水稻抗性蛋白OsRRK1抗褐飞虱机理分析

doi:10.16819/j.1001-7216.2020.0406

中国水稻科学 ›› 2020, Vol. 34 ›› Issue (6): 512-519.DOI: 10.16819/j.1001-7216.2020.0406

水稻抗性蛋白OsRRK1抗褐飞虱机理分析

马银花¹, 李萍芳¹, 董文静¹, 易松望¹, 杨芳², 杜波², 金晨钟^1,^*()

¹ 湖南人文科技学院湖南省农田杂草防控技术与应用协同创新中心/农药无害化应用省高校重点实验室, 湖南娄底 417000
² 武汉大学生命科学学院杂交水稻国家重点实验室, 武汉 430072

收稿日期:2020-04-25 修回日期:2020-05-18 出版日期:2020-11-10 发布日期:2020-11-10
通讯作者: 金晨钟
基金资助:
湖南省自然科学基金青年项目（2019JJ50281）;湖南省教育厅优秀青年项目（18B455）;杂交水稻国家重点实验室（武汉大学）开放课题基金资助项目(KF201901);娄底市应用技术与开发项目（33319013）;湖南省应用特色学科（植物保护）资助项目;湖南人文科技学院卓越人才培养计划资助项目（校教通 2017 第 74 号）;湖南省高等学校科技创新团队(201937924)

Mechanism Analysis of Rice Resistance Protein OsRRK1 Againstthe Brown Planthopper

Yinhua MA¹, Pingfang LI¹, Wenjing DONG¹, Songwang YI¹, Fang YANG², Bo DU², Chenzhong JIN^1,^*()

¹ Hunan Provincial Collaborative Innovation Center for Field Weeds Control/ Key Laboratory of Harmless Application of Pesticide in Hunan Higher Education, Hunan University of Humanities, Science and Technology, Loudi417000,China
² College of Life Sciences, Wuhan University /National Key Laboratory of Hybrid Rice,Wuhan 430072,China

Received:2020-04-25 Revised:2020-05-18 Online:2020-11-10 Published:2020-11-10
Contact: Chenzhong JIN

摘要/Abstract

摘要： 目的 OsRRK1（Rop-interacting receptor-like kinase 1）蛋白是水稻中第一个被研究的类胞质受体激酶RLCKⅥ家族蛋白。在调控水稻叶片的卷曲和对褐飞虱的防御中都起着重要作用,但是其调控机理尚不清楚。本研究试图进一步探究OsRRK1基因抗褐飞虱的机理。方法利用酵母双杂交的方法分析OsRRK1蛋白与OsLecRK（lectin-like receptor kinase）、OsLecRK1、OsLecRK2、OsLecRK3的互作关系,OsLecRK是抗褐飞虱基因BPH15区间的候选基因,是水稻先天免疫系统中一个重要成员,既参与水稻先天免疫反应,包括对褐飞虱、白叶枯病和稻瘟病的抗性,又参与水稻的发育过程。OsLecRK1、OsLecRK2、OsLecRK3的基因簇组成抗褐飞虱基因BPH3,含BPH3基因的水稻具有广谱、持久的抗虫性。同时利用DNAMAN软件分析OsLecRK蛋白与OsLecRK1、OsLecRK2、OsLecRK3的同源性。结果 DNAMAN的分析结果显示OsLecRK与OsLecRK1、OsLecRK2、OsLecRK3的同源性都很高,蛋白一致性在50%以上;酵母双杂交结果显示OsRRK1与OsLecRK、OsLecRK2和OsLecRK3互作。结论 OsRRK1通过与抗褐飞虱基因BPH15和BPH3候选基因的相互作用参与水稻抗褐飞虱过程。

关键词: 水稻, OsRRK1, 褐飞虱, 酵母双杂交系统

Abstract:

【Objective】OsRRK1 (Rop-interacting receptor-like kinase 1) protein in rice is the first RLCK VI family protein in rice that has been studied.It plays an important role inregulating the curling of rice leaves and the defense against brown planthoppers. However, the mechanism of its regulation of resistance to the brown planthopperremains unclear. Weattempt to further explore the mechanism of OsRRK1gene resistance to the brown planthoppers.【Method】The interaction between OsRRK1 protein and OsLecRK (lectin-like receptor kinase), OsLecRK1, OsLecRK2 and OsLecRK3 was analyzed by yeast two-hybrid method.OsLecRK is a candidate gene forBPH15 range of anti-brown planthopper, and is an important member of rice innate immune system. OsLecRK is involved innot only rice innate immune response, including resistance to brown planthoppers, white leaf blight and rice blast, but alsothe development of rice. The gene clusters of OsLecRK1, OsLecRK2 and OsLecRK3 constitute the anti-brown planthopper gene BPH3. Rice containing the BPH3 gene has a broad spectrum and lasting insect resistance. Meanwhile, DNAMAN software was used to analyze the homology of OsLecRK with OsLecRK1, OsLecRK2 and OsLecRK3.【Result】The analysis results of DNAMAN showed that OsLecRK has high homology with OsLecRK1, OsLecRK2 and OsLecRK3, and the proteinsshared more than 50% identity.The results of yeast two-hybrid analysis showed that OsRRK1 interacted with OsLecRK, OsLecRK2 and OsLecRK3.【Conclusion】OsRRK1 participates in the process of resistance to the brownplanthoppersthroughinteracting with BPH15 and BPH3 candidate genes.

