中国农业科学Issue(5)：895-902,8.DOI:10.3864/j.issn.0578-1752.2014.05.006
致病疫霉NLP家族基因PITG_10839的克隆和功能分析
Cloning and Functional Analysis of Gene PITG_10839 of NLP Family in Phytophthora infestans
摘要
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.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PCRKey words
Phytophthora infestans/NLP gene family/active site/functional analysis/real time PCR引用本文复制引用
赵冬梅,杨志辉,徐进,朱杰华,朱丽丹..致病疫霉NLP家族基因PITG_10839的克隆和功能分析[J].中国农业科学,2014,(5):895-902,8.基金项目
现代农业产业技术体系建设专项 （）
