绿色棉纤维类黄酮3',5'羟基化酶基因GhF3'5'H的克隆及实时定量表达分析

Cloning and Quantitative Analysis by Real-Time RT-PCR of GhF3'5'H in the Natural Green Cotton

以发育18 d的天然绿色棉及其近等基因系白色棉纤维细胞为材料,利用cDNA-AFLP结合cDNA末端快速扩增技术,克隆了1个绿色棉纤维优势表达基因的全长cDNA。序列分析结果表明,该cDNA全长1873bp,含有一个编码509个氨基酸残基的开放阅读框。Blast分析表明,该基因的编码产物为一个类黄酮3',5'羟基化酶,将该基因命名为GhF3'5'H,序列提交到GenBank,登录号为GU062184。实时荧光定量PCR检测结果显示:GhF3'5'H在绿色棉不同组织中的表达量均显著高于其近等基因系白色棉,而且主要在纤维细胞中表达。在纤维发育过程中,GhF3'5'H在纤维发育的早期表达量最高,并随纤维发育逐渐降低。推测该基因可能在绿色棉纤维色素前体物质的形成中发挥作用。

cDNA-amplified fragment length polymorphism（cDNA-AFLP）was employed to analyze genes that are specifically or preferentially expressed in color cotton fiber cells.Using rapid amplification of cDNA ends（RACE）,the complete cDNA se-quence of GhF3＇5＇H gene was isolated from green cotton fiber at the 18 day post anthesis（DPA）.The full-length of this cDNA clone was 1873 bp,and its largest open reading frame encoded 509 amino acids.The gene was designated as GhF3＇5＇H（Gen-Bank accession： GU062184）.Fluorescence real time quantitative PCR method were used to study the expression of GhF3＇5＇H gene in different development stages.Real-time PCR analysis showed that GhF3＇5＇H specially expressed in naturally green cot-ton fibers prior to white cotton fiber,its expression level was much higher in green cotton than that in white cotton,and it shrinked in proportion to fiber development.It is suggested that GhF3＇5＇H gene may involve in the formation of its precursor and play an important role in green pigment development in natural green cotton fibers.