陆地棉转录因子GhNAC7的克隆及功能分析  

Molecular Cloning and Functional Analysis of GhNAC7 in Upland Cotton(Gossypium hirsutum L.)

作　　者：郑学伟[1,2] SHAH Syed Tariq 范术丽[2] 魏恒玲[2] 庞朝友[2] 李鸿彬[1] 喻树迅[2] 

机构地区：[1]石河子大学生命科学学院,新疆石河子832000 [2]中国农业科学院棉花研究所/棉花生物学国家重点实验室,河南安阳455000

出　　处：《中国农业科学》2017年第3期426-436,共11页Scientia Agricultura Sinica

基　　金：国家棉花产业技术体系建设专项(CARS-18)

摘　　要：【目的】从陆地棉中克隆GhNAC7,分析其结构和功能,研究其在棉花不同组织中以及叶片不同发育时期的表达量。并转入拟南芥进一步探究其在棉花叶片衰老过程中的作用。【方法】利用中国农业科学院棉花研究所棉花生物学国家重点实验室建立的棉花衰老叶片cDNA文库中的序列,获得1个含有NAM结构域的EST,使用Oligo6.71设计引物,重新在陆地棉叶片cDNA中进行克隆。使用Gene Structure Display Server软件分析GhNAC7结构,使用在线工具Plant CARE分析启动子序列,利用在线工具Gen Scan进行氨基酸序列翻译。同时,利用拟南芥基因组数据库(TAIR)进行序列比对,选取得分较高的NAC家族基因,使用MEGA 6.06软件和Gene Doc软件进行进化树分析和氨基酸比对。以XbaⅠ和SacⅠ为酶切位点构建35S::GhNAC7-GFP融合表达载体,分析其在洋葱表皮细胞中的瞬时表达,进行亚细胞定位。利用实时荧光定量PCR技术分析GhNAC7在棉花不同组织、不同叶片发育时期以及在200μmol·L^(-1) ABA调控下的表达量。通过构建p GhNAC7-GUS融合表达载体并转拟南芥,分析其启动子特异性。以Eco RⅠ和SalⅠ为酶切位点,利用p BI101和p BI121载体,分别构建融合表达载体并转拟南芥进行过表达分析。【结果】从陆地棉中成功克隆GhNAC7,其全长为1 064 bp,包含3个外显子,2个内含子。生物信息学分析结果表明,GhNAC7开放阅读框为834 bp,可编码277个氨基酸,其蛋白质分子量为31.35 k D,等电点为9.22。结构域分析表明其属于NAC转录因子的NAM亚家族,进化树分析显示GhNAC7与ANAC041、ANAC083同源性最高,其中,GhNAC7与ANAC083结构域位置均为17—58 aa。其启动子核心元件包含一系列与衰老、激素、胁迫相关的顺式作用元件。亚细胞定位表明其蛋白为核蛋白。组织特异性表明GhNAC7在真叶、子叶、花、花药和衰老真叶中均明显表达,其中在衰老的真叶中表达量最高。启动�[Objective] The primary objectives of this experiment are to clone GhNAC7 gene, analyze its structure, detect its expression in different tissues of cotton and at different developmental leaf senescence stages. Furthermore, its function in cotton leaf senescence was further studied through transforming GhNAC7 gene into Arabidopsis. [Method] Based on the cotton senescent leaves cDNA library, which was built by State Key Laboratory of Cotton Biology of Institute of Cotton Research of CAAS, this gene was cloned from upland cotton using an expressed sequence tag （EST） containing NAM domain after designing primer using Oligo 6.71. Gene Structure Display Sever was conducted to analyze its structure, PlantCARE was used on-line to study its promoter sequence, and GenScan was simultaneously performed to translate amino acid on-line. Meanwhile, NAC family genes with higher scores were chosen after aligning sequence from Arabidopsis in TAIR. Afterwards MEGA 6.06 was used to display evolutionary relationships of the gene and GeneDOC was conducted to perform sequence alignment of amino acids. Via constructing 35S：：GhNAC7-GFP fusion expression vector with Xba I and Sac I restriction sites, subcellular localization of GhNAC7 was studied by transient expression analysis of onion epidermal cells. Expression profiles of GhNAC7 in various tissues, in response to 200 μmol.L-1 ABA treatment and developmental leaf senescence stages were investigated through quantitative real-time PCR （qRT-PCR）. Its promoter specificity was conducted by transforming into Arabidopsis thaliana after constructing pGhNAC7-GUS fusion expression vector. Simultaneously, using pBI101 and pBI121 with EcoR I and Sal I restriction sites were used to construct fusion expression vectors, and then over-expression analysis was performed by transforming GhNAC7 into Arabidopsis thaliana. [Result] In this study, a novel gene GhNAC7 was successfully cloned from upland cotton （Gossypium hirsutum L.）. Its full-length was 1 064 bp with three exons and two i

关 键 词：陆地棉 NAM结构域 叶片衰老 GhNAC7 过表达 

分 类 号：S562[农业科学—作物学]