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[1]武广珩,傅仙玉.利用CRISPR/Cas9技术编辑水稻负调控抗病基因OsEDR1及基因功能分析[J].应用与环境生物学报,2019,25(06):1375-1380.[doi:10.19675/j.cnki.1006-687x.2019.05007]
 WU Guangheng,** & FU Xianyu.Editing rice negative regulation resistance gene OsEDR1 by CRISPR/Cas9 and analysis of its function[J].Chinese Journal of Applied & Environmental Biology,2019,25(06):1375-1380.[doi:10.19675/j.cnki.1006-687x.2019.05007]
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利用CRISPR/Cas9技术编辑水稻负调控抗病基因OsEDR1及基因功能分析
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
25卷
期数:
2019年06期
页码:
1375-1380
栏目:
研究论文
出版日期:
2019-12-30

文章信息/Info

Title:
Editing rice negative regulation resistance gene OsEDR1 by CRISPR/Cas9 and analysis of its function
作者:
武广珩傅仙玉
1福建省生态产业绿色技术重点实验室 武夷山 354300 2武夷学院生态与资源工程学院 武夷山 354300 3武夷学院茶与食品学院 武夷山 354300
Author(s):
WU Guangheng1 2** & FU Xianyu3
1 Provincial Key Laboratory of Eco-industrial Green Technology, Wuyishan 354300, China 2 College of Ecology and Resources Engineering, Wuyi University, Wuyishan 354300, China 3 College of Tea and Food Science, Wuyi University, Wuyishan 354300, China
关键词:
EDR1蛋白激酶基因编辑过表达白叶枯病CRISPR/Cas9技术
Keywords:
EDR1 protein kinase gene editing over-expression bacterial blight CRISPR/Cas9 technology
分类号:
Q943 : S511
DOI:
10.19675/j.cnki.1006-687x.2019.05007
摘要:
拟南芥EDR1(Enhanced Disease Resistance1),一个Raf样MAPKKK蛋白激酶,通过级联蛋白激酶途径MKK4/MKK5-MPK3/MPK6负调控水杨酸诱导的免疫防卫反应. 为研究水稻OsEDR1基因的功能,利用CRISPR/Cas9技术创制OsEDR1的功能缺失突变体的同时,构建过表达载体pTCK303-Ubi-OsEDR1转化水稻,获得转基因植株,最后接种白叶枯病菌. 此外,也构建pBI1.4t-35S-OsEDR1表达载体并转化拟南芥,分析转基因植株的白粉菌抗病性. 结果表明利用CRISPR/Cas9技术创制了单碱基插入的OsEDR1功能缺失突变体;白叶枯病原菌Xoo PXO99的接种实验表明,该功能缺失突变体与日本晴相比,病斑长度减少约50%,增强了水稻对Xoo PXO99的抗性. 定量PCR和接种实验结果显示在日本晴中过量表达OsEDR1增强了水稻对Xoo PXO86的感病性. 在拟南芥edr1突变体中异源表达OsEDR1无法抑制edr1突变体对白粉菌的抗病和白粉菌诱导的细胞死亡. 综上所述,OsEDR1负调控水稻对白叶枯的抗病反应和自发的细胞死亡,可能与拟南芥所依赖的防御反应信号通路不同相关;结果可为下一步深入研究EDR1基因的作用机制和在水稻育种中的应用提供参考. (图4 表1 参29)
Abstract:
EDR1 (Enhanced Disease Resistance1) in Arabidopsis thaliana is a Raf-like MAPKK protein kinase that negatively regulates salicylic acid-induced defense response and controls plant immune defense response through MKK4/MKK5–MPK3/MPK6. In order to study the function of OsEDR1 gene in rice, we developed a loss-of-function mutant of OsEDR1 using CRISPR/Cas9 technology. Over-expression vectors pTCK303-Ubi-OsEDR1 and pBI1.4t-35S-OsEDR1 were constructed to transform rice and Arabidopsis to obtain transgenic plants. Finally, bacterial blight and powdery mildew were inoculated to analyze the resistance. The results showed that CRISPR/Cas9 technology created loss-of-function mutant of OsEDR1 with single base insertion. The inoculation experiment of Xoo PXO99 showed that the length of lesion of loss-of-function mutant was reduced by about 50% compared with that of Nipponbare, which enhanced resistance of rice to Xoo PXO99. Quantitative PCR and inoculation experiment results showed that over-expression of OsEDR1 in Nipponbare could enhance susceptibility to Xoo PXO86. The heterologous expression of OsEDR1 in Arabidopsis edr1 mutant could not compromise resistance of edr1 mutant to powdery mildew and cell death induced by powdery mildew. In conclusion, OsEDR1 negatively regulates resistance to bacterial blight and spontaneous cell death in rice, but it may be different from defense signaling in Arabidopsis. These results will be helpful to further study the mechanism of EDR1 gene and its application in rice breeding.

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更新日期/Last Update: 2019-12-25