【摘要】 【目的】克隆了1个在棉纤维发育次生壁加厚期优势表达的R2R3类MYB (v-myb avian myeloblastosis viral oncogene homolog, MYB)转录因子基因Gh MYB52(Gh_A12G2460)。本研究旨在分析该MYB转录因子在陆地棉纤维次生壁发育过程中的作用。【方法】通过一步克隆法,从陆地棉遗传标准系TM-1中克隆了1个在纤维次生壁加厚期优势表达的R2R3类MYB转录因子基因Gh_A12G2460,进化树分析结果表明Gh_A12G2460与拟南芥中AtMYB52基因的相似度最高,因此将其命名为Gh MYB52。通过进化树构建、氨基酸序列多重比对、定量聚合酶链式反应(Polymerase chain reaction,PCR)、烟草瞬时转化、酵母双杂交等对其基因结构、编码产物的结构、表达特征、亚细胞定位以及互作蛋白进行了分析。【结果】GhMYB52基因的开放阅读框长672 bp,编码223个氨基酸残基,预测蛋白的相对分子质量为26.06 kDa,等电点为9.83。qRT-PCR结果表明,Gh MYB52基因在开花后15～25 d的棉纤维中优势表达。亚细胞定位结果显示,GhMYB52蛋白定位于细胞核,符合转录因子的特征。酵母转化结果显示,GhMYB52蛋白具有强烈的转录激活活性,并且与1个NAC类转录因子GhFSN1有强烈互作。【结论】GhMYB52是1个在棉花纤维次生壁加厚时期优势表达的R2R3类MYB转录因子,它可能通过与NAC类转录因子互作,形成蛋白复合物参与棉花纤维次生壁加厚过程。本研究为进一步从分子水平上验证GhMYB52基因在棉花纤维发育过程中的生物学功能奠定了基础。更多还原

【Abstract】 [Objective] In this study, we cloned the R2 R3 MYB(v-myb avian myeloblastosis viral oncogene homolog, MYB)transcription factor gene Gh_A12 G2460, which is highly expressed in the second cell wall(SCW) development stage during cotton fiber formation, and preliminarily investigated its functional role in fiber development at SCW stage. [ Method] The Gh_A12 G2460 gene was isolated from Gossypium hirsutum acc. TM-1 by one-step cloning strategy. Sequence alignment showed it is similar to At MYB52 in Arabidopsis, so it was renamed as GhMYB52. The gene structure, protein structure,expression profile, subcellular location and protein-protein interaction were analyzed by phylogenetic analysis, multiple alignments of amino acid sequences, qRT-PCR, Agrobacterium tumefaciens-mediated transient transformation system of tobacco, and yeast two hybridization assay. [Result] The results showed that Gh MYB52 contains a 672 bp open reading frame,encoding 223 amino acid residues, with a predicted nuclear localization signal region. qRT-PCR results indicated that GhMYB52 is predominantly expressed in the fiber at 15―25 DPA(days post anthesis), suggesting it may be an SCW-associated gene. GhMYB52 localized in the nucleus examined by tobacco transient expression, consistent with the characteristics of transcriptional factors. Yeast transformation test demonstrated it has transcriptional activation activity. Further protein-proteininteraction analysis revealed that Gh MYB52 strongly interacts with Gh FSN1, a NAC transcription factor. [Conclusion] These results demonstrated that Gh MYB52 is a nuclear-localized transcription activator, which might participate in the SCW process of cotton fiber development by forming a corresponding regulatory network with NAC transcription factors. This study lays a foundation for further verification of the biological function of Gh MYB52 in cotton fiber development at the molecular level.更多还原