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类固醇5α-还原酶(DET2),被认为是油菜素类固醇物质(BRs)生物合成的限速酶。为了明确BRs在棉花纤维发育中的作用,本研究利用RT-PCR方法从纤维组织中获得基因开放阅读框(ORF)全长,经测序验证、Blast比对与同源性分析发现该序列编码的蛋白质与其他植物的DET2蛋白具有较高的相似性,故命名为Gb DET2基因。生物信息学分析结果表明,Gb DET2基因ORF长777 bp,可编码1个包含258个氨基酸的蛋白。氨基酸多序列比对发现Gb DET2序列保守性较高,具有的DET2的典型特征。通过物种间系统发育树可以看出Gb DET2与Gh DET2进化关系最接近,二者属于同一进化分支。实时荧光定量PCR分析表明,Gb DET2基因在纤维发育的不同时期中均有表达,在开花后5 d的胚珠、10 d的纤维中其表达量最高。研究结果显示Gb DET2基因在棉花纤维的起始和快速伸长过程中发挥着重要的作用。
Steroid 5 alpha -reductase (DET2) is considered the rate-limiting enzyme for brassinosteroid (BRs) biosynthesis. In order to clarify the role of BRs in the development of cotton fiber, we used RT-PCR to obtain the full-length ORF (full-length open reading frame) of the gene from the fibrous tissue. After sequencing validation, Blast analysis and homology analysis, Proteins and other plants DET2 protein has a high similarity, so named Gb DET2 gene. Bioinformatics analysis showed that the ORF of Gb DET2 gene was 777 bp long and encoded a protein of 258 amino acids. Multiple amino acid sequence alignment revealed that the Gb DET2 sequence is highly conserved with the typical features of DET2. The phylogenetic tree between species shows that Gb DET2 has the closest evolutionary relationship with Gh DET2 and both belong to the same evolutionary branch. Quantitative real-time PCR analysis showed that Gb DET2 gene was expressed in different stages of fiber development, and its expression level was the highest at ovule 5 days and 5 days after flowering. The results show that the Gb DET2 gene plays an important role in the initiation and rapid elongation of cotton fiber.