生长素抑制基因OsARP1调控水稻抗病性的分子机制研究

Epidemic disease caused by the loss-of-function of resistance (R) genes resulted in heavy losses in rice production. The use of the regulators on R gene signaling pathways has been considered to be the new strategy to obtain lasting and broad-spectrum resistance. Our previous study found that the expression of auxin-repressed gene OsARP1 was strongly induced during the defense response mediated by rice resistance gene Pid3. In this project we intend to: 1) Construct OsARP1 over-expression and knock-out genetic modified (GM) plants in the susceptible material TP309 and the Pid3-containing material TP309-Pid3. 2) Detect the responses of the GM plants against the Pid3 identification pathogen, and clarify the relationship between OsARP1 and rice blast resistance. 3) Examine the changes of salicylic acid, jasmonic acid, ethylene and auxin, and the expressions of the genes on resistance and auxin signaling pathways in the GM plants after inoculation, clarify the pathways that used by OsARP1 during resistance regulation. 4) Characterize the responses of the GM plants against different pathogens. The aim of this project is to study the molecular mechanism of rice resistance regulated by OsARP1, and provide basis for the use of OsARP1 in breeding rice varieties with lasting and broad-spectrum resistance in the future.

抗病基因失效导致的病害流行给水稻生产带来重大损失，利用抗病信号通路基因获得持久广谱抗性是育种新策略。我们前期研究发现生长素抑制基因OsARP1在抗稻瘟病基因Pid3介导的抗病反应中被强烈诱导。本项目拟：1）以水稻感病材料TP309和转入了Pid3的抗病材料TP309-Pid3为背景，构建OsARP1过表达和敲除转基因植株；2）检测转基因植株对Pid3鉴别菌的抗性，阐明OsARP1与稻瘟病抗性的关系；3）检测转基因植株在病菌处理后水杨酸、茉莉酸、乙烯和生长素的变化，以及抗病信号通路和生长素信号通路关键基因的表达，解析OsARP1调控水稻抗病性的信号途径；4）利用不同病菌检测OsARP1转基因植株的抗谱。本项目旨在探讨OsARP1调控水稻抗病性的分子机制，为将来利用其进行水稻广谱抗病育种提供研究基础。

为了使作物获得广谱、持久的抗性，发掘抗病信号通路基因的抗病功能是重要的研究方向。水稻全基因组芯片对稻瘟病诱导的水稻早期抗病反应的转录组研究结果显示生长素抑制基因OsARP1表达上调。本研究中我们对感病材料TP309和转入了Pid3基因的抗病材料TP309-Pid3在四叶期接种Pid3鉴别菌Zhong10-8-14，并对0h、24h、48h、72h、96h和120h的OsARP1的表达情况进行了检测，结果显示对TP309接菌后OsARP1的表达被诱导，至120h表达量达到最高，与0h相比升高了5.3倍，对TP309-Pid3接菌后OsARP1的表达也被诱导，并在48h时表达量达到最高，比0h升高7.2倍，说明OsARP1在抗病反应中诱导更早，表达量更高。接下来我们在TP309-Pid3中利用CRISPR/Cas9技术对OsARP1进行了基因编辑，选取OsARP1功能缺失纯合突变体，采用打孔接种方式接种稻瘟病菌Zhong10-8-14，结果显示OsARP1功能缺失纯合突变体叶片上的病斑增大2倍以上，相对真菌生物量平均增加7.5倍，说明OsARP1在Pid3介导的抗性中具有重要作用。我们还检测了感病品种中花11背景下OsARP1过表达转基因植株对稻瘟病菌Zhong10-8-14的抗性，结果显示过表达植株叶片上的病斑比野生型减小了24%，叶片相对真菌生物量减少44%，表明在中花11背景下过表达OsARP1可提高水稻对稻瘟病的抗性。我们对中花11背景的OsARP1敲除植株的种子萌发和幼苗生长情况进行了检测和统计分析，结果显示转基因植株的萌发率和幼苗株高均明显低于野生型，表明水稻OsARP1的缺失明显影响了水稻的生长发育。通过本研究我们解析了OsARP1在水稻抗病性和生长发育中的作用，为水稻抗病育种提供了理论参考。