大豆浅棕茸毛色基因Td的克隆与功能分析

Cloning and Functional Analysis of Td Gene Responsible for Light Tawny Pubescence in Soybean

大豆的茸毛色由T和Td两个基因控制。在显性T位点下,Td和td分别形成棕色和浅棕色茸毛表型。影响茸毛色形成的黄酮化合物由黄酮合成酶基因FNS Ⅱ-1和FNS Ⅱ-2催化合成。本研究旨在定位并克隆Td基因。遗传连锁分析结果表明Td基因由单个基因控制,定位于3号染色体上。基因组的序列比对及测序分析结果表明,td位点在R2R3-MYB转录因子(Glyma.03G258700)编码区均存在突变,导致翻译提前终止。与野生型Harosoy-T相比,基因Glyma.03G258700,FNS Ⅱ-1和FNS Ⅱ-2在Clark-td茸毛中的表达水平极低。基因FNS Ⅱ-1和FNS Ⅱ-2启动子区均包含与MYB蛋白结合的顺式调控元件。这些结果表明,野生型Glyma.03G258700可能与FNS Ⅱ基因的启动子结合,正调控其表达,提高黄酮含量,形成棕色茸毛。相反,突变型的Glyma.03G258700不能与FNS Ⅱ基因的启动子结合,黄酮含量降低,形成浅棕茸毛。因此,推测Td位点的基因为Glyma.03G258700,编码一个R2R3型MYB转录因子。

Pubescence color of soybean is controlled by two genes,Tand Td.In the presence of a dominant Tallele,dominant and recessive alleles of the Tdlocus generate tawny and light tawny(or near-gray)pubescence,respectively.Flavones,responsible for pubescence color,are synthesized viatwo copies of flavone synthase Ⅱ genes(FNS Ⅱ-1 and FNS Ⅱ-2).This study was conducted to map and clone the Tdgene.Genetic linkage analysis results revealed that Tdgene is controlled by a single gene,located on chromosome 3.Sequence alignment and sequencing analysis of the genome revealed an association between premature stop codons in Glyma.03 G258700(R2R3 MYB transcription factor)and recessive td allele.Compared to wild type Harosoy-T,Clark-tdproduced extremely small amounts of transcripts of Glyma.03 G258700,FNS Ⅱ-1,and FNS Ⅱ-2 in pubescence.The promoter of FNS Ⅱ-1 and FNS Ⅱ-2 shared cis-acting regulatory elements for binding of MYB proteins.These results suggest that the wild-type of Glyma.03G258700 protein may bind to the promoter of FNS Ⅱ genes and upregulates their expression,resulting in increased flavone content and deeper pubescence color.In contrast,mutated Glyma.03G258700 protein may fail to upregulate the expression of FNS Ⅱ genes,resulting in decreased flavone content and dilute pubescence color.These results strongly suggest that soybean Glyma.03 G258700 encoding the R2R3 MYB transcription factor corresponds to the Tdgene.