中国农业科学 ›› 2007, Vol. 40 ›› Issue (6): 1166-1173 .

• 植物保护 • 上一篇    下一篇

辣椒疫霉elicitin基因的克隆及其原核表达产物功能分析

李 俊,王开峰,张正光,王源超,郑小波   

  1. 南京农业大学植物保护学院/农业部病虫监测与治理重点开放实验室
  • 收稿日期:2006-03-06 修回日期:1900-01-01 出版日期:2007-06-10 发布日期:2007-06-10
  • 通讯作者: 张正光

Cloning of a gene encoding elicitin from Phytophthora capsici and bioacitivity of prokaryotic expression of the gene

  

  1. 南京农业大学植物保护学院/农业部病虫监测与治理重点开放实验室
  • Received:2006-03-06 Revised:1900-01-01 Online:2007-06-10 Published:2007-06-10

摘要: 【目的】克隆Phytophthora capsici的elicitin基因并对其原核表达产物的生物活性进行研究。【方法】根据疫霉菌elicitin基因序列的保守性设计了2条引物,采用RT-PCR从P. capsici中克隆到长度为357 bp的cDNA片段。【结果】将该cDNA在网上进行分析表明该基因的编码产物为P. capsici的elicitin,即capsicin;该蛋白质N端前20个氨基酸为信号肽,等电点为4.2。同时将该蛋白质的氨基酸序列与Genbank中登录的14个elicitin进行聚类分析,发现所克隆的elicitin与Phytophthora drechsleri的α-elicitin及Phytophthora megasperma的α-elicitin聚为一组,表明该elicitin蛋白质属于α-elicitin。经southern杂交分析表明,该基因在P. capsici基因组中最少存在两个拷贝。该基因的原核表达产物能诱导野生型烟草Xanthi产生过敏反应和对TMV与Phytophthora nicotianae产生系统获得抗性,且能诱导该烟草的PR-1a、PR-1b和PR-1c基因的表达;但是它只能诱导不能积累水杨酸的转nahG基因烟草NahG产生过敏反应,不能诱导其PR-1a、PR-1b和PR-1c的表达。【结论】该SA不参与capsicin诱导的HR,而参与其诱导的系统获得抗性。

关键词: 辣椒疫霉, elicitin, 过敏性反应, 系统获得抗性

Abstract: Elicitin is an extracellular elicitor protein form Phytophthora species induced hypersensitive response in the genus Nicotiana. A cDNA with length of 357 bp encoding capsicin, was amplified from Phytophthora capsici by reverse transcript PCR (RT-PCR) based on high homology of elicitin genes from various Phytophthora spp. The gene codes a predicted 118 amino acids protein containing a 20-amino acid signal peptide of that is processed before secretion and with a calculated pI of 4.2. By compared with published 14 elicitin sequences of Phytophthhora spp in Genbank, capsicin belongs to α-elicitin. Southern blot analysis indicated that capsicin genes occur at least two copies in P. capsici. Bacterial expression of the cloned elicitin gene as translational funsion protein yielded a biologically active protein capable of inducing a hypersensitive response in wile-type tobacco, suggesting that Phytophthora-specific posttranslational modifications of elicitin are not required for its activity. The elicitor induced HR of a consistent shape and size on tobacco plants expressing the bacterial gene nahG. Salicylate hydroxylase is encodec by nahG and inactivates SA by converting it into catechol. The mutant NahG does not accumulate SA. HR appears to be mediated by a SA-independent signaling pathway. And elicitin treatment resulted in enhanced resistance of wild-type tobacco plants to infection by black shank fungus, P. nicotianae, and TMV. And the capsicin could induce expression of an SAR marker gene encoding PR-1a was suppressed in NahG plants. These results indicate that SA mediates SAR but not HR in tobacco treated with capsicin. During plant-elicitor interactions, HR and SAR may be regulated by distinct signal pathways, or SA may function as an intermediate signal upstream of SAR but downstream of HR, and HR may not be a direct defense mechanism against pathogen infection.

Key words: Phytophthora capsici, elicitin, hypersensitive response, systemic acquired resistance