黄瓜耐镉相关基因CsNAC019的克隆及表达分析

发布时间：2018-02-02 06:20

本文关键词： 黄瓜 Cd CsNAC 基因克隆表达分析　出处：《中国农业科学》2017年10期 　论文类型：期刊论文

【摘要】：【目的】研究黄瓜耐镉(Cd)分子机制,结合生物信息学分析获得一个NAC转录因子家族基因CsNAC019,对其进行克隆和表达分析,为进一步将该基因用于黄瓜耐Cd品种的改良与选育提供试验基础。【方法】通过PCR技术扩增CsNAC019全长;利用NCBI和DNAMAN进行序列比对和保守结构域分析;利用Expasy、TMHMM等在线软件进行氨基酸组成、稳定系数、亲水系数分析;通过Plant CARE在线工具进行启动子序列分析;采用MEGA 6.0软件根据NJ方法,构建系统进化树;采用q RT-PCR技术检测Cd胁迫后黄瓜CsNAC019在叶中的表达情况,并用DPS 7.05数据处理系统软件进行方差及显著性分析。【结果】CsNAC019含有典型的NAC保守结构域。通过BLAST分析比对,该基因在氨基酸序列上与拟南芥NAC019存在60%的同源性,两者在从N端到C端有A、B、C、D、E 5个氨基酸相对保守区的序列高度一致。该基因CDS序列全长为960 bp,编码319个氨基酸,编码蛋白质分子量为35.66 k D,理论等电点为8.72,不稳定系数为68.18,平均亲水系数为-0.483。启动子分析表明,除含有真核生物启动子固有的TATA和CAAT元件外,CsNAC019的启动子序列还存在G-box、ABRE、W-box、P-box、TCA-element、TC-richrepeats、HSE等多种响应逆境胁迫的顺式元件。系统进化分析表明,CsNAC019与甜瓜、桃、苹果、柑桔、葡萄、大豆等15种植物NAC转录因子有64%—96%的同源性,其中与甜瓜的NAC蛋白同源性最高,为96%。通过q RT-PCR分析发现,与对照相比,CsNAC019在Cd胁迫条件下表达量明显上调(8.2倍)。【结论】CsNAC019为黄瓜Cd胁迫诱导表达基因,推测其可能通过调节下游基因的表达调控黄瓜对Cd胁迫的应答。
[Abstract]:[objective] to study the molecular mechanism of cadmium tolerance in cucumber and obtain a NAC transcription factor gene CsNAC019 by bioinformatics analysis. In order to provide the experimental basis for further application of this gene to the improvement and breeding of Cd-tolerant cucumber varieties. [methods] the full-length CsNAC019 was amplified by PCR. Sequence alignment and conserved domain analysis were carried out by NCBI and DNAMAN. The amino acid composition, stability coefficient and hydrophilic coefficient were analyzed by using the on-line software Expasyy TMHMM. The promoter sequence was analyzed by Plant CARE online tool. The phylogenetic tree was constructed by using MEGA 6.0 software according to NJ method. Q RT-PCR technique was used to detect the expression of CsNAC019 in cucumber leaves under CD stress. The variance and significance were analyzed with DPS 7.05 data processing system software. [results] CsNAC019 contained typical conserved NAC domain. The results were compared by BLAST analysis. The amino acid sequence of this gene is 60% homology with Arabidopsis NAC019. The sequence of E 5 amino acids was highly consistent with that of conserved region. The CDS sequence of the gene was 960 BP, encoding 319 amino acids and encoding a molecular weight of 35.66 KD. The theoretical isoelectric point is 8.72, the instability coefficient is 68.18, and the average hydrophilic coefficient is -0.483.Promoter analysis shows that. In addition to the TATA and CAAT elements inherent in eukaryote promoters, the promoter sequence of CsNAC019 also contains G-boxbox ABRERE-W-box P-box. TCA-element-TC-rich repeat HSE and other cis-elements in response to stress stress. Phylogenetic analysis showed that CsNAC019 and muskmelon, peach. There was 64-96% homology of NAC transcription factors in 15 plants, such as apple, citrus, grape and soybean, and the highest homology was found with NAC protein in muskmelon. It was found by Q RT-PCR analysis that compared with the control. The expression of CsNAC019 was significantly up-regulated under CD stress. [conclusion] CsNAC019 is the gene that induces the expression of Cucumber under CD stress. It is speculated that it may regulate the response of cucumber to CD stress by regulating the expression of downstream genes.
【作者单位】：东北农业大学园艺园林学院/农业部东北地区园艺作物生物学与种质创制重点实验室;
【基金】：国家自然科学基金青年基金(31101545) 黑龙江省普通本科高等学校青年创新人才培养计划(UNPYSCT-201500) 东北农业大学“青年才俊”项目(14QC07);东北农业大学“学术骨干”项目(16XG05)
【分类号】：Q943.2;S642.2
【正文快照】： 【研究意义】黄瓜(Cucumis sativus L.)是中国设施蔬菜生产中的主要栽培作物之一,由于温室大棚内化肥、农药等的连年使用,黄瓜易受到重金属的毒害,其中,镉(Cd)污染较为严重。近些年,Cd污染事件频发,例如,2009年8月在湖南浏阳以及2012年1月在广西龙源出现的Cd污染事件等。Cd是

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