西瓜抗枯萎病相关基因ClMYB转录因子的克隆及表达分析

摘要

Objective]The objective of this study is to clone ClMYB, which is a gene encoding MYB transcription factor, analyze its sequence features and expression characteristics, and to provide new insights into the understanding of the molecular mechanism in response of watermelon plants toFusarium oxysporum f. sp.niveum infection.[Method]In a recent study, a novel EST of MYB transcription factors was obtained from data analysis of microarray and suppression subtractive hybridization (SSH) of incompatible interaction between watermelon andF. oxysporum f. sp.niveum. cDNAsequenceof theMYB was isolated and cloned using cDNA from watermelon roots withF. oxysporum f. sp.niveum infection by RT-PCR method, designated asClMYB. The conserved domains and sequence features of ClMYB protein were analyzed by bioinformatics methods. Multiple sequence alignments and a phylogenetic tree of homologous species were done between ClMYB protein and its homologous ones from other species using MEGA5.0. The subcellular localization of ClMYBwas analyzedusing fusion expression vector PYBA1332-GFP. Then ORF of the cloned gene was inserted into the expression vector pCzn1 byNde I andXba I digestion. The recombinant plasmid was transformed intoE. coliArctic Express system and induced expression by 0.5 mmol·L-1 IPTG for 4 h. The expression of the fusion protein was detected by SDS-PAGE. The expression pattern of target genes in watermelon withF. oxysporum f. sp. niveum were detected by real-time fluorescent quantitative PCR (qRT-PCR).[Result]Using RT-PCR method, cDNA sequence ofClMYB (GenBank: KT751229) was cloned from watermelon (PI296341-FR) root. Sequence alignment and bioinformatics analysis revealed that the deduced amino acids of ClMYB had common characteristics ofMYB transcription factors with two MYB domains of R2 and R3 at the N-terminal and highly variation at the C-terminal. Phylogenetic tree analysis suggested that the encoded protein had the closest genetic relationship with Cucumis melo MYB (GenBank: XM_008440304) andCucumis sativusMYB (GenBank: XM_011652633). Subcellular localization results showed thatClMYB protein was located in the nucleus which belongs to a typical transcription factor. The expression of fusion protein was obtained by inducing with IPTG inE. coli and its relative molecular weight was 36 kD. qRT-PCR showed that the expression ofClMYB was induced byF. oxysporum f. sp.niveum, and appeared peak earlier and higher in PI296341-FR than Black diamond. MeJA at 50μmol·L-1could significantly improve the resistance of Black diamond to F. oxysporum f. sp.niveum and induce the expression ofClMYB. In PI296341-FR, the expression ofClMYB was also induced by MeJA, while its expression level was lower than Black diamond.[Conclusion]ClMYB is a typical R2R3-MYB transcription factor and is located in the nucleus. The expression of fusion protein was obtained by prokaryotic expression and its relative molecular weight is 36 kD. The expression ofClMYB was induced byF. oxysporum f. sp. niveum and JA. It is speculated thatClMYB may be involved in JA-mediated resistance signal transduction network ofF. oxysporum f. sp.niveum. It might play an important role in disease resistance ofCitrullus lanatus.%【目的】克隆西瓜抗枯萎病相关基因 ClMYB转录因子，进行生物信息学及表达模式分析，为进一步解析ClMYB在西瓜抗枯萎病机制中的作用提供理论依据。【方法】根据西瓜与枯萎病菌不亲和互作的抑制差减文库和Microarray数据分析，获得与西瓜抗枯萎病相关的基因ClMYB，采用RT-PCR技术分离克隆ClMYBcDNA全长序列；应用生物信息学方法分析该基因的保守结构域及序列特征；使用MEGA5.0对ClMYB蛋白序列及其同源序列进行多序列比对，并构建同源物种间系统进化树；采用GFP标记的方法进行亚细胞定位，分析编码蛋白表达位置；将该基因片段通过Nde I和Xba I双酶切连接至原核表达载体pCzn1，重组质粒转化至大肠杆菌Arctic Express，经终浓度为0.5 mmol·L-1IPTG诱导4 h，用SDS-PAGE分析融合蛋白的表达；利用实时荧光定量PCR方法检测目的基因在西瓜与枯萎病菌互作中及在茉莉酸（jasmonate，JA）诱导下的表达情况。【结果】利用RT-PCR方法从西瓜野生材料PI296341-FR根系组织中克隆该基因片段（GenBank：KT751229），序列比对及生物信息学分析表明，其基因编码的氨基酸序列具有MYB转录因子R2R3型的典型特征，其N端具有R2、R3两个MYB结构域，C端高度变异。多序列比对及进化树分析表明，该基因编码的蛋白与甜瓜MYB（GenBank：XM_008440304）和黄瓜MYB（GenBank：XM_011652633）同源性最高，其编码的氨基酸一致性达到86%，这与它们同属葫芦科植物有关；亚细胞定位显示ClMYB定位于细胞核，为典型的转录因子；成功构建了该基因的原核表达载体pCzn1-ClMYB，转化至大肠杆菌得到36 kD左右蛋白；ClMYB受枯萎病菌诱导，在高抗枯萎病菌材料PI296341-FR中，相对感病品种表达量高峰出现的早，且表达量高。50μmol·L-1的MeJA处理可以显著提高感病材料Black diamond对枯萎病的抗病水平，同时诱导ClMYB表达，与抗病材料PI296341-FR相比表达趋势一致，但表达量更高。【结论】ClMYB为典型的R2R3-MYB转录因子，亚细胞定位于细胞核中；基因的原核表达得到36 kD的融合蛋白，在西瓜中的表达受枯萎病菌和茉莉酸诱导，推测ClMYB可能参与JA介导的西瓜抗枯萎病防卫反应的信号通路，在西瓜抗病中起一定的作用。