四川农业大学学报 ›› 2021, Vol. 39 ›› Issue (3): 297-306.doi: 10.16036/j.issn.1000-2650.2021.03.004

所属专题: 水稻研究专题

• 特别策划:水稻研究专题 • 上一篇    下一篇

利用R基因RRS1/RPS4探索水稻原生质体瞬时表达转化体系的研究

李丽, 赵彦丰, 吴琪琦, 黄燕*   

  1. 四川农业大学生命科学学院,四川雅安625014
  • 收稿日期:2020-11-09 出版日期:2021-06-28 发布日期:2021-07-05
  • 通讯作者: *黄燕,博士,副教授,主要从事植物抗病分子机理研究, E-mail: 14116huangyan@sicau.edu.cn。
  • 作者简介:李丽,硕士研究生。
  • 基金资助:
    四川农业大学创新训练计划(201810626006); 四川省科技创新苗子工程(2020056)

Exploring the Transient Expression and Transformation System of Rice Protoplasts by Using R Gene RRSl/RPS4

Li Li, ZHAO Yanfeng, WU Qiqi, HUANG Yan*   

  1. College of Life Science, Sichuan Agricultural University, Ya'an 625014, Sichuan, China
  • Received:2020-11-09 Online:2021-06-28 Published:2021-07-05

摘要: 【目的】将拟南芥TNL型R基因RRS1/IRPS4转入水稻原生质体中,探索瞬时表达转化体系的最佳条件,并分析AtRRS1/AtRPS4对水稻内源ETI下游抗病基因表达的影响,为揭示水稻EDS1是否参与ETI反应途径提供实验依据。【方法】以日本晴两叶一心期幼苗为植物材料,设计不同梯度的PEG浓度(30%、35%、40%)、转化时的质粒浓度(0.5、1、2μg/μL)以及PEG介导的转化温度(0、18、37℃),利用Box- Benhnken设计三因素三水平来分析不同参数组合条件下对转化效果的影响,并对原生质体转化效率进行预测和试验验证。随后,利用该瞬时系统表达拟南芥TNL型R基因RRS1/IRPS4,并通过检测水稻内源抗病标志基因的表达水平来揭示其对水稻抗病能力的影响。【结果】经过试验验证得到水稻原生质体转化最佳条件:PEG浓度36.16%、质粒浓度1.72 μg/μL转化温度37℃,该组合条件下转化效率可达75%;利用该优化体系开展的瞬转研究发现拟南芥TNL型R基因/基因对RRS1/RPS4能显著提高水稻原生质体中抗病相关基因的表达。【结论】构建了高效的水稻原生质体瞬时转化系统,并以此为平台,研究揭示了拟南芥TNL型R基因RRS1/RPS4可能通过水稻内源的EDS1去诱导ETI下游抗病免疫反应的发生。

关键词: 水稻, 原生质体, 瞬时表达, R基因, 转化体系

Abstract: 【Objective】 The Arabidopsis TNL type R gene RRS1/RPS4 was transferred into rice protoplasts to explore the optimal conditions for transient expression transformation system,and the effect of these R genes on the expression of endogenous ETI downstream disease resistance genes in rice was analyzed to provide experimental basis for revealing whether OsEDS1 is involved in ETI response pathway. 【Method】 Seedlings in two leaf and one heart stage of Nipponbare were used as plant materials to explore the optimal combination of conditions for PEG-mediated transformation. By designing different gradients of PEG concentration(30%,35%,40%),and PEG-mediated transformation temperature(0,18,37℃),Box-Benhnken was used to design three factors and three levels to analyze the effect of different parameter combinations on the transformation effect,and the protoplast transformation efficiency was predicted and experimentally verified. Subsequently,the Arabidopsis TNL type R gene RRS1/RPS4 was single/cotransformed into the rice protoplast transient expression system,and their influence on rice disease resistance was revealed by detecting the expression level of rice endogenous disease resistance marker genes.【Result】 The optimal conditions verified by experiments for rice protoplast transformation were as follows:PEG concentration 36.16%,plasmid concentration 1.72μg/μL,transformation temperature 37℃,and the conversion efficiency can reach 75% under the combined conditions. Transient studies using this optimized system found that the Arabidopsis TNL type R gene/gene pair RRS1/RPS4 can significantly increase the expression of disease resistance-related genes in rice protoplasts.【Conclusion】 In thisstudy,an efficient protoplast transient transformation system was established and used as a platform,this study revealed that the Arabidopsis TNL type R gene RRS1/RPS4 may induce downstream immune response through the endogenous EDS1 in rice.

Key words: rice, protoplast, transient expression, R gene, transformation system

中图分类号: 

  • S511