杜氏盐藻甘油醛-3-磷酸脱氢酶基因在棉花中的转化及分子检测  

Transformation of Dunaliella salina Glyceraldehyde-3-phosphate Dehydrogenase Gene into Cotton and Molecular Detection

作　　者：孔静静[1] 陆许可[1] 赵小洁[1] 阴祖军[1] 王帅[1] 王德龙[1] 王俊娟[1] 樊伟丽[1] 张德超[1] 张天豹 叶武威[1] 

机构地区：[1]中国农业科学院棉花研究所,棉花生物学国家重点实验室,安阳455000

出　　处：《分子植物育种》2015年第2期301-309,共9页Molecular Plant Breeding

基　　金：转基因生物新品种培育科技重大专项(2011ZX08005-004)资助

摘　　要：根据NCBI核酸数据库中杜氏盐藻耐盐基因甘油醛-3-磷酸脱氢酶基因(dunaliella salina glyceraldehyde-3-phosphate dehydrogenase,Ds GAPDH)的c DNA全长序列信息,本研究利用RT-PCR技术扩增克隆该基因,获得完整全长为1 131 bp并编码376个氨基酸的多肽的ORF序列。生物信息学分析表明:Ds GAPDH编码的蛋白与拟南芥、蒺藜苜蓿、玉米和葡萄等物种高度同源,分别达到90%、92%、96%和97%。构建的系统发育树结果显示,该基因编码的蛋白与团藻中该蛋白的亲缘关系最为接近。该蛋白主要由四种二级结构即α-螺旋、β-折叠、转角和无规则卷曲等组成。构建p BI121-Ds GAPDH植物表达载体,以非抗虫棉F12A、F12B和HU-749513为材料,利用基因枪活体技术转化棉花花粉,并对T0代种子进行分子检测和相关序列验证,成功获得两株阳性转基因植株,为基因枪活体转化技术研究奠定了一定的实践基础,同时也为棉花耐盐机制的研究提供了新材料。According to the NCBI nucleotide database,the salt-tolerance gene complete sequence of Dunaliella salina glyceraldehyde-3-phosphate dehydrogenase（Ds GAPDH） was cloned by RT-PCR,and the full open reading frame（ORF） of Ds GAPDH gene was 1 131 bp,encoding 376 amino acids.Bioinformatics analysis presented that the Ds GAPDH protein encoded shares similarity of 90%,92%,96% and 97% with that from Arabidopsis thaliana,Medicago truncatula,Zea mays and Vitis vinifera respectively.Phylogenetic tree analysis showed that Ds GAPDH protein has the closest relationship to Volvox aureus.Secondary structure prediction indicated that the Ds GAPDH protein should be composed of α-helix,β-sheet,turn and random coil.Meanwhile,an eukaryotic expression vector p BI121-Ds GAPDH was constructed and then was transformed into the pollens of non-Bt cotton accessions（F12A,F12 B and HU-749513） by gene bombarding alive technique.The seeds of T0 generation were detected and validated by molecular identification and sequencing analysis.The results exhibited that two cotton individuals were positive and were confirmed that transformed plant successfully harbored with Ds GAPDH gene.Therefore,these results would have great significance in laying a practical foundation for gene Bombarding alive transformation technology as well as providing new materials for the further study of salt-tolerant mechanism in cotton.

关 键 词：杜氏盐藻 甘油醛-3-磷酸脱氢酶基因 陆地棉 转化 分子检测 

分 类 号：S562[农业科学—作物学]