2021年4月23日 星期五
胡萝卜WRKY69基因(DcWRKY69)的克隆及其对不同植物生长调节剂的响应
Cloning of WRKY69 gene in Daucus carota var. sativa (DcWRKY69) and its response to different plant growth regulators
2019年 第28卷 第4期 页码[1-10]    下载全文[4.2MB]  
摘要

 以胡萝卜品种‘黑田五寸’(Daucus carota var. sativa ‘Heitian Wucun’)为研究对象,采用RT-PCR技术从叶片cDNA中克隆获得DcWRKY69基因片段,并对该基因及其编码的氨基酸序列进行生物信息学分析;同时,采用RT-qPCR技术比较了0.1 mmol·L-1茉莉酸甲酯(MeJA)、1.0 mmol·L-1水杨酸(SA)、0.1 mmol·L-1赤霉素(GA3)和0.1 mmol·L-1脱落酸(ABA)处理12 h内叶中DcWRKY69基因相对表达量的差异。结果表明:DcWRKY69基因包含1个长度为783 bp的开放阅读框(ORF),编码260个氨基酸;DcWRKY69转录因子氨基酸序列在第52至第110位含有1个WRKY结构域,其锌指结构类型为C2H2型,理论相对分子质量为30 030,理论等电点为pI 4.78;该转录因子含有34个磷酸化位点,无信号肽和跨膜结构,亲水性氨基酸较多,且酸性氨基酸比例最高(27%);该转录因子的二级结构由α-螺旋、β-折叠、延伸链和随机卷曲组成,比例分别为21.92%、4.23%、11.92%和61.92%。序列比对和系统进化树分析结果表明:WRKY转录因子在植物进化过程中具有高度的保守性。RT-qPCR分析结果表明:经MeJA、SA、GA3和ABA处理后,DcWRKY69基因相对表达量的变化趋势不同,分别在处理后8、12、1和4 h达到峰值,且显著高于对照(处理0 h)。研究结果显示:DcWRKY69转录因子属于Ⅱ类WRKY转录因子,为亲水性蛋白,且可能属于非分泌蛋白;DcWRKY69基因可能参与胡萝卜对不同植物生长调节剂的响应过程,但其对不同植物生长调节剂的响应模式存在差异。

Abstract

Taking Daucus carota var. sativa ‘Heitian Wucun’as research object, DcWRKY69 gene fragment was cloned from cDNA in leaf by RT-PCR technology, and bioinformatic analysis of this gene and its coding amino acid sequence was carried out; meanwhile, difference in relative expression level of DcWRKY69 gene in leaf treated by 0.1 mmol·L-1 methyl jasmonate (MeJA), 1.0 mmol·L-1 salicylic acid (SA), 0.1 mmol·L-1 gibberellin (GA3) and 0.1 mmol·L-1 abscisic acid (ABA) within 12 h was compared by RT-qPCR technology. The results show that DcWRKY69 gene contains an open reading frame. (ORF) with length of 783 bp, which encodes 260 amino acids. The amino acid sequence of DcWRKY69 transcription factor contains a WRKY domain in position from 52nd to 110th, its zinc finger structure type is C2H2 type, with theoretical relative molecular mass of 30 030 and theoretical isoelectric point of pI 4.78. This transcription factor contains 34 phosphorylation sites but without signal peptide and transmembrane structure, and has many hydrophilic amino acids with the highest percentage (27%) of acidic amino acid. The secondary structure of this transcription factor consists of α-helix, β-sheet, extended strand and random coil, their percentages are 21.92%, 4.23%, 11.92% and 61.92%, respectively. The sequence alignment and phylogenetic tree analysis results show that WRKY transcription factor is highly conserved in plant evolutionary process. RT-qPCR analysis result shows that after treatments of MeJA, SA, GA3 and ABA, the change trend of relative expression level of DcWRKY69 gene is different, which reaches the peak value after treatment for 8, 12, 1 and 4 h, respectively, and is significantly higher than the control (treatment for 0 h). It is suggested that DcWRKY69 transcription factor belongs to type Ⅱ WRKY transcription factor, it is hydrophilic protein and probably belongs to nonsecretory protein. DcWRKY69 gene may be involved in response process of D. carota var. sativa to different plant growth regulators, but there are differences in its response mode to different plant growth regulators.

关键词胡萝卜; WRKY69转录因子; 基因克隆; 序列分析; 植物生长调节剂; 基因表达
Key wordsDaucus carota var. sativa Hoffm.; WRKY69 transcription factor; gene cloning; sequence analysis; plant growth regulator; gene expression
作者张榕蓉, 王雅慧, 李彤, 丁旭, 徐志胜, 熊爱生
所在单位南京农业大学园艺学院 作物遗传与种质创新国家重点实验室
农业农村部华东地区园艺作物生物学与种质创新重点实验室, 江苏 南京 210095
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基金项目国家自然科学基金资助项目(31872098); 江苏省自然科学基金杰出青年基金项目(BK20130027); 江苏高校优势学科建设项目(PAPD)