黄瓜耐镉相关基因CsNAC019的克隆及表达分析

Cloning and Expression Analysis of Cadmium Tolerance Related Gene CsNAC019 in Cucumber

【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。

[ Objective ] To understand the molecular mechanism of Cd tolerance in cucumber, a NAC transcription factor CsNACO19 was obtained by using bioinformatics analysis. CsNAC019 was cloned and its expression was analyzed under Cd stress. The results of the present study will provide an experimental foundation for breeding of cucumber cultivars with high capacity for Cd tolerance. [Method] The full length sequence of CsNACO19 was cloned by PCR. Sequence comparison and conserved domain were analyzed by NCBI and DNAMAN. Amino acid composition, stability coefficient, and hydrophilic coefficient were analyzed by online software Expasy and TMHMM. The promoter region was analyzed by online software PlantCARE. Phylogenic tree was constructed by MEGA 6.0 according to the NJ method. The real-time PCR （qRT-PCR） was used to analyze the expression pattern of CsNAC019 under Cd treatments. The variance and significance were analyzed by DPS 7.05. [Result] CsNACO19 contained a typical conserved NAC domain. BLAST analysis revealed that CsNACO19 had 60% homology with NAC019 in the amino acid sequence, and the five relatively conserved regions of its amino acid sequences, A, B, C, D and E, were highly consistent between them from the N-terminal to the C-terminal. The full-length CDS of CsNACO19 was 960 bp, which encoded 319 amino acids with 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. Besides the eukaryotic promoter TATA and CAAT natural elements, promoter analysis showed that the promoter sequence of CsNACO19 also had cis elements in response to stress, such as G-box, ABRE, W-box, P-box, TCA-element, TC-richrepeats, and HSE, etc. Phylogenetic analysis showed that CsNAC019 had 64%-96% homology with NAC transcription factors of other 15 plants （melon, peach, apple, citrus, grape and soybean, etc）. CsNAC019 had the highest homology, 96%, with the NAC sequence of melon, qRT-PCR analysis revealed that the expression of CsNAC019 was significantly increased （8.2 folds） compared with the control under Cd tolerance condition. [Conclusion] CsNACO19 is a Cd tolerance induced gene. It was speculated that CsNAC019 may regulate the cucumber to response to Cd tolerance by regulating the expression of downstream genes.