一个棉花微管结合蛋白基因GhSP1L5的克隆及表达分析

Cloning and Expression Analysis of a Cotton Microtubule Associated Protein Gene of GhSP1L5

利用RT-PCR技术从处于快速伸长发育时期的棉花纤维组织中克隆得到Spiral1-Like(SP1L5)基因(GhSP1L5),其开放读码框为315 bp,编码105个氨基酸的蛋白质,生物信息学分析表明,GhSP1L5蛋白N端和C端较为保守,并含有SCOP结构域,是典型的微管结合蛋白。进化树分析表明该基因与SP1L5家族基因的亲缘关系较近。构建原核表达载体pET28a-GhSP1L5,转化大肠杆菌BL21(DE3)并进行体外诱导表达,通过SDS-PAGE检测目的蛋白的表达,获得分子质量约为12 kD的重组蛋白。QRTPCR结果分析表明GhSP1L5基因在开花后15和20 dpa的纤维组织中表达量较高,在棉纤维发育的其他时期及根、茎、叶组织中表达量较低。

A cotton Spirall-Like5 （ GhSP1L5 ） full-length open reading frame （ ORF ） cDNA was cloned from fast elongating fiber tissue by RT-PCR method, The GhSPIL5 gene contains 315 bp necleotides and codes a protein of 105 amino acids. Through sequence function analysis and homology sequence alignment, GhSPIL5 protein includes several microtubule associated protein binding sites and SCOP domains. Phylogenetic tree analysis showed that GhSP1L5 has a similar relationship with SP1L5 protein family. Prokaryotic expression vector pET28a- GhSP1L5 was constructed and transformated into E.coli BL21 （ DE3 ） . Recombinant GhSP1L5 protein was obtained after induction by IPTG and SDS-PAGE analysis witha MW of -12 kD. Quantitative RT-PCR analysis showed that the GhSPIL5 gene expresses highly at 15 dpa and 20 dpa fiber tissues compared with the expressions in root, stem, leaf and fiber tissuse of other development stages. GhSPIL5 cloning and expression analysis establish a basis for its further functions in cotton fiber development.