人参中苯甲酸胁迫响应基因WRKY7的克隆与表达分析(1)
[摘要]WRKY转录因子是高等植物中最重要的转录因子基因家族之一，其在植物形态建成、生长发育以及应对生物(病菌、害虫等)、非生物(干旱、盐、冷、热等)胁迫过程中发挥重要作用。该研究根据人参化感自毒物质苯甲酸诱导的人参转录组数据，设计全长扩增引物，利用RTPCR方法获得1条编码WRKY转录因子的基因，暂命名为WRKY7，并对其进行TA克隆、测序和序列分析。该基因cDNA全长1 216 bp，其开放阅读框(open reading frame， ORF)为1 014 bp，编码337个氨基酸。序列分析及系统进化分析表明，该蛋白属于WRKY家族第Ⅲ类成员，与西洋参种的WRKY6转录因子亲缘关系最近，同源性为87%。实时荧光定量PCR分析发现，苯甲酸胁迫后WRKY7的表达量显著升高，推测该基因可能参与了人参响应苯甲酸胁迫的过程，为进一步研究人参自毒胁迫响应的分子机制奠定了基础。
[关键词]人参； WRKY转录因子； 化感自毒作用； 克隆； 序列分析
[Abstract]WRKY transcription factor is one of the most important transcription factor families widely existing in higher plants， which playing critical role in plant morphogenesis， development， biotic (including phytopathogens， pests etc.) and abiotic (drought， salt， chilling， high temperature， etc.) stress. In the present work， primers used to amplify fulllength gene encoding WRKY transcription factor were designed based on the transcriptome data of P. ginseng that induced by benzoic acid， one of the most important autotoxins identified from root exudates and rhizosphere soil of P. ginseng. Then， a WRKY gene， temporarily named as WRKY7， was confirmed by RTRCR. Furthermore， sequencing and sequence analysis of WRKY7 was conducted. Results indicated that， the full length cDNA of WRKY7 was 1 216 bp， the open reading frame (ORF) of which was 1 014 bp， encodes 337 amino acids. Homologous analysis and phylogenetic tree showed that， WRKY7 belonged to the Ⅲ category of WRKY families， which showing 87% similarity to WRKY6 in P. quinquefolius. Realtime PCR results showed that the expression of WRKY7 in P. ginseng induced by benzoic acid was upregulated markedly than the control， so we speculated that WRKY7 was involved in the response to benzoic acid stress， which will be helpful for further research on the molecular mechanism of ginseng plant response to benzoic acid stress.
, http://www.100md.com
[Key words]Panax ginseng； WRKY transcription factors； autotoxicity； cloning； bioinformatic analysis
轉录因子是一类含有DNA结合域的蛋白质，能识别基因上游特异的核苷酸序列，形成转录复合体从而激活或抑制靶基因的表达，WRKY是植物十大转录因子家族之一。这类转录因子最早发现于甘薯[1]，后相继从20余种植物中分离出WRKY转录因子[2]。WRKY蛋白的共同特点是含有60个氨基酸组成的WRKY结构域，在N端有一段高度保守的WRKYGQK多肽序列，其是WRKY结构域的核心，如果上述氨基酸发生变异，将直接影响WRKY转录因子的DNA结合活性[3]。根据WRKY转录因子结构域的数量和锌指结构，可将WRKY转录因子分为3类[4]：第Ⅰ类含有2个WRKY结构域，其锌指结构为C2H2型；第Ⅱ类含有1个WRKY结构域，其锌指结构为C2H2型；第Ⅲ类含有1个WRKY结构域，其锌指结构为C2HC型。
