大豆两个MYB 转录因子基因的克隆及表达分析.DOC

大豆两个MYB 转录因子基因的克隆及表达分析 杨文杰1,2，杜 海3，方 芳4，杨婉身1，吴燕民2，唐益雄2 （1四川农业大学生命科学与理学院，四川雅安 625014；2中国农业科学院生物技术研究所，北京 100081；3四川农业大学玉米研究所， 四川雅安 625014；4哈尔滨工业大学（威海）海洋学院，山东威海 264209） 摘要：【目的】克隆新的植物MYB转录因子基因，进行序列分析，并对其功能进行初步鉴定。【方法】RT-PCR法结合RACE-PCR法分离克隆MYB基因cDNA全长序列；酵母系统检测其转录激活活性；半定量RT-PCR检测目的基因在植物体中的表达情况，及对类黄酮代谢途径中生物合成酶的影响。【结果】根据植物中MYB基因DNA结合域保守区设计简并引物，以大豆品种中豆27的叶片为材料，RT-PCR扩增出两个MYB基因同源片段；据此设计基因特异引物，通过RACE-PCR分离克隆出两个新的MYB基因GmMYBZ1、GmMYBZ2。酵母系统检测表明，GmMYBZ2具有转录激活功能，β－半乳糖苷酶活性为10.35 U；半定量RT-PCR在中豆27的茎和叶中检测到GmMYBZ1的表达，而GmMYBZ2在植物的根、茎、叶及未成熟种子中均有表达；对转基因烟草的RT-PCR检测结果显示，GmMYBZ2的表达可抑制类黄酮代谢途径中PAL、C4H、4CL、CHS、CHI、F4H及FLS等生物合成酶基因的表达。【结论】从大豆栽培品种中豆27中克隆出了两个新的MYB基因GmMYBZ1、GmMYBZ2；功能研究表明，GmMYBZ2可能参与植物类黄酮合成调控。 关键词：大豆；MYB转录因子；基因表达调控 Cloning and Characterization of Two New MYB Transcription Factor Genes from Soybean YANG Wen-jie1,2, DU Hai3, FANG Fang4, YANG Wan-shen1, WU Yan-min2, TANG Yi-xiong2 (1College of Life and Science, Sichuan Agricultural University , Ya’an 625014, Sichuan; 2Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081; 3 Maize Research Institute, Sichuan Agricultural University , Ya’an 625014, Sichuan; 4Marine College, Harbin Institute of Technology at Weihai, Weihai 264209, Shandong) Abstract: 【Objective】enes encoding new MYB transcription factors were to be cloned and their function to be clarified.【Method】 RT-PCR was used to isolate cDNA sequences of MYB genes and to analyze their expression in plants The transcriptional activities of the MYB proteins were detected by yeast expression system.【Result】 A pair of degenerate primers were designed according to the conserved regions which encode the MYB DNA binding domains in plant MYB genes. Two 168 bp fragments were amplified from the leaves of soybean cultivar ZhongDou-27 using RT-PCR. Two new MYB genes, GmMYBZ1 and GmMYBZ2, were isolated by RACE-PCR. The transcriptional activation ability of GmMYBZ2 protein was confirmed by the yeast system with β-galactosidase activity of