甘薯响应蔓割病病原菌侵染的IbWRKY7基因克隆与表达分析

Cloning and expression analysis of IbWRKY7 gene in response to Fusarium oxysporum f. sp. batatas infection in sweet potato

关键词:  甘薯  IbWRKY7  蔓割病  表达模式  基因克隆

Key words:  sweet potato   IbWRKY7  Fusarium wilt  expression pattern  gene cloning

摘要:

为探究IbWRKY7基因在甘薯响应蔓割病病原菌侵染过程中的表达模式，以高抗蔓割病品种‘鄂薯11’为供试材料，克隆到1个新的WRKY家族基因IbWRKY7，进行生物信息学分析；并对‘鄂薯11’和蔓割病敏感品种‘栗子香’在蔓割病病原菌侵染后IbWRKY7基因的表达模式进行分析。结果显示，IbWRKY7的CDS序列全长为945 bp，编码314个氨基酸；推测其编码不稳定的亲水性蛋白，蛋白分子质量为33.92 kU，pI 9.82，含有1个WRKY七肽保守序列和1个C₂HC型锌指结构。IbWRKY7基因上游2 000 bp的启动子区域内存在多种类型的顺式作用元件，如植物激素应答元件Myb、胁迫响应元件MYC和MYB等。多序列比对及系统进化树分析结果表明，IbWRKY7蛋白与日本牵牛花InWRKY7和三裂叶薯ItWRKY7亲缘关系最近。亚细胞定位预测显示IbWRKY7蛋白定位于细胞核。实时荧光定量PCR结果表明，与蔓割病病原菌侵染0 h相比，侵染2、4、12、24、48、72、96和120 h后，‘鄂薯11’的IbWRKY7基因表达量均显著提高(P<0.05)；而‘栗子香’的IbWRKY7基因表达量除侵染24 h外的其他7个时间点均显著高于0 h。侵染2~48 h，‘鄂薯11’的IbWRKY7基因表达量显著高于‘栗子香’。双因素方差分析结果表明，不同品种和侵染时间点的IbWRKY7基因表达量均存在显著差异。综上，甘薯IbWRKY7基因上游2 000 bp的启动子区域含有12种激素应答和胁迫应答相关的顺式作用元件。IbWRKY7具有WRKY家族的典型结构特征，属于第Ⅲ类WRKY蛋白，氨基酸序列与同源物种相似度高，进化上高度保守。IbWRKY7基因受蔓割病病原菌侵染诱导表达，且在不同蔓割病抗性的甘薯品种中表达量差异显著。

Abstract:

To investigate IbWRKY7 expression pattern of sweet potato in response to Fusarium oxysporum f. sp. batatas(Fob)infection, a novel WRKY family gene IbWRKY7 was cloned from high resistance Fusarium wilt variety ‘Eshu 11'. Bioinformatics analysis and the expression levels of IbWRKY7 in ‘Eshu 11' and Fob-susceptible cultivars ‘Lizixiang' under infection by Fob were carried out. The result of bioinformatics analysis showed that the full length of IbWRKY7 CDS sequence was 945 bp, encoding 314 amino acids. It was presumed that IbWRKY7 encodes an unstable hydrophilic protein, and its molecular weight was 33. 92 kU. The isoelectric point was 9. 82, and contained one WRKYGQK and one C₂HC type zinc-finger domain. Different promoter cis-acting elements such as plant hormone response elements Myb(Myeloblastosis), stress response element MYB(Myeloblastosis), and MYC(Myelocytometosis)were found in the 2 000 bp promoter region upstream of IbWRKY7 gene. The multiple sequences alignment and phylogenetic tree analysis revealed that IbWRKY7 protein was the closest to the InWRKY7 of Ipomoea nil and the ItWRKY7 of Ipomoea triloba. The results of subcellular localization prediction showed that IbWRKY7 protein was a nucleus localized protein. The results of real-time quantitative PCR analysis indicated that the expression level of IbWRKY7 in ‘Eshu 11' was significantly increased(P<0. 05)at 2, 4, 12, 24, 48, 72, 96, and 120 h compared with 0 h under Fob infection. The expression of IbWRKY7 in ‘Lizixiang' was significantly higher than that of 0 h at 7 other time points except 24 h under Fob infection. The expression level of IbWRKY7 in ‘Eshu 11' was significantly higher than that of ‘Lizixiang' from 2 to 48 h under Fob infection. The results of two-factor ANOVA showed that IbWRKY7 expression levels were significant different among different cultivars and different infection time points. To sum up, the 2 000 bp promoter region upstream of IbWRKY7 contains 12 cis-acting elements related to hormone and stress response. IbWRKY7 has typical structural characteristics of the WRKY family, which belongs to Ⅲ-type WRKY protein, and its amino acid sequence was highly similar to those homologous species and highly conserved during evolution. The expression of IbWRKY7 could be induced by Fob infection and there were significant differences among sweet potato varieties with different Fusarium wilt resistance.