GA信号转导因子LsRGL1在叶用莴苣高温抽薹中的作用机制研究

In our country the cultivation area of lettuce is small a decade ago, but it is developing fast currently. Lettuce presently holds the second position in planting area in Beijing.Lettuce is adapted to cool climate. Premature bolting caused by the high temperature in summer seriously reduces its quality, in worse cases total harvest failure. Lettuce is the typically high-temperature vernalization vegetable among leafy and root vegetables. In previous researches, we obtained one DELLA protein closely related to lettuce bolting using RNA-Seq analysis. This project is aimed to study the gene’s function and its mechanism in high temperature bolting and flowering. We will construct the over-expression vector 、RNAi vector and CRISPR/Cas9 vector of LsRGL1, transfer these vectors into leaf lettuce material" PS-11 ". then we can obtain transgenic plants. The growth characteristics of transgenic plants were investigated, such as flowering time. The endogenous hormone content and related gene expression patterns in developmental stages will be detected. We also want to do RNA-Seq using the transgenic plants to investigate some genes related with lettuce bolting. We hope to explore the molecular mechanism of bolting in high temperature, thus we can regulate the bolting time and avoid the premature bolting phenomenon.

叶用莴苣，俗名生菜，目前在全国各地广泛栽培，种植面积逐年扩大，在北京已成为播种面积仅次于番茄的第二大蔬菜。其性喜冷凉气候，夏季高温极易造成“先期抽薹”，困扰生产。项目组在前期研究中，对两份具有极端高温抽薹性差异材料的苗期叶片做了转录组分析，研究结果显示，一条赤霉素信号转导相关的DELLA家族基因在两份材料中的相对表达差异极其显著。本项目将克隆这条基因，通过构建过表达、RNAi、CRISPR/Cas9表达载体，转入叶用莴苣材料“PS-11”，获得转基因植株；调查转基因植株的抽薹开花时间、花茎形态等植株生长特性；检测各发育时期、各部位内源激素含量及相关基因表达模式；提取转基因植株抽薹前后的总RNA进行转录组测序等研究，分析该条基因的功能及其在叶用莴苣响应高温后抽薹开花过程中的作用机制。该项目将在基因水平探索叶用莴苣高温抽薹开花的分子机制，以期达到调控其抽薹期，避免“先期抽薹”现象的目的。

叶用莴苣是我国特色农业调整、生态环境改良及农民增收方面的重要产业。叶用莴苣以设施栽培为主，栽培过程中容易遇到高温情况。高温会造成叶用莴苣快速抽薹，从而使其叶质粗老，食之略有苦味，且叶片容易发生腐烂。因此，高温抽薹是限制叶用莴苣产业发展的重要问题。研究发现LsRGL1基因在高温胁迫下能够对叶用莴苣发育过程中起作用，可以作为叶用莴苣抽薹的调控者。本研究构建了RGL1的TRV载体(TRV2-LSRGL1)、过表达载体(OE-LSRGL1)和RNAi载体(RNAi-LSRGL1)，将其导入叶用莴苣热敏性品种“PS-11”中，获得转基因植株。结果表明，将获得转基因植株进行T1代筛选后，通过高温处理，发现OE-LSRGL1能够延迟叶用莴苣的开花时间，并且相对于WT茎更短，LsRGL1的相对表达量显著上升；RNAi-LSRGL1能够加快叶用莴苣的开花时间，并且相对于WT茎更长，LsRGL1的相对表达量显著下降；石蜡切片中观察到的花芽分化结果与表型一致。通过转录组测序后我们注意到，KEGG在“植物激素信号转导”中富集，并且参与生长素 (IAA)和脱落酸 (ABA)信号转导在过表和沉默株系中都有富集，而参与赤霉素 (GA)、乙烯 (Eth)在RNAi-LSRGL1株系中显著富集更多。同时Y1H试验结果显示，LsRGL1能够结合LsGID1、LsSLY1基因启动子。这些结果表明，LsRGL1是一个可以抑制GA反应的转录调控因子，对叶莴苣抽薹具有负调控作用。