WRKY转录因子与植物生长发育、衰老及环境胁迫响应密切相关。当植物遭受生物胁迫时，WRKY蛋白通过多通路、多层次调控基因表达，进而调节植物对胁迫信号的转导途径[5]。例如：AtWRKY33超表达可提高拟南芥对灰霉病和黑斑病的抗病性[6]。干旱、高温、物理损伤等非生物胁迫同样可以激发WRKY基因高表达，以帮助植物抵御外界不良环境[7]。例如：拟南芥AtWRKY18，AtWRKY33，AtWRKY40，AtWRKY46表达量增加能提高植株耐热性[8]；大豆GmWRKY21过量表达可增强拟南芥的抗寒能力[9]。WRKY转录因子在植物逆境信号传递过程中发挥重要作用，1个WRKY基因往往能响应多种胁迫信号，而多个WRKY基因之间则形成复杂的基因调控网络。, 百拇医药(王梓 李勇 王蓉 丁万隆)
[关键词]人参； WRKY转录因子； 化感自毒作用； 克隆； 序列分析
[Abstract]WRKY transcription factor is one of the most important transcription factor families widely existing in higher plants， which playing critical role in plant morphogenesis， development， biotic (including phytopathogens， pests etc.) and abiotic (drought， salt， chilling， high temperature， etc.) stress. In the present work， primers used to amplify fulllength gene encoding WRKY transcription factor were designed based on the transcriptome data of P. ginseng that induced by benzoic acid， one of the most important autotoxins identified from root exudates and rhizosphere soil of P. ginseng. Then， a WRKY gene， temporarily named as WRKY7， was confirmed by RTRCR. Furthermore， sequencing and sequence analysis of WRKY7 was conducted. Results indicated that， the full length cDNA of WRKY7 was 1 216 bp， the open reading frame (ORF) of which was 1 014 bp， encodes 337 amino acids. Homologous analysis and phylogenetic tree showed that， WRKY7 belonged to the Ⅲ category of WRKY families， which showing 87% similarity to WRKY6 in P. quinquefolius. Realtime PCR results showed that the expression of WRKY7 in P. ginseng induced by benzoic acid was upregulated markedly than the control， so we speculated that WRKY7 was involved in the response to benzoic acid stress， which will be helpful for further research on the molecular mechanism of ginseng plant response to benzoic acid stress.
, http://www.100md.com
[Key words]Panax ginseng； WRKY transcription factors； autotoxicity； cloning； bioinformatic analysis
轉录因子是一类含有DNA结合域的蛋白质，能识别基因上游特异的核苷酸序列，形成转录复合体从而激活或抑制靶基因的表达，WRKY是植物十大转录因子家族之一。这类转录因子最早发现于甘薯[1]，后相继从20余种植物中分离出WRKY转录因子[2]。WRKY蛋白的共同特点是含有60个氨基酸组成的WRKY结构域，在N端有一段高度保守的WRKYGQK多肽序列，其是WRKY结构域的核心，如果上述氨基酸发生变异，将直接影响WRKY转录因子的DNA结合活性[3]。根据WRKY转录因子结构域的数量和锌指结构，可将WRKY转录因子分为3类[4]：第Ⅰ类含有2个WRKY结构域，其锌指结构为C2H2型；第Ⅱ类含有1个WRKY结构域，其锌指结构为C2H2型；第Ⅲ类含有1个WRKY结构域，其锌指结构为C2HC型。
WRKY转录因子与植物生长发育、衰老及环境胁迫响应密切相关。当植物遭受生物胁迫时，WRKY蛋白通过多通路、多层次调控基因表达，进而调节植物对胁迫信号的转导途径[5]。例如：AtWRKY33超表达可提高拟南芥对灰霉病和黑斑病的抗病性[6]。干旱、高温、物理损伤等非生物胁迫同样可以激发WRKY基因高表达，以帮助植物抵御外界不良环境[7]。例如：拟南芥AtWRKY18，AtWRKY33，AtWRKY40，AtWRKY46表达量增加能提高植株耐热性[8]；大豆GmWRKY21过量表达可增强拟南芥的抗寒能力[9]。WRKY转录因子在植物逆境信号传递过程中发挥重要作用，1个WRKY基因往往能响应多种胁迫信号，而多个WRKY基因之间则形成复杂的基因调控网络。, 百拇医药(王梓 李勇 王蓉 丁万隆)