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为了分析 WRKY40 抗病抗逆作用,以湖北海棠[Malus hupehensis(Pamp)Rehd.]为试材,利用特异引物进行 RT-PCR 基因克隆,利用实时荧光定量 PCR 方法分析组织中基因的表达及低温、高盐、干旱、PEG6000 胁迫和外源 IAA、ABA 处理对基因表达的影响。结果表明,克隆到的片段长度为 1 240 bp,包含一个完整的开放阅读框,长 999 bp,编码 333 个氨基酸。该基因与拟南芥 AtWRKY40 同源,故命名为 MhWRKY40b,GenBank 登录号为 JX112913。基因表达分析表明,MhWRKY40b 在根、茎、叶等组织均有表达,其中在花萼、花辨、果实中表达量较高。该基因明显受低温、高盐、干旱胁迫诱导表达,最高相对表达量可达 20 倍以上;而受 PEG、苹果白粉病菌及外源 ABA 和 IAA 轻微诱导,最高相对表达量在10 倍以下。MhWRKY40b 除了在抗白粉病方面与 AtWRKY40 具有相似功能外,可能还在湖北海棠的抗寒、抗盐、抗旱过程中起到比较重要的作用。
In order to analyze the resistance to WRKY40, the RT-PCR gene was cloned using specific primers from Malus hupehensis (Pamp) Rehd. The gene expression and the effects of low temperature were analyzed by real-time fluorescence quantitative PCR. Effects of high salt, drought, PEG6000 stress and exogenous IAA, ABA treatment on gene expression. The results showed that the cloned fragment was 1 240 bp in length and contained a complete open reading frame with a length of 999 bp encoding 333 amino acids. This gene is homologous to Arabidopsis AtWRKY40, so named MhWRKY40b, GenBank accession number is JX112913. Gene expression analysis showed that MhWRKY40b was expressed in the tissues of roots, stems and leaves, among which, the expression level of MhWRKY40b was higher in calyx and flower. The gene was obviously induced by low temperature, high salt and drought stress. The highest relative expression level was up to 20 times. However, it was slightly induced by PEG, powdery mildew of apple and exogenous ABA and IAA, the highest relative expression was less than 10 times. MhWRKY40b, besides having similar function to AtWRKY40 in resistance to powdery mildew, may play a more important role in the process of cold resistance, salt tolerance and drought resistance of Begonia.