一个新的棉纤维表达蛋白cDNA的克隆、表达及功能初步分析

Cloning and Characterization of A Novel Cotton Fiber Expressed Protein (GhCFE)cDNA

通过陆地棉高品质纤维种质系7235的棉纤维混合cDNA文库随机测序,得到一个开放阅读框(open reading frame,ORF)完整的棉纤维表达蛋白(GhCFE,GenBank登录号:DQ073045)cDNA序列。该cDNA序列全长1274bp,最大的ORF为996bp,编码332个氨基酸,其理论上的等电点pI=6.14,分子量Mw=37.7KD。它的N-末端存在一个长度为27个氨基酸残基的信号肽。RT-PCR分析表明,该基因在根、茎中不表达,在叶片中微弱表达,在不同发育时期的纤维细胞中均表达,且在纤维伸长期表达量最高。进化树显示GhCFE与已报导的3个棉纤维表达蛋白cDNA有很高的同源性。通过构建酵母表达载体,发现该基因对酵母细胞的伸长和细胞壁加厚没有显著影响。利用pBI121质粒构建了含35S启动子的正义表达载体和含棉纤维特异表达启动子E6的正义和反义表达载体,正在通过农杆菌介导的遗传转化,开展该基因的功能验证。

Through randomly sequencing the cotton fiber cDNA library from line 7235 with elite fiber quality in Gossypium hirsuturn L. , a cDNA clone encoding cotton fiber expressed protein （designated as GhCFE,GenBank accession number： DQ073045） was isolated. The full length of this cDNA clone was 1274bp, and its largest open reding frame encoded 332 amino acids. The putative protein of this gene had an isoelectric point of 6.14 and a calculated molecular weight of 37.7KD. It had a signal peptide with 27 amino acid residues in N-terminal. RT-PCR analysis indicated this gene did not express in root or stem but only had a weak expression in leaf, while it could be detected in ovules and fiber cells of different developing periods, especially in elongating fibers. Phylogenetic tree showed GhCFE had high homologies with other CFEs reported before. We cloned GhCFE into the fission yeast （S. pornbe） vector pREPSN and detected that the gene had no significant effect on elongating cells or thickening cell wall in the transformed yeast. Further, Sense expression vector containing 35S promoter and both sense and antisense expression vectors containing E6 promoter using pBI121 plasmid were constructed, and the work of transferring these recombined vectors into Gossypiurn hirsuturn L.by Agrobacterium tumefaciens is ongoing.