橡胶树抗病相关基因HbNPR1的克隆及其表达分析

Cloning and Expression Analysis of Resistance-related Gene HbNPR1 from Hevea brasiliensis

前期利用cDNA-AFLP技术分离获得了一个与橡胶树抗棒孢霉落叶病反应相关的差异表达基因片段EST-IAN-188,通过Blast比对分析发现,该基因片段与植物抗病相关NPR基因家族中的NPR1具有高度的同源性。将该片段与橡胶树基因组序列进行比对,设计引物进行PCR扩增,得到了一个橡胶树NPR1基因的cDNA和基因组序列,命名为HbNPR1基因。基因序列分析显示,该基因编码区全长(CDS)1 374 nt,含有两个外显子和一个内含子,编码457个氨基酸,蛋白分子量为51.0 ku,等电点5.82,具有BTB/POZ、ANK锚蛋白重复序列、DUF和NPR1-like C等4个结构域。qRT-PCR定量分析发现,HbNPR1基因在橡胶叶片中的表达丰度最高,特别是古铜期叶片;多主棒孢病菌(Corynespora cassiicola)诱导下HbNPR1基因在抗病品种(IAN873)的表达丰度明显高于感病品种(PR107);另外,SA、MeJA、ET处理下均能诱导HbNPR1基因的表达,SA处理后的表达量丰度最高。本研究初步表明,HbNPR1基因可能参与橡胶树抗病信号途径的调控和寄主对病原菌侵染的防御反应。

A resistance related differential expression fragment of rubber tree to Corynespora leaf fall disease was obtained in pre-work, and named as EST-IAN-188. Blast showed that the fragment had high homology with NPR1, a plant resistance NPR gene family member. Based on the Hevea brasiliensis genome database, the long fragment including EST-IAN-188 was gained and Hevea NPR1 gene was cloned, named as HbNPR1. Sequence analysis revealed that the CDS length of HbNPR1 was 1 374 nt, encoding a protein of 457 amino acids with an average molecular weight of 51.0 ku, and a pI value of 5.82. Exon/intron structure analysis revealed 2 exons and 1 introns in HbNPR1 genomic sequence. The protein had four conserved domains of BTB/POZ, ANK anchored protein repeat sequences, DUF and NPR1-like C. The expression pattern of HbNPR1 in rubber plants was assessed by qRT-PCR. The tissue-specific expression analysis indicated that the HbNPR1 was mainly expressed in leaves, especially in the bronze blade. When C. cassiicola infected, the expression level of HbNPR1 was significantly higher in resistant rubber germplasm（IAN873） than in susceptible rubber germplasm（PR107）. The expressions of HbNPR1 were up-regulated by ethylene, salicylic acid and Methyl jasmonate treatment, especially by salicylic acid. The results preliminarily show that HbNPR1 gene may be involved in rubber tree resistance signal pathway.