水稻P450基因Oscyp71Z2增强稻瘟病抗性的机制

doi:10.3864/j.issn.0578-1752.2014.13.001

摘要/Abstract

摘要： 【目的】分析水稻细胞色素P450基因Oscyp71Z2在稻瘟病抗性中的作用，并解析其介导的水稻抗稻瘟病机制。【方法】根据高抗稻瘟病转hrf1水稻NJH12表达谱结果，筛选到一个表达量高达114.6倍的细胞色素P450基因Oscyp71Z2（NCBI登录号：Os07g0217600）。根据Oscyp71Z2全长编码区序列设计引物，以水稻模式粳稻品种日本晴的cDNA为模板，通过PCR扩增Oscyp71Z2全长编码区序列。将所得的cDNA序列经测序后，与双元表达载体pVec8连接，构建超量表达载体pVec8::Oscyp71Z2。采用冻融法将构建的超量表达载体pVec8::Oscyp71Z2质粒转入农杆菌菌株EHA105，并利用农杆菌转化法获得Oscyp71Z2超表达水稻。常规PCR技术检测T4代Oscyp71Z2超量表达水稻中选择标记基因潮霉素磷酸转移酶编码基因（hygromycin phosphotransferase gene，hph），并通过qRT-PCR检测超量表达水稻中目的基因Oscyp71Z2的表达量。进一步对阳性超量表达水稻（T4、T5和T6）进行稻瘟病表型鉴定，并检测植保素含量变化（T4）。【结果】RT-PCR和qRT-PCR结果显示，水稻细胞色素P450基因Oscyp71Z2在高抗稻瘟病的转hrf1水稻中有较高的表达，与表达谱数据一致。通过生物信息学分析Oscyp71Z2全长1 554 bp，含有2个细胞色素P450保守结构域，是CYP71Z2亚家族成员。将Oscyp71Z2成功连接到双元载体pVec8，利用农杆菌介导法获得Oscyp71Z2超量表达水稻。超量表达水稻株系在各生育期、株高等性状，与非转基因水稻日本晴基本一致。超量表达株系中Oscyp71Z2的表达量较野生型有显著升高。超量表达水稻的穗颈瘟抗性等级维持在1—2级，表现为抗病或者中抗，而野生型水稻的穗颈瘟等级为3—4级，表现为感病，Oscyp71Z2在水稻中超量表达显著增强了对穗颈瘟的抗性。Oscyp71Z2超量表达株系每克新鲜叶片中植保素MA和PB含量平均是328和124 ng，相对于野生型的85和42 ng•g-1新鲜叶片有显著升高。Oscyp71Z2超量表达株系中涉及到植保素MA和PB合成通路的4个关键基因的表达量较野生型有显著地升高。【结论】水稻细胞色素P450基因Oscyp71Z2通过调控植保素生物合成通路，赋予水稻稻瘟病抗性。

关键词: 水稻 , P450基因Oscyp71Z2 , 植保素 , 抗病性 , 稻瘟病

Abstract: 【Objective】The objective of this study is to analyze the role of cytochrome P450 gene Oscyp71Z2 in resistance to blast in rice, and expose the resistance mechanism mediated by Oscyp71Z2.【Method】Based on the transcriptome profile of the transgenic rice line NJH12 expressing hrf1 from Xoo, a resistance-related gene showing 114.6-fold increase of expression level was identified. The gene is the cytochrome P450 gene, named as Oscyp71Z2 (NCBI No.: Os07g0217600). Oscyp71Z2 coding sequence was cloned from rice cultivar Nipponbare transcript by PCR. To better understand the function of P450 gene Oscyp71Z2, binal transformation vectors pVec8::Oscyp71Z2 for Oscyp71Z2 overexpression were made, pVec8::Oscyp71Z2 plasmid was transformed into Agrobacterium tumefaciens strain EHA105 by freezing and thawing, and Oscyp71Z2-overexpressing rice was obtained by A. tumefaciens method. The selective marker hygromycin phosphotransferase gene hph was detected in T4 Oscyp71Z2-overexpressing rice by normal PCR, and the relative expression level of Oscyp71Z2 in transgenic rice was analyzed by qRT-PCR. Resistance phenotypes to Magnaporthe oryzae in positive Oscyp71Z2-overexpressing rice (T4, T5 and T6) were identified at booting stage. And phytoalexins accumulation of Oscyp71Z2-overexpressing rice (T4) was quantified.【Result】RT-PCR and qRT-PCR data showed that the expression level of cytochrome P450 gene Oscyp71Z2 in hrf1 transgenic rice with resistance to blast was higher than that in wild type rice, which was consistent with the Oscyp71Z2 data from transcriptome profile. Oscyp71Z2 gene cDNA is 1 554 bp, contains two cytochrome P450 domains by bioinformation analysis, which belongs to the CYP71Z subfamily. The full-length cDNA of Oscyp71Z2 was successfully ligated into binary vector pVec8, and transformed into rice Nipponbare genome mediated by A. tumefaciens. The rice growth periods, plant height and other traits in Oscyp71Z2-overexpressing rice were consistent with that in wild type rice. The transcripts of Oscyp71Z2 in Oscyp71Z2-overexpressing rice obviously increased, compared with that in wild type. The resistance degree to panicle blast in wild type was 3-4, showed susceptibility. However, the resistance degree to panicle blast in Oscyp71Z2-overexpressing rice was 1-2, showed resistance or middle resistance. The overexpression of Oscyp71Z2 in rice significantly enhanced the resistance to panicle blast. The accumulation levels of phytoalexin MA and MB in fresh leaves of Oscyp71Z2-overexpressing rice were 328 and 124 ng•g-1, respectively, which were higher than that of 85 and 42 ng•g-1 in wild type fresh leaves, respectively. Furthermore, the relative expression level of four key genes (OsCPS2, OsCPS4, OsKSL7 and OsKSL4) involve in phytoalexin MA and PB synthesis pathway in Oscyp71Z2-overexpressing rice was obviously increased.【Conclusion】The overexpression of cytochrome P450 gene Oscyp71Z2 in rice conferred resistance to panicle blast via activation of phytoalexins biosynthesis pathway.

Key words: rice , P450 gene Oscyp71Z2 , phytoalexins , disease resistance , blast

李文奇1, 王芳权1, 王军1, 范方军1, 朱金燕1, 杨杰1, 邵敏2, 仲维功1. 水稻P450基因Oscyp71Z2增强稻瘟病抗性的机制[J]. 中国农业科学, 2014, 47(13): 2485-2493.

LI Wen-Qi-1, WANG Fang-Quan-1, WANG Jun-1, FAN Fang-Jun-1, ZHU Jin-Yan-1, YANG Jie-1, SHAO Min-2, ZHONG Wei-Gong-1. Study on Resistance Mechanism to Blast Mediated by P450 Gene Oscyp71Z2 in Rice[J]. Scientia Agricultura Sinica, 2014, 47(13): 2485-2493.

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