巴西橡胶树HbRPW8基因克隆与表达分析

Cloning and Expression Analysis of HbRPW8 Gene from Hevea brasiliensis

RPW8(resistance to powdery mildew locus 8)是对植物多种病害,尤其白粉病具有抗性的广谱抗病基因位点,通过对橡胶树RPW8基因进行克隆及表达分析,为橡胶树抗白粉病机制解析和分子育种等方面打下基础。以橡胶树‘热研73397’为材料,采用RT-PCR克隆HbRPW8基因编码区(CDS)序列;对其进行生物信息学分析,并利用qRT-PCR方法,测定7种激素和白粉菌侵染不同时间、不同病害等级处理后RPW8基因表达量。该基因cDNA全长570 bp,编码189个氨基酸,该蛋白的分子量为21.73 kDa,等电点为9.23,脂肪系数为86.30,总平均亲水性指数为-0.404,为亲水性蛋白,共有8个磷酸化位点,无跨膜域和信号肽,定位于细胞核,具有RPW8的保守结构域,α-螺旋和无规则卷曲是HbRPW8蛋白二级结构的主要元件,分别占氨基酸序列的70.90%和22.22%。亲缘关系显示,HbRPW8基因与木薯RPW8基因相似性最高。过氧化氢(H_(2)O_(2))、水杨酸(SA)、乙烯利(ETH)、赤霉素(GA)和脱落酸(ABA)能迅速诱导HbRPW8转录本的表达,并在处理后0.5 h达到最高水平,分别约为对照的14倍、6倍、75倍、2.5倍和4倍;茉莉酸甲酯(MeJA)处理后6h达到最高水平,是对照的10倍左右;生长素处理后HbRPW8转录本显著下调;随着白粉菌侵染时间和病害等级的增加,HbRPW8的表达量均呈现先上升后下降的趋势。研究结果初步表明,HbRPW8可能参与橡胶树的抗病反应机制,为橡胶树抗病育种的研究提供参考。

RPW8(resistance to powdery mildew locus 8) is a broad-spectrum resistance gene locus to many plant diseases, especially powdery mildew. In this study, the cloning and expression analysis of RPW8 gene in Hevea brasiliensis were carried out to lay the foundation for the analysis of powdery mildew resistance mechanism and molecular breeding.HbRPW8 gene coding region(CDS) was cloned from rubber tree ‘Reyan 73397’ by RT-PCR. Bioinformatics analysis was carried out, and qRT-PCR method was used to determine the expression of RPW8 gene after seven hormones and powdery mildew infection at different times and disease grades. The full-length cDNA of HbRPW8 was 570 bp, encoding 189 amino acids. The molecular weight of HbRPW8 was 21.73 kDa, the isoelectric point was 9.23, the fat coefficient was 86.30, and the total average hydrophilicity index was-0.404, which was a hydrophilic protein with eight phosphorylation sites. The protein was located in the nucleus without transmembrane domain and signal peptide, and had a conserved domain of RPW8. The main components of the secondary structure of HbRPW8 protein were α-helix and random coil, accounting for 70.90% and 22.22% of the amino acid sequence, respectively. Phylogenetic relationships analysis showed that HbRPW8 gene had the highest similarity with cassava RPW8 gene. Hydrogen peroxide, salicylic acid, ethephon, gibberellin and abscisic acid could rapidly induce the expression of HbRPW8 transcript, and reached the highest level at 0.5 h after treatment, which was about 14, 6, 75, 2.5 and 4 folds of the control, respectively;methyl jasmonate treatment reached the highest level at 6 h after treatment, about 10 times that of the control;HbRPW8transcript was significantly down-regulated after auxin treatment;with the increase of infection time and disease grade of powdery mildew, the expression level of HbRPW8 increased first and then decreased. The results of this study preliminarily showed that HbRPW8 may be involved in the resistance response mechanism of rubber tree, providing a reference for the study of rubber tree resistance breeding.