达分析表明基因在根、茎、叶、雄花和雌花中均有表达,在叶中表达量最高。SAD氨基酸序列同源比对发现,Gb SAD与其他被子植物的序列相似度较高,表明Gb SAD在进化上较保守。将银杏Gb SAD克隆到原核表达载体p ET-32a(+)中,转化大肠杆菌(Escherichia coli)BL21(DE3)并诱导表达。结果表明,Gb SAD蛋白在1 mmol·L^(-1)IPTG的诱导下,培养2 h即能实现融合蛋白的高效表达,为进一步纯化目的蛋白及研究其功能奠定了基础。We studied the expression pattern of Gb SAD under abiotic stress,and cultured the callus of ginkgo in the MS liquid media supplied with Na Cl and different exogenous hormones. The expression of Gb SAD was induced by exogenous SA（ 100μmol·L^-1）,but not altered when exposed to exogenous ABA（ 100 μmol·L^-1）,ETH（ 40 μmol·L^-1） or Me JA（ 100μmol·L^-1）. The expression of Gb SAD was slightly down-regulated by Na Cl treatment. Gb SAD expressed in various tissues,and had the highest expression level in leaves. The deduced amino acid sequence of Gb SAD shared a higher identity with SAD from other angiosperms,which indicated that Gb SAD was remarkably conservative throughout evolution. We cloned Gb SAD into prokaryotic expression vector p ET-32a（ ＋）,and then transformed the recombined plasmid into Escherichia coli BL21（ DE3） to induce its expression. The fusion protein was expressed to a high level at 1 mmol·L^-1IPTG induction for 2 h,which was used for further purification and functional analysis of Gb SAD.

关 键 词：银杏 GbSAD基因 非生物胁迫 原核表达 

分 类 号：Q786[生物学—分子生物学]