大豆GmNCED1-2的非生物胁迫诱导表达及其转基因烟草鉴定

Expression of soybean GmNCED1-2 induced by abiotic stress and the identification of GmNCED1-2 transgenic tobacco

9-顺式-环氧类胡萝卜素双加氧酶(NCED)是脱落酸(ABA)合成途径中的关键酶。本研究从大豆中克隆GmNCED1-2,其开放阅读框全长1818 bp,编码605个氨基酸,预测蛋白质相对分子质量6.693×104,等电点8.00。GmNCED1-2蛋白含有1个RPE65超家族结构域、2个保守序列MIAHPKxDP和HDFAITE以及4个保守的组氨酸残基。GmNCED1-2蛋白的亚细胞定位预测结果显示其位于叶绿体中。蛋白质系统进化分析结果表明GmNCED1-2蛋白与CrNCED1蛋白和MhNCED3蛋白的亲缘关系较近。实时荧光定量PCR结果表明GmNCED1-2在叶中的表达量最高。脱落酸、干旱、高盐、高温和低温胁迫下GmNCED1-2的表达量均升高。顺式作用元件预测分析结果表明GmNCED1-2启动子含有2个ABRE,3个ARE,1个TC-rich repeats和1个TGACG-motif顺式作用元件。将GmNCED1-2构建植物表达载体并转化烟草,获得3棵转基因烟草植株。烟草根长和生物量测定结果显示,GmNCED1-2的过表达抑制了转基因烟草根的伸长及植株的生长。

The 9-cis-epoxycarotenoid dioxygenase(NCED)is the key rate-limiting enzyme in abscisic acid(ABA)synthesis pathway.GmNCED1-2 was cloned from soybean in this research.The open reading frame(ORF)of GmNCED1-2 was 1818 bp in length,encoding 605 amino acids,with a predicted protein molecular weight of 6.693×104 and a theoretical isoelectric point of 8.00.GmNCED1-2 protein contained one RPE65 superfamily domain,two conserved sequences MIAHPKxDP and HDFAITE,and four conserved histidine residues.The subcellular localization of GmNCED1-2 protein was predicted to be located in chloroplasts.Phylogenetic analysis showed that GmNCED1-2 protein was closely related to CrNCED1 protein and MhNCED3 protein.The results of real-time quantitative PCR showed that the expression level of GmNCED1-2 was the highest in leaves.The expression of GmNCED1-2 increased under abscisic acid,drought,salt,heat and cold stresses.Cis-element prediction showed that the promoter region of GmNCED1-2 contained two ABRE,three ARE,one TC-rich repeats and one TGACG-motif.GmNCED1-2 was constructed into plant expression vector and transformed into tobacco.Three transgenic tobacco plants were obtained.The root length and biomass of wild type tobacco and GmNCED1-2 transgenic tobacco were measured.The results showed that the overexpression of GmNCED1-2 inhibited the root elongation and plant growth of transgenic tobacco.