ABH1在乙烯受体ETR1响应乙烯信号中的调节机制解析

As a gaseous hormone, ethylene regulates plant growth and development, as well as plant response to abiotic and biotic stresses. Ethylene receptor is the key player which is responsible for ethylene perception and initiation of ethylene signal transduction pathway. Therefore, it is crucial to elucidate the regulatory mechanism of ethylene receptor function. Previously, RTE1, a positive regulator of ETR1, was reported to be able to interact with ETR1 physically. Using EMS as a mutagen, we screened the novel regulator of ethylene receptor in etr1-2 population. We found that mutation of ABH1 gene could confer ethylene sensitivity in etr1-2, implying that ABH1 is a candidate involving in regulation of ethylene receptor ETR1. It has been documented that ABH1 is a subunit of cap-binding complex (CBC) and plays important role in RNA precursor processing, such as 5’ caping and exon-intron splicing. In this project, we try to figure out if and how ABH1 regulates function of ethylene receptor. The role of ABH1 in ethylene perception will be test to find out if ABH1 specifically modulates ETR1. The transcriptome between etr1-2 and etr1-2 abh1 will be compared to unravel the difference in RNA precusor splicing, especially for the transcript of ethylene receptor and signaling genes. In addtion, the 5’ caping process will be monitored in both etr1-2 and etr1-2 abh1 by RLM-RACE to find out the possible ABH1-targeted genes which are involving in regulation of ETR1-depedent ethylene perception. This work will provide novel evidences for the regulatory mechanism of ethylene receptor and extend our knowledge of the ethylene perception and signaling, and potentially benefit us from the genetic improvement of the economic plants in the future.

乙烯影响植物生长发育的多个环节以及植物对生物与非生物胁迫的响应。乙烯受体是感受乙烯、开启乙烯信号转导的首要因子，明晰其调节机制是了解乙烯信号转导的重要内容。之前仅发现乙烯受体ETR1的一个调节因子RTE1可与其直接互作，在转录后水平对ETR1进行调节，更多乙烯受体的调节因子以及其在不同层面对乙烯受体的调节机制尚未得到挖掘。申请者所在的研究团队通过EMS化学诱变筛选和图位克隆，得到了乙烯受体ETR1的一个可能的调节因子ABH1。已知ABH1参与RNA前体剪切与5’端加帽等转录加工过程。据此，本课题拟分析ABH1对乙烯受体ETR1的调控是否具有特异性，以及ABH1是否影响ETR1和乙烯信号转导途径中其他组分的内含子剪切和5’加帽修饰的过程，从而较为系统地了解ABH1在RNA转录加工层面对ETR1的分子调节机制，进而为深入了解乙烯受体的分子调控和乙烯信号转导机制提供新的试验数据和理论基础。

本项目通过EMS化学诱变筛选到了乙烯受体ETR1的一个可能的调节因子ABH1，利用CRISPR技术以及Get-way等方法构建了ABH1的RNAi、过表达等各种载体，并进行了遗传转化。拟南芥abh1突变体的乙烯“三重反应”分析显示，abh1突变体下胚轴缩短的百分比要明显大于野生型，这说明ABH1对乙烯感受的敏感性和响应程度有明显影响。abh1突变体应答ABA的反应显示，随着ABA浓度的增加abh1和野生型WT的萌发率均呈现降低趋势，abh1降低明显，显著低于野生型，这说明abh1确实对ABA超敏感，响应了ABA的调控。在拟南芥野生型和突变体中通过Real-time PCR检测基因的表达检测，结果显示ABH1明显影响了ABA信号转导中关键因子ABI1、ABI2、CHLH、KAT1、Rboth和PYR的表达，这暗示ABA信号可能以复杂的机制影响乙烯信号受体的响应转导，并且他们的影响可能是相互的。乙烯信号转导下游响应因子ERFs的表达在abh1、abi1-1突变体和野生型中的表达存在明显差异，这暗示ABA信号途径影响乙烯下游响应因子的表达，且ABH1和ABI1对其的影响并不相同，ABH1可能以一种依赖或不依赖乙烯受体调节的方式影响乙烯信号的响应和传递。ABH1是否影响乙烯受体ETR1以及其他乙烯关键因子的可变剪接的分析显示，ABH1可能影响了ETR1的5-7号外显子的可变剪接，这暗示ABH1对ETR1的影响可能通过可变剪接调控来完成。本研究发现了ABH1在乙烯信号转导中的新功能。. 本项目还发现烟草中乙烯不敏感突变体增强了干旱胁迫耐性，为植物耐旱性突变体的筛选提供了一种新的方法。拟南芥脂转运蛋白LTP1通过与乙烯受体调节因子RTE1相互作用在乙烯信号转导中发挥作用，发现了LTP1在乙烯信号转导中的的新的功能。拟南芥中CPR5通过与ETR1受体协同作用调节乙烯信号转导，发现了CPR5可以影响乙烯信号转导的新功能。. 上述结果为深入了解乙烯受体的分子调控和乙烯信号转导机制提供新的数据和理论基础。项目实施以来，发表SCI论文3篇，待发表SCI论文1篇，培养硕士研究生5名。