稻瘟病抗病蛋白Pigm-1互作蛋白的筛选与鉴定

doi:10.3864/j.issn.0578-1752.2025.06.001

中国农业科学 ›› 2025, Vol. 58 ›› Issue (6): 1043-1051.doi: 10.3864/j.issn.0578-1752.2025.06.001

• 作物遗传育种·种质资源·分子遗传学 • 下一篇

稻瘟病抗病蛋白Pigm-1互作蛋白的筛选与鉴定

晋艺丹¹^,²(), 何旎清¹(), 程朝平¹, 林少俊¹, 黄凤凰¹, 白康呈¹^,², 张涛¹^,², 汪文潇¹^,², 余敏祥¹, 杨德卫¹()

¹ 福建省农业科学院水稻研究所，福州 350018
² 福建农林大学农学院，福州 350002

收稿日期:2024-12-26 接受日期:2025-01-20 出版日期:2025-03-25 发布日期:2025-03-25
通信作者:
杨德卫，E-mail：dewei-y@163.com
联系方式: 晋艺丹，E-mail：stml093015@163.com。何旎清，E-mail：heniqing430@163.com。晋艺丹和何旎清为同等贡献作者。
基金资助:
福建省公益项目(2023R1021006); 国家自然科学基金(32202324); 国家自然科学基金(32402387); 国家自然科学基金项目延伸研究项目(GJYS202302); 福建省农业科学院自由探索项目(ZYTS2023001); 5511协同工程项目(XTCXGC2021001); 福建省农业科学院对外合作项目(DKBF-2024-12); 福建省自然科学基金(2024J01171); 福建省自然科学基金(2022J01450)

Screening and Identification of Pigm-1 Interaction Proteins for Disease Resistance of Rice Blast

JIN YiDan¹^,²(), HE NiQing¹(), CHENG ZhaoPing¹, LIN ShaoJun¹, HUANG FengHuang¹, BAI KangCheng¹^,², ZHANG Tao¹^,², WANG WenXiao¹^,², YU MinXiang¹, YANG DeWei¹()

¹ Institute of Rice, Fujian Academy of Agricultural Sciences, Fuzhou 350018
² College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002

Received:2024-12-26 Accepted:2025-01-20 Published:2025-03-25 Online:2025-03-25

摘要/Abstract

摘要：

【目的】稻瘟病是水稻最重要的病害之一，稻瘟病抗性基因Pigm-1具有广谱抗性，但其抗病的遗传机制还不清楚，通过筛选并鉴定Pigm-1信号通路中的关键蛋白，为水稻抗病育种提供重要的理论依据。【方法】通过同源重组的方法构建诱饵载体pGBKT7-Pigm-1-CC^1-576，检测诱饵蛋白自激活活性，同时，将pGBKT7和pGBKT7-Pigm-1-CC^1-576质粒分别单独转化Y2H Gold酵母菌株检测诱饵蛋白毒性；利用稻瘟病菌诱导后构建的水稻酵母cDNA文库筛选水稻抗病R蛋白Pigm-1的互作蛋白，并通过Rice Information GateWay（RIGW）比对与注释测序结果；利用荧光素酶互补（Luc）、免疫共沉淀（Co-IP）和酵母双杂交试验对Pigm-1与OsbHLH148进行互作验证；运用实时荧光定量PCR（qRT-PCR）对互作蛋白OsbHLH148对应的基因进行组织表达分析。【结果】自激活性检测显示，共转化诱饵蛋白pGBKT7-Pigm-1-CC^1-576与AD质粒没有自激活现象，毒性检测结果表明，诱饵蛋白对酵母细胞毒性较小或没有毒性；通过筛选酵母文库，获得124个可能与Pigm-1互作的蛋白，涉及乙烯合成、赤霉素合成、活性氧清除、酶代谢等相关蛋白，还有功能未知蛋白；利用Luc、Co-IP和酵母双杂交试验验证了Pigm-1-CC^1-576与OsbHLH148具有相互作用；进一步分析发现OsbHLH148受稻瘟病菌诱导表达；OsbHLH148在水稻6周龄的叶片中表达量最高。【结论】获得可能与Pigm-1互作的124个蛋白，其中OsbHLH148与Pigm-1-CC^1-576具有互作关系，推测OsbHLH148可能在Pigm-1介导的稻瘟病抗性中发挥作用。

关键词: 水稻, 稻瘟病, Pigm-1, 互作蛋白, OsbHLH148

Abstract:

【Objective】 Rice blast is one of the most devastating diseases of rice production. A broad-spectrum disease resistance gene Pigm-1 was identified but its functional pathway and interactors are unknown. The screening and identification of key proteins in the Pigm-1 signaling pathway will provide an important theoretical basis for rice disease resistance breeding. 【Method】 In this study, the decoy protein pGBKT7-Pigm-1-CC^1-576 vector was constructed to detect the decoy protein self-activation, and the toxicity of the decoy protein was detected by separately transforming the plasmid pGBKT7 and pGBKT7-Pigm-1-CC^1-576 into Y2H Gold yeast. The rice disease resistance R protein Pigm-1 was screened by cDNA expression yeast library induced by rice blast fungus. The sequencing results were compared and annotated by Rice Information GateWay (RIGW). The interaction of OsbHLH148 protein was verified by Luc, Co-IP and yeast two-hybrid assays, and the tissue expression of the corresponding gene of the interaction protein OsbHLH148 was analyzed by qRT-PCR. 【Result】 The self-activation test showed that the decoy protein pGBKT7-Pigm-1-CC^1-576 did not self-activate when cotransformed with the AD plasmid, and the toxicity analysis showed that the decoy protein had little or no toxicity to yeast cells. A total of 124 proteins that may interact with Pigm-1 were obtained by screening the yeast library, and among these proteins, there are ethylene synthesis related, gibberellin synthesis related, active oxygen species clearly related, enzyme metabolism related, and some function unknown. The interaction between Pigm-1-CC^1-576 and OsbHLH148 was verified by Luc, Co-IP and yeast two-hybrid methods. Further analysis showed that OsbHLH148 can be induced by blast fungus infection, and the tissue expression analysis showed that OsbHLH148 expression level was the highest in rice leaves at 6 weeks. 【Conclusion】 In this study, 124 proteins that may interact with Pigm-1 were obtained. One of these proteins, OsbHLH148, was selected and verified to interact with Pigm-1-CC^1-576. Suggesting that OsbHLH148 may be involved in Pigm-1 mediated resistance of rice blast.

Key words: rice, rice blast, Pigm-1, interacting protein, OsbHLH148

晋艺丹, 何旎清, 程朝平, 林少俊, 黄凤凰, 白康呈, 张涛, 汪文潇, 余敏祥, 杨德卫. 稻瘟病抗病蛋白Pigm-1互作蛋白的筛选与鉴定[J]. 中国农业科学, 2025, 58(6): 1043-1051.

JIN YiDan, HE NiQing, CHENG ZhaoPing, LIN ShaoJun, HUANG FengHuang, BAI KangCheng, ZHANG Tao, WANG WenXiao, YU MinXiang, YANG DeWei. Screening and Identification of Pigm-1 Interaction Proteins for Disease Resistance of Rice Blast[J]. Scientia Agricultura Sinica, 2025, 58(6): 1043-1051.

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稻瘟病抗病蛋白Pigm-1互作蛋白的筛选与鉴定

Screening and Identification of Pigm-1 Interaction Proteins for Disease Resistance of Rice Blast

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