陆地棉纤维发育相关基因GhEXPs的分析及表达研究

Analysis and expression of GhEXPs related to fiber development in Gossypium hirsutum L.

【目的】扩展蛋白是1种细胞壁蛋白,能够松弛细胞壁从而促进细胞伸长。本研究旨在挖掘棉花纤维发育过程中特异的扩展蛋白基因。【方法】利用转录组测序和定量PCR(Polymerase chain reaction)研究棉花扩展蛋白基因的表达模式,并运用生物信息学的方法分析扩展蛋白进化关系、预测三维结构和关键氨基酸位点。【结果】以陆地棉cDNA(Complementary DNA,互补DNA)为模板,克隆得到4个棉花扩展蛋白基因GhEXPA1d、GhEXPA4b、GhEXPA23b和GhEXPB3a,开放阅读框长度分别为768 bp、795 bp、798 bp和804 bp。进化树分析表明,GhEXPA1d、GhEXPA4b、GhEXPA23b属于EXPA(α-expansin)亚家族,GhEXPB3a属于EXPB(β-expansin)亚家族。组织特异性表达分析表明它们是纤维发育特异基因;定量PCR验证了这4个GhEXPs都是在纤维发育的起始期和伸长期表达量较高。根据序列比对以及三维结构模型预测了4个棉花扩展蛋白的关键氨基酸位点,分别是苯丙氨酸、色氨酸和酪氨酸,均为芳香族氨基酸,在三维结构模型中处于同一个平面上。【结论】获得的4个棉花扩展蛋白基因在棉花纤维发育起始期和伸长期特异表达,为进一步阐明GhEXPs对棉花纤维发育的分子机制提供了参考。

[Objective]The expansins(EXPs)are a kind of cell wall proteins,which can loosen the cell wall and promote cell elongation.The purpose of this study is to explore the specific expansin genes in cotton fiber development.[Methods]The expression pattern of four GhEXPs was analyzed in different tissues using the RNA-seq and qRT-PCR(Quantitative real-time polymerase chain reaction)technology,respectively.The phylogenetic tree,three-dimensional structure prediction of GhEXPs,and locations of key amino acid were analyzed by bioinformatics.[Results]The four GhEXPs,GhEXPA1d,GhEXPA4b,GhEXPA23b,and GhEXPB3a,were cloned from the upland cotton cDNA(complementary DNA),the intact ORF(Open read frame)of them are 768 bp,795 bp,798 bp and 804 bp,respectively.The evolutionary analysis showed that GhEXPA1d,GhEXPA4b and GhEXPA23b belonged to the EXPA(α-expansin)subfamily,and GhEXPB3a belongs to EXPB(β-expansin)subfamily.Tissue-specific expression analysis indicated that the four genes were specific-expressed genes in fiber development,and predominantly expressed in the early stage of fiber development,and the reliability of the results was verified by qRT-PCR.According to sequence alignment and three-dimensional structure prediction,we have predicted the key amino acid residues of four GhEXPs,including the phenylalanine,tryptophan and tyrosine.The results showed that they were all aromatic amino acids and located the same surface.[Conclusion]The four GhEXPs specifically expressed at the initiation and elongation stages of cotton fiber development which provided a reference for further elucidating the molecular mechanism of GhEXPs during cotton fiber development.