棉花GhSP1L基因克隆与表达分析

Cloning and Expression Analysis of a Cotton Gh SP1L cDNA

【目的】棉花Spiral1-LIKE(SP1L)基因(Gh SP1L)的克隆、功能序列分析及原核表达。【方法】通过RT-PCR从棉纤维中克隆Gh SP1L基因,进行Gh SP1L蛋白的功能结构域分析,构建原核表达载体p ET28a-GhSP1L,进行蛋白体外诱导表达,SDS-PAGE分析蛋白的表达情况;RT-PCR检测Gh SP1L基因的表达特征。【结果】从陆地棉纤维组织中克隆得到Gh SP1L基因的全长c DNA,其开放读码框为318 bp,编码106个氨基酸的蛋白质,理论分子质量为11.76 k Da。对氨基酸序列进行生物信息学分析,其N端和C端较为保守,含有SCOP结构域,属于SP1L家族蛋白,能够在其他微管蛋白协助下与微管结合,SPR1的定位可能与其结合的微管结合蛋白相关。构建了p ET28a-Gh SP1L原核表达载体,并通过体外诱导表达后获得分子质量约为12 k Da左右的重组蛋白Gh SP1L;半定量RT-PCR结果表明,Gh SP1L基因开花后3 d的纤维组织中表达量较高。【结论】Gh SP1L基因的克隆、序列分析和表达,重组蛋白的诱导表达,为深入研究该基因在棉纤维发育中的作用奠定了基础。

【Objective】Cloning a cotton Gh SP1 L gene from fiber tissue,analyzing sequence function structure domain and Gh SP1 L expression characteristics during the fiber development. 【Method 】Gh SP1 L gene was cloned from fiber tissue through RT- PCR method. A series of bioinformatics software were used to analyze sequence characteristics. The recombinant protein Gh SP1 L was obtained by transforming p ET28a- Gh SP1 L vector into E. Coli BL21（ DE3） with in vitro induction,and SDS- PAGE analysis. Gh SP1 L gene expression analysis was performed by semi- quantitative RT- PCR method. 【Result】A cotton Gh SP1 L full- length c DNA was cloned from fiber tissue. Gh SP1 L c DNA contains a 318 bp open reading frame coding a ~ 12 k Da protein of 108 amino acids. Gh SP1 L protein contains several SCOP functional domains. Phylogenetic tree analysis showed that Gh SP1 L belonged to the SP1L1 protein family with the function of binding to microtubule through assistance of other microtubule protein,which inferred that the location of SPR1 was related with its binding microtubule protein. 12 k Da recombinant protein Gh SP1 L were obtained by SDS- PAGE analysis; Semi-quantitative RT- PCR analysis showed that the Gh SP1 L gene expressed highly at 3- dpa fiber tissue,and was closely related to the process of cotton fiber initiation development.【Conclusion】Gh SP1 L cloning,functional sequence analysis and expression analysis established a basis for its further functions in cotton fiber development.