芝麻硬脂酸脱饱和酶基因SiSAD的克隆及功能验证  

Cloning and Functional Characterization of Sesame SiSAD Gene

作　　者：周瑢[1] 刘盼[1] 黎冬华[1] 张艳欣[1] 王林海[1] 张秀荣[1] 魏鑫 ZHOU Rong;LIU Pan;LI DongHua;ZHANG YanXin;WANG LinHai;ZHANG XiuRong;WEI Xin(Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture,Wuhan 430062;College of Life Sciences,Shanghai Normal University,Shanghai 200234)

机构地区：[1]中国农业科学院油料作物研究所/农业部油料作物生物学与遗传育种重点实验室,武汉430062 [2]上海师范大学生命科学学院,上海200234

出　　处：《中国农业科学》2019年第10期1678-1685,共8页Scientia Agricultura Sinica

基　　金：创新工程(CAAS-ASTIP-2013-OCRI);国家自然科学基金(31671282);武汉市科技计划(2018020401011303);上海市青年科技启明星计划(19QA1406500)

摘　　要：【目的】对芝麻△9硬脂酰-ACP脱饱和酶基因SiSAD(△9 stearoyl acyl-carrier-protein desaturase)进行克隆与表达分析,并转入拟南芥,探究其在油酸合成过程中的作用,为芝麻油酸含量的遗传改良提供分子基础。【方法】提取中芝13叶片的总RNA,反转录为cDNA。根据芝麻基因组数据库中的SiSAD序列信息(序列号为SIN_1008977)设计引物,以cDNA为模板,通过RT-PCR克隆获得SiSAD编码区序列,并与参考基因组序列进行比较。利用InterPro进行保守结构域分析,获得SiSAD蛋白的保守结构域。利用BLAST对SiSAD蛋白进行同源对比,获得SiSAD的同源蛋白质。采用邻接法构建系统进化树,获得芝麻SAD蛋白与橄榄、牵牛花、蓖麻、莴苣、葡萄、柑橘、拟南芥等植物SAD蛋白的亲缘关系。通过荧光定量PCR检测SiSAD在2个芝麻品种中芝33和中丰芝一号的根、茎、叶、蕾和种子中的相对表达量,分析SiSAD的表达特异性。将SiSAD连接过表达载体,通过农杆菌介导法转化野生型拟南芥(Col-0),筛选阳性后代,对T3代转基因和野生型的拟南芥种子中硬脂酸和油酸相对含量进行测定,分析SiSAD的功能。【结果】成功获得SiSAD的编码区序列,与参考基因组序列一致,全长为1 152 bp,编码383个氨基酸,SiSAD蛋白的分子量为43 kD,等电点为6.18。发现SiSAD蛋白含有一个保守结构域,属于脂肪酸去饱和酶家族成员,与其他植物的SAD蛋白质序列的同源性较高,暗示SiSAD在不同物种中的功能可能比较保守。系统进化分析显示,芝麻SAD蛋白与牵牛花和橄榄的SAD蛋白处于同一分支,进化关系较近,与蓖麻、拟南芥、柑橘的SAD蛋白亲缘关系较远。荧光定量PCR结果表明,SiSAD在芝麻种子中的表达量远远高于其他组织,有显著的组织特异性。成功构建了SiSAD的过表达载体,通过农杆菌介导法转化拟南芥,结果表明SiSAD成功导入拟南芥中,而且转录水平很高。对T3代转基因拟南�【Objective】 Sesame SiSAD (△9 stearoyl acyl-carrier-protein desaturase) gene was cloned and the expression of it was detected. It was transformed into Arabidopsis to investigate its role in the oleic acid synthesis. This study aims to provide molecular basis for the genetic improvement of sesame oleic acid content.【Method】Total RNA was extracted from leaf of the variety Zhongzhi13 and then was reverse transcripted into cDNA. Using the primers that designed according to the reference genome, the coding region sequence of SiSAD was obtained by RT-PCR. The sequence was further compared with the reference genome. The conserved motifs of SiSAD protein were identified by InterPro and the homologous proteins of SiSAD were recognized by BLAST. A phylogenetic tree of SiSAD from sesame, Olea europaea var. sylvestris, Ipomoea nil, Ricinus communis, Lactuca sativa, Vitis vinifera, Citrus sinensis and Arabidopsis thaliana was constructed by neighbor-joining method to reveal the relationship of SiSAD protein in these species. Expression profiles of SiSAD in roots, stems, leaves, buds and seeds at two varieties Zhongzhi33 and Zhongfengzhi No.1 were investigated. The SiSAD gene was linked to a 35S vector and transformed into Arabidopsis by the Agrobacterium tumefaciens-mediated floral dip method. Based on the qRT-PCR detection, successful transformed Arabidopsis individuals were selected from the progenies. The stearic acid and oleic acid content in the seeds of transgenic T3 Arabidopsis seeds and Col-0 were detected and function of SiSAD was concluded.【Result】 Total coding region sequence of SiSAD was cloned and the sequence was the same as the reference genome. It consisted of 1 152 nucleotides encoding a protein of 383 amino acids with a calculated molecular mass of 43 kD and a predicted pI of 6.18. We found that SiSAD gene contained one conserved function domain, which had been identified as a signature motif within the fatty acid desaturase family members. The similarity of SiSAD proteins from different species

关 键 词：芝麻 SiSAD 过表达 油酸 功能验证 

分 类 号：S565.3[农业科学—作物学]