中国农业科学 ›› 2014, Vol. 47 ›› Issue (5): 895-902.doi: 10.3864/j.issn.0578-1752.2014.05.006

• 植物保护 • 上一篇    下一篇

致病疫霉NLP家族基因PITG_10839的克隆和功能分析

 赵冬梅, 杨志辉, 徐进, 朱杰华, 朱丽丹   

  1. 河北农业大学植物保护学院,河北保定 071000
  • 收稿日期:2013-08-12 出版日期:2014-03-01 发布日期:2013-09-26
  • 通讯作者: 朱杰华,E-mail:zhujiehua356@126.com
  • 作者简介:赵冬梅,E-mail:zhaodongm03@126.com
  • 基金资助:

    现代农业产业技术体系建设专项(CARS-10-P12)

Cloning and Functional Analysis of Gene PITG_10839 of NLP Family in Phytophthora infestans

 ZHAO  Dong-Mei, YANG  Zhi-Hui, XU  Jin, ZHU  Jie-Hua, ZHU  Li-Dan   

  1. College of Plant Protection, Agricultural University of Hebei, Baoding 071000, Hebei
  • Received:2013-08-12 Online:2014-03-01 Published:2013-09-26

摘要: 【目的】分析致病疫霉(Phytophthora infestans)NLP(Necrosis- and ethylene-inducing like proteins)家族基因PITG_10839的致坏死功能及所编码蛋白的活性位点对其功能的影响。【方法】以PITG_10839作为研究对象,选inf1作为正对照,以致病疫霉菌株HK09-19的总RNA为模板,通过RT-PCR获得基因的cDNA全长序列。将所得cDNA与表达载体pBI121连接,构建所选基因的重组表达载体,并采用冻融法将其转入农杆菌LBA4404中。利用农杆菌介导的瞬时表达体系在本生烟(Nicotiana benthamiana)中对PITG_10839的表达和功能进行分析。利用over-lap PCR方法定点突变基因PITG_10839的活性位点,同时构建活性位点突变后的重组表达质粒,并通过农杆菌注射接种本生烟来研究各活性位点在基因坏死功能中所起的作用。通过荧光定量PCR分析致病疫霉游动孢子侵染马铃薯叶片后不同时期PITG_10839的表达模式。【结果】获得了PITG_10839和inf1的全长cDNA,分别为405及357 nt。同时,成功构建了PITG_10839和inf1的真核表达载体pBI121-10839和pBI121-inf1,并成功转入农杆菌。将含有重组质粒pBI121-10839的农杆菌注射接种本生烟,5 d后接种叶片开始逐渐黄化,7 d后叶片出现典型的皱缩坏死症状。然而含有对照重组质粒pBI121-inf1的农杆菌在注射接种3 d后,本生烟叶片便开始出现萎蔫,5 d时则出现坏死症状,7 d时坏死症状严重。进一步对PITG_10839编码蛋白的活性位点进行突变,发现D9A、E22A和D20A 3个氨基酸位点的突变使基因PITG_10839所编码蛋白的致坏死功能丧失,叶片仅出现轻微的黄化现象,然而H17A位点氨基酸的突变对该基因的致坏死功能没有影响,注射接种7 d后,仍然可以使本生烟叶片出现皱缩坏死的症状。同时,荧光定量PCR表明,致病疫霉游动孢子接种后不同侵染阶段PITG_10839的表达呈现出了不同水平的增加。接种后12 h至36 h,表达量变化水平稳定,增加了20倍左右;48 h时其表达量急剧上升至350倍左右;随后表达量开始下降,但仍高于接种36 h时的表达水平。【结论】致病疫霉NLP家族基因PITG_10839具有致使烟草叶片坏死的功能,并确定了影响该基因所编码蛋白致坏死功能的氨基酸活性位点。

关键词: 致病疫霉 , 诱导坏死基因家族 , 活性位点 , 功能分析 , 实时荧光定量PCR

Abstract: 【Objective】 The objective of this study is to analyze function of gene PITG_10839 of NLP (Necrosis- and ethylene-inducing like proteins) family in Phytophthora infestans.【Method】The full-length cDNA of gene PITG_10839 and inf1 (as a positive control in the following experiments) were obtained by RT-PCR. Then the fragments were inserted into binary expression vector pBI121 to produce recombinant plasmids, which were transferred into Agrobacterium LBA4404 by freeze thawing method. Functional analysis of PITG_10839 was conducted through heterologous expression in Nicotiana benthamiana by agro-infiltration method. Site-directed mutagenesis of PITG_10839 was carried out by over-lap PCR. Recombinant plasmids of mutants were constructed and expressed in N. benthamiana to determine the roles of mutated sites. The expression pattern of PITG_10839 was analyzed using reverse-transcribed real-time PCR. 【Result】The full-length cDNA of PITG_10839 and inf1 were 405 and 357 nt, respectively. Those fragments were successfully ligated into binary vector pBI121 and transformed into Agrobacterium LBA4404, separately. After agro-infiltration, the leaves began to exhibit yellowing symptoms on 5th day post inoculation (dpi) and then showed serious collapse and necrosis symptoms on 7th dpi, whereas the inf1 of P. infestans elicited wilting on 3rd dpi and caused leaf death on 5th dpi. Furthermore, three amino acid mutants of D9A, E22A and D20A only caused slightly yellow symptoms in leaves, which suggested the important roles related to the necrosis function of PITG_10839. While mutant of H17A site amino acid did not affect the function of PITG_10839, and led to the same serious collapse symptom as inf1 after 7 dpi. Real-time PCR analysis showed that PITG_10839 was expressed after P. infestans zoospore inoculation into potato leaves and exhibited different increased trends. The relative expression level was up-regulated stably at 12 to 36 hours post inoculation (hpi) and the expression peak appeared at 48 hpi, which was up-regulated about 350 times. Then the expression level decreased, but it was still higher than that of 36 hpi.【Conclusion】PITG_10839 of NLP gene family was capable of causing leaf necrosis in N. benthamiana, and the amino acid active sites of protein coded by PITG_10839 have been ascertained.

Key words: Phytophthora infestans , NLP gene family , active site , functional analysis , real time PCR