棉花FAD2-1基因的5'UTR内含子与启动子的顺式作用元件鉴定及转录调控作用研究

In Gossypium, the Δ12-desaturase enzyme gene FAD2-1 is highly expressed and seed-specific, and it encodes the key enzyme that catalyzes the conversion of oleic acid to linoleic acid in the developing seed. The expression level of FAD2-1 determines the nutritional properties and oxidative stability of cottonseed oil. It is essential for understanding the mechanism of FAD2-1 expression to identify and analyze upstream regulatory regions of FAD2-1, the promoter and 5'UTR intron. We have previously cloned the upstream sequence of FAD2-1. In this study, we would further analyze the effect of deletion of the promoter and 5'UTR intron of FAD2-1 on gene expression in transgenic plants to reveal the regulatory mechanism of FAD2-1 gene and explore the physiological significance of the formation of cottonseed fatty acid composition. The full length and the PCR-mediated DNA deletion sequences of the 5'UTR intron and promoter were fused with GUS gene, respectively. The different expression vectors were transformed into tobacco and cotton by Agrobacterium-mediated genetic transformation.The GUS gene expression patterns would be detected at different developmental stages of transgenic plants and their offspring involved in abiotic stresses and phytohormones responses. The expression activity, the developmental stage and tissue specificity of GUS would be comparatively analyzed, and the genetic stability of GUS in transgenic plants offspring would be also evaluated. The important cis-regulatory elements in 5'UTR intron and promoter region would therefore be identified. The results would clear the spatial and temporal expression patterns of the promoter and 5'UTR intron and their effects on gene expression regulation, and elucidate the synergistic effects between the 5'UTR intron and promoter and their utility value in molecular plant breeding. It would also provide scientific basis for improving cottonseed oil quality.

棉花中，FAD2-1基因是控制种子发育阶段油酸向亚油酸转变的关键基因，其表达水平决定了棉籽油的营养价值与氧化稳定性。对FAD2-1上游序列启动子及5'UTR内含子的关键转录调控元件的识别是理解其表达机制的必要步骤。为了探索FAD2-1表达调控的分子机制和棉籽脂肪酸品质形成的生理学意义，本项目在前期克隆获得的棉花FAD2-1上游序列基础上，利用DNA序列缺失法，分别与GUS基因融合构建表达载体，并用农杆菌介导转化烟草与棉花；进而检测GUS基因在转基因植株及其子代不同发育时期、非生物胁迫及激素处理等条件下的表达模式；比较分析GUS的表达活性、表达的组织特异性、发育阶段性及遗传稳定性等。从而鉴定出5'UTR内含子与启动子区重要顺式调控元件，明确5'UTR内含子与启动子的时空表达特性及对基因表达的影响；阐明5'UTR内含子与启动子的协同效应及其在分子育种上的利用价值；为改良棉籽油品质提供科学基础。

棉花中，GhFAD2-1基因是控制种子发育阶段油酸向亚油酸转变的关键基因，对GhFAD2-1上游序列启动子及5'UTR内含子的转录调控元件的识别是理解其表达机制的必要步骤。本研究利用5'RACE 技术，克隆GhFAD2-1的5'UTR序列，结合棉花基因组序列，进而克隆GhFAD2-1基因启动子与5'UTR内含子，并分析其作用元件。结果表明：棉花A、D基因组的GhFAD2-1 5'启动子全长分别为1349bp、1279bp；两者有238个碱基差异。启动子序列中具有种子特异的表达元件，以及与激素和胁迫相关的应答元件等。棉花A、D基因组的GhFAD2-1 5' UTR中各含一内含子序列，全长分别为1103bp、1111bp。GhFAD2-1 mRNA的5' UTR为77bp，转录的起点碱基为T；5' UTR内含子剪切位点分别是AA-GG、CA-GC。内含子包括一些典型的与光响应相关的元件，以及与激素和胁迫相关的应答元件。通过PCR技术扩增GhFAD2-1 5'全长及缺失序列，结合Gateway技术构建出表达载体：pFAD2-12467bp::GUS、pFAD2-12219bp::GUS、pFAD2-12165bp::GUS、pFAD2-11642bp::GUS、pFAD2-11348bp::GUS、pFAD2-11279bp::GUS、pFAD2-12163bp::GUS、pFAD2-11799bp::GUS、pFAD2-11428bp::GUS、pFAD2-11119bp::GUS、pFAD2-11111bp::GUS。通过农杆菌介导的遗传转化，获得相应的转基因拟南芥植株。通过比较分析转基因植株中GUS的表达活性、组织特异性、发育阶段性等表明，GhFAD2-1启动子主要在种子发育的中后期表达，但在花药与胚珠发育时期也有较弱的表达。GhFAD2-1的5’UTR内含子具有启动子活性；并且通过与GhFAD2-1启动子之间的协同效应，发挥了转录增强子功能。从序列缺失驱动GUS基因表达的结果表明，-300 element (TGHAAARK)元件对基因的表达量起着重要的调控作用；一定数目的E-Box (CANNTG)等元件的累计与基因表达量成正比。研究结果将有助于对GhFAD2-1的时空表达调控有更深入的了解，也可望为植物的遗传转化提供高效率的特异启动子和高效特异的增强子元件。

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