Key words: rice, OsRRK1, brown planthopper, yeast two-hybrid system

中图分类号:

Q786

马银花, 李萍芳, 董文静, 易松望, 杨芳, 杜波, 金晨钟. 水稻抗性蛋白OsRRK1抗褐飞虱机理分析[J]. 中国水稻科学, 2020, 34(6): 512-519.

Yinhua MA, Pingfang LI, Wenjing DONG, Songwang YI, Fang YANG, Bo DU, Chenzhong JIN. Mechanism Analysis of Rice Resistance Protein OsRRK1 Againstthe Brown Planthopper[J]. Chinese Journal OF Rice Science, 2020, 34(6): 512-519.

图/表 6

表1 用于构建载体的引物

Table 1 Primers used in vectors constructed.

引物 Primer	引物序列（5'-3'） Primer sequence（5'-3'）	限制性内切酶酶切位点 Restriction endonuclease site
AD-LecRKF	CGGAATTCATGGTTGCTCTGCTACTCTTT	EcoR I
AD-LecRKR	CGGAATTCTTAATGAGAAAACAGAGGATTACATTCC	EcoR I
AD-LecRK1F	TCCCCCGGGATGGTTGCTCTGCTACTCTTTCCC	Sma I
AD-LecRK1R	TCCCCCGGGCTATGGAAGTGAGCTGATGAAGG	Sma I
AD-LecRK2F	TCCCCCGGGTATGGCACCTATCCTGTTCTTGCCC	Sma I
AD-LecRK2R	TCCCCCGGGTCATGCGAGTGAACTGATATAGG	Sma I
AD-LecRK3F	TCCCCCGGGTATGGCTCATCTCCTGTTCTTGCCC	Sma I
AD-LecRK3R	TCCCCCGGGTCATGCAAGTGAACTGATATAGG	SmaI
BK-OsRRK1F	CGGAATTCATGAGGCCTCTGTACCTGCGCAGC	EcoR I
BK-OsRRK1R	CGGAATTCCTAATTGCTCAAAGATGATGAGCA	EcoR I
T7	TAATACGACTCACTATAGGGCGA
3′AD	AGATGGTGCACGATGCACAG
3′BD	TTTTCGTTTTCCTAAGAGTC

图1 OsLecRK与OsLecRK1-OsLecRK3蛋白结构域预测 S_TKc—丝氨酸/苏氨酸蛋白激酶催化结构域。

Fig.1. Prediction of OsLecRK and OsLecRK1-OsLecRK3 protein domains. S_TKc, Serine/Threonine protein kinasescatalytic domain.

图2 OsLecRK与OsLecRK1-OsLecRK3蛋白序列比对

Fig. 2. Alignment of OsLecRK and OsLecRK1-OsLecRK3.

图3 OsLecRK与OsLecRK1-OsLecRK3进化分析

Fig. 3. Phylogenetic tree analysis of OsLecRK and OsLecRK1-OsLecRK3.

图4 利用酵母双杂交对OsRRK1与OsLecRK蛋白互作的验证 pGBKT7-53/ pGADT7-T：阳性对照;pGBKT7-Lam/ pGADT7-T：阴性对照。DDO：SD/-Leu/-Trp（缺乏亮氨酸和色氨酸）双缺;TDO：SD/-Leu/-Trp/-His（缺乏亮氨酸,色氨酸和组氨酸）三缺。

Fig. 4. The interaction between OsRRK1 and OsLecRK protein was verified by yeast two-hybridization. pGBKT7-53/pGADT7-T, Positive control; pGBKT7-Lam/pGADT7-T, Negative control. DDO, SD/-Leu/-Trp; TDO, SD/-Leu/-Trp/-His.

图5 OsRRK1与OsLecRK1-OsLecRK3蛋白互作的验证 DDO：SD/-Leu/-Trp（缺乏亮氨酸和色氨酸）双缺;TDO：SD/-Leu/-Trp/-His（缺乏亮氨酸,色氨酸和组氨酸）三缺。

Fig. 5. Confirmation of positive interactions between OsRRK1 and OsLecRK1-OsLecRK3. DDO: SD/-Leu/-Trp; TDO: SD/-Leu/-Trp/-His.

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水稻抗性蛋白OsRRK1抗褐飞虱机理分析

Mechanism Analysis of Rice Resistance Protein OsRRK1 Againstthe Brown Planthopper

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