大豆两个MYB转录因子基因的克隆及表达分析

Cloning and Characterization of Two New MYB Transcription Factor Genes from Soybean

【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10.35U;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。

[Objective] Genes 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 13-galactosidase activity of 10.39 U. Results of the semi-quantitative RT-PCR analysis showed that GmMYBZ2 was expressed in roots, stems, leaves and immature seeds of soybean, whereas the expression of GmMYBZ1 was detected only in the stems and leaves. Semi-quantitative RT-PCR analysis indicated that GmMYBZ2 may also repress the expression of some flavonoid biosynthetic genes, such as PAL, C4H, 4CL, CHS, CHI, F4H and FLS. [ Conclusion] Two novel genes, GmMYBZ1 and GmMYBZ2 encoding MYB transcriptional factors were isolated and characterized from soybean cultivar ZhongDou-27. Functional study of these two genes showed that GmMYBZ2 may be involved in the regulation of flavonoid biosynthesis.