Cloning of flavonoid 3'-hydroxylase gene (GhF3'H) and expressional characteristics of several genes associated with pigment synthesis in brown cotton (Gossypium hirsutum L.) fibers | 棕色棉类黄酮3'-羟化酶基因(F3'H)的克隆及色素合成途径中相关基因表达特性研究

Chinese. 为探讨花色苷途径在彩棉色素形成中的作用及彩棉色素形成规律，本研究根据葡萄(Vitis vinifera)的类黄酮3'-羟化酶(flavonoid 3'-hydroxylase, F3'H)基因全长cDNA序列blast所得棉花(Gossypium hirsutum)的EST序列(GenBank登录号: DT545210, CO071403和 BG447485)设计引物，以开花后16 d的新彩棉5号(XC-5)纤维为材料，采用RACE(rapid amplification of cDNA ends)和RT-PCR方法分离得到了2个类黄酮3'-羟化酶基因的全长cDNA序列，长度为1 761和1 892 bp，均含有一个97~1 629 bp、长度为1 533 bp的开放阅读框，编码510个氨基酸，将这两个序列命名为GhF3'H-1和GhF3'H-2，分别提交GenBank，登录号为HM598123和HM598124，此2个序列编码区完全相同，仅在3'非翻译区(UTR)存在片段长短的差异。半定量RT-PCR检测花色苷合成途径中查尔酮合成酶(chalcone synathase, CHS)基因、F3'H、类黄酮3',5'-羟化酶(flavonoid 3', 5'-hydroxylase, F3'5'H)基因在不同色泽的棕色品种中时空表达特性，结果显示，CHS和F3'5'H基因在白棉和彩棉中花、纤维和胚珠中均有着较高水平的表达，而F3'H基因主要在开花后1 d胚珠中有一定表达，在纤维和花瓣中基本不表达。研究结果认为，棕色棉纤维色素形成与花色苷的合成可能存在相关性，进一步揭示了彩棉色素形成的基本机制，为用基因工程手段改良彩棉色彩提供了基础。

English. The purpose of this research is to explore the role of anthocyanidin synthesis pathway in pigmentation of brown cotton fibers. Two full-length cDNAs encoding flavonoid 3' -hydroxylase gene (F3'H) was cloned from XC-5 fibers of 16 days post anthesis (DPA) by rapid amplification of cDNA ends (RACE) and RT-PCR using specific primers based on the Gossypium hirsutum EST sