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【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。启动子特异性分析表明,其GUS活性在衰老的叶片中最强。在拟南芥中过表达该基因,转基因植株比野生型表现出明显的衰老症状。荧光定量PCR分析表明,ABA处理后6 h GhNAC7明显上调表达,并在48 h表达量达到最高,这表明ABA可调控GhNAC7表达从而调节棉花叶片衰老。【结论】GhNAC7可以促进棉花叶片衰老并受ABA的调控。
【Objective】 GhNAC7 was cloned from upland cotton and its structure and function were analyzed. The expression of GhNAC7 in different tissues of cotton and leaf at different developmental stages was studied. And transferred to Arabidopsis to further explore its role in cotton leaf senescence process. 【Method】 One EST with NAM domain was obtained by using the sequence in the cDNA library of cotton senescence leaves established by the State Key Laboratory of Cotton Biology, Institute of Cotton Science, Chinese Academy of Agricultural Sciences. Oligo 6.71 primers were designed to re-establish cotton leaves on upland cotton leaves Clone in cDNA. The GhNAC7 structure was analyzed using the Gene Structure Display Server software, the promoter sequence was analyzed using the Plant CARE online tool, and the amino acid sequence translated using the online tool Gen Scan. At the same time, sequence alignment was performed using the Arabidopsis thaliana genome database (TAIR). NAC family genes with higher scores were selected and phylogenetic tree analysis and amino acid alignment were performed using MEGA 6.06 software and Gene Doc software. The 35S :: GhNAC7-GFP fusion expression vector was constructed by XbaⅠ and SacⅠ restriction sites, transient expression in onion epidermal cells was analyzed and subcellular localization was performed. The expression of GhNAC7 in different tissues of cotton, different leaf development stages and under the regulation of 200μmol·L -1 ABA was analyzed by real-time fluorescence quantitative PCR. The promoter specificity was analyzed by constructing the pGhNAC7-GUS fusion expression vector and transforming Arabidopsis thaliana. Using EcoRI and SalI as restriction sites, pBI101 and pBI121 vectors were used to construct the fusion expression vector and overexpressed in Arabidopsis. 【Result】 GhNAC7 was successfully cloned from G. hirsutum and its full length was 1 064 bp, containing 3 exons and 2 introns. Bioinformatics analysis showed that the open reading frame of GhNAC7 was 834 bp, encoding 277 amino acids. The molecular weight of GhNAC7 was 31.35 kD and the isoelectric point was 9.22. Phylogenetic analysis showed that the NAM subfamily belonged to the NAC transcription factor. Phylogenetic analysis showed that GhNAC7 had the highest homology with ANAC041 and ANAC083, and the GhNAC7 and ANAC083 domains were all 17-58 aa. Its promoter core element contains a series of cis-acting elements related to aging, hormones and stress. Subcellular localization indicates that the protein is a nuclear protein. Tissue specificity showed that GhNAC7 was highly expressed in true leaves, cotyledons, flowers, anther and true senescent leaves, of which the highest expression was in true senescent leaves. Promoter specificity analysis showed that GUS activity was the strongest among the aged leaves. The gene was overexpressed in Arabidopsis, and the transgenic plants showed obvious senescence symptoms than the wild type. Quantitative real-time PCR analysis showed that GhNAC7 was up-regulated at 6 h after ABA treatment and reached its peak at 48 h, indicating that ABA can regulate GhNAC7 expression and regulate senescence of cotton leaves. 【Conclusion】 GhNAC7 can promote senescence of cotton leaves and be regulated by ABA.