MsMYB转录因子对紫花苜蓿耐铝毒的调控和机制研究

The red soil in south of china are generally acid, and soil pH is lower than 5.0. The acid soil strongly affect alfalfa (Medicago sativa L.) growth due to high activity of aluminum cation in the acid soil, consequently restrict alfalfa production in south of china. This project will clone seven MsMYB transcription factors, which were significantly expressed in aluminum (Al) stressed alfala seedling, transfer these seven MsMYB genes to alfalfa callus，and induce the callus to take roots. The effects of aluminum on alfalfa growth and biosynthesis of flavonoid and lignin in the callus roots will be studied under treatments with different Al concentrations. The main research contents of the project included: 1) Effect of the seven MsMYB transcription factors on regulating Al tolerance of alfalfa; 2) Effect of the seven MsMYB transcription factors on regulating biosynthesis of flavonoid and lignin in Al-stressed alfalfa, and on regulating key gene expressions of biosynthesis of flavonoid and lignin in Al-stressed alfalfa; 3) Screen and test of downstream target genes which directly act with the seven MsMYB trasncription factors in Al-stressed alfalfa. The downstream target genes are taking part in the biosynthesis of flavnonid and lignin; 4) The localization and expression changes of the seven MsMYB transcript factors in root tissue of Al-stressed alfalfa; 5) The mechnisms in the aspects of antioxidant physiology and cell structure for alfalfa to adapt Al toxicity regulated by the seven MsMYB transcript factors under Al-stressed condition. The purpose of the project are wish to find regulatory function of the seven MsMYB transcription factors on alfalfa adapting Al toxicity, and to understand mechnism of the seven MsMYB genes on regulating Al tolerance of alfalfa in the aspects of molecular, antioxidant physiology and cellar structure. The results of the study could be helpful to assist the work for breeding new alfalfa varity with high ability of aluminum tolerance.

我国南方红壤区土壤普遍呈现强酸性，导致土壤中铝离子活性增加，严重抑制紫花苜蓿生长，制约紫花苜蓿在南方种植。本课题通过对铝胁迫下差异表达显著的、功能未知的7个紫花苜蓿MsMYB转录因子基因进行克隆，并利用发根农杆菌将MsMYB基因转入紫花苜蓿愈伤，诱导愈伤根系。在不同铝浓度处理下，研究转基因愈伤根系耐铝毒性能和黄酮与木质素合成代谢的变化及其机理。主要研究内容包括：MsMYB基因对紫花苜蓿耐铝毒的调控作用；MsMYB基因对黄酮和木质素合成及其关键调控基因表达的影响；MsMYB转录因子调控黄酮和木质素合成的下游靶基因筛选与验证；MsMYB基因在根组织的定位和表达变化；以及MsMYB基因调控紫花苜蓿耐铝毒的抗氧化生理和细胞学机理，揭示7个MsMYB对紫花苜蓿耐铝毒功能的调控作用及其调控紫花苜蓿耐铝毒的分子、抗氧化生理和细胞机理。

南方红壤土高活性Al离子对紫花苜蓿生长产生明显的抑制作用。本研究从紫花苜蓿中克隆了MsMYB206和MsMYB741转录因子基因，通过正向遗传和反向遗传）二种方式，以及获得紫花苜蓿发根和全株苗二种转基因材料，对二个转录因子调控的下游基因、互作蛋白和耐铝毒功能进行了研究，发现这两个转录因子能够直接调控苜蓿类黄酮代谢通路中相关基因，其中，MsMYB206与MsMYB450.1形成复合体，共同正向调控下游MsF3'H基因表达，导致转基因紫花苜蓿FLS，CHS，PAL，IFS，4CL，CHI，COMT, F3H及F3'H 9个参与类黄酮代谢的基因明显上调，而DFR基因表达量明显下调，转基因苜蓿根系总黄酮含量明显增加，铝含量明显下降，耐铝毒能力明显提高，但RNAi苜蓿株系的根系总黄酮含量和铝含量未发生明显变化。MsMYB741直接与类黄酮代谢通路的2个关键基因MsCHI和MsPAL1启动子结合，正向调控MsCHI和MsPAL1二个基因表达，其中，与MsCHIpro的结合位点位于-236~-153bp处的ABRE（cis-acting element involved in the abscisic acid responsiveness，ACGTG）顺式作用元件，与MsCHIpro的结合位点为MYB元件（CAACCA）。同时，MsMYB741受上游MsABF2转录因子直接调控。转MsMYB741苜蓿中F3H，FLS，PAL，IFS，CHS，4CL，COMT及CHI基因明显上调，而DFR和F3'H基因表达量呈现下调趋势，过表达发根中总黄酮含量显著高于对照，铝含量明显低于对照，而在干扰表达发根中总黄酮含量显著低于对照，铝含量未发生明显变化，甚至高于对照，转基因苜蓿的耐铝毒性能明显增加。本研究结果对揭示紫花苜蓿对Al胁迫的应答机理和培育耐铝毒苜蓿品种具有一定的理论价值和指导意义。