大豆MYB（v-myb avian myeloblastosis viral oncogene homolog）转录因子基因对大豆异黄酮合成调控的研究

There is the highest isoflavone content in soybean and the isoflavone gets very important physiological function and amplication value. It is beneficial for enhancing soybean isoflavone content when proving the MYB transcription factor regulate the soybean isoflavone biosynthesis, because the MYB transcription factor participate in the flavonoid metabolism. Two new MYB transcription factor localized in cell nucleus would enhance flavonoid content significant which were cloned by our research group, our study will proves the regulation mechanism of soybean isoflavone biosynthesis by MYB transcription factor furthermore on the basis of our former research foundation. Our study will analysis relativity between the two gene's tissue expression characteristic and genistein、daidzein、glycitein cotent using real-time fluorescent quantitative PCR in order to prove the soybean biosynthesis direction regulated by this two gene. Gel shift assay results will reveal the cis-acting element which specific binding to the MYB transcription factor. The effector and reporter constructs were procuced to co-transfect into tobacco leaf, get the key enzyme which activated by this two MYB transcription factors and regulating the soybean isoflavonoid biosynthesis according to the GUS assay. After getting the T3 generation soybean, detect the correlation between expression situation of the key enzyme which regulating isoflavonoid biosynthesis and genistein、daidzein and glycitein content for the purpose of proving regulation mechanism of soybean isoflavonoid by MYB transcription factor. Screening the protein interact with MYB transcription factor by yeast two-hybrid technology, and regulate the soybean isoflavonoid biosynthesis when the MYB transcription factor interact with the protein. At finally, proved the regulation mechanism of the soybean isoflavonoid biosynthesis by MYB trasnscription factor and get high content transgenic soybean. All of this will lay the theoretical foundation for MYB transcription factor improving the effective application of soybean isoflavone and provide effective genetic resources for soybean directional improvement.

异黄酮具有重要的生理功能和应用价值，在大豆中含量最高。MYB 转录因子参与类黄酮代谢，探明其调控机制有利于人为提高大豆异黄酮含量。本课题在克隆了2个新的能显著提高类黄酮含量的MYB转录因子的基础上，欲进一步探明MYB转录因子对大豆异黄酮合成的调控机制。应用QPCR技术分析目的基因的组织表达特性与异黄酮含量的相关性，初步探明目的基因对大豆异黄酮合成的调控方向；采用凝胶阻滞法分析与MYB蛋白特异结合的顺式作用元件；用报告载体与效应载体共转化烟草叶片，根据其瞬时表达探明能被目的基因激活的大豆异黄酮合成关键酶；分析转目的基因T3代株系中大豆异黄酮合成关键酶基因的表达特性及与其异黄酮含量的相关性，探明目的基因对异黄酮合成的调控；应用酵母双杂交技术筛选与MYB转录因子基因的互作蛋白，研究相关蛋白互作时对大豆异黄酮的调控。最终探明MYB转录因子基因对异黄酮合成的调控机理，同时获得高异黄酮转基因大豆株系。

本研究利用RT-PCR技术从大豆品种（吉林32）中首次克隆了GmMYB12B2基因（基因银行注册号码为JF510467）,同时克隆了GmMYB12a。对克隆的基因转录活性和组织特异性表达进行了检测，同时对其与植物类黄酮合成途径中关键酶的调控关系进行分析研究，为明确MYB转录因子对植物类黄酮合成的调控机制提供理论依据，为高异黄酮含量的大豆新品种培育提供有利工具。.研究得到以下结果：（1）利用RT-PCR技术从大豆中首次克隆了GmMYB12B2基因，通过氨基酸序列比对发现，它含有两个MYB结构域，属于典型的R2R3-MYB转录因子；（2）构建GmMYB12a与GmMYB12B2基因的原核表达载体pET-28a-GmMYB12a及pET-28a- GmMYB12B2，分别转入大肠杆菌Rosetta中，在28℃、1.2mM IPTG诱导6h后，可获得纯度较高的目的蛋白，构建酵母效应质粒pGBK7-GmMYB12a及pGBK7- GmMYB12B2，酵母表达结果显示GmMYB12B2具有转录激活活性，GmMYB12a不具有转录激活功能或功能不明显；（3）利用半定量RT-PCR分析了两个转录因子对各种胁迫的应答情况，结果显示，在紫外辐射、高盐胁迫下，随着处理时间的延长，两个基因的表达量逐渐增加，且GmMYB12B2的表达量升高较GmMYB12a显著。两基因均对低温、干旱、ABA没有响应；（4）通过亚细胞定位预测及农杆菌介导转化洋葱表皮细胞的瞬时表达，表明两基因均定位于细胞核中；（5）GmMYB12B2在根及成熟的种子中表达量较高，随着种子发育成熟，其表达量也随之升高，GmMYB12a与GmMYB12B2表达模式基本一致，只是其表达量相对较低。两个基因在大豆发育时期的表达规律与大豆异黄酮的积累规律基本一致；（6）对GmMYB12a与GmMYB12B2进行拟南芥和大豆的遗传转化，证明二者均正调控植物的类黄酮生物合成，并可提高转基因拟南芥的盐及紫外线辐射耐受能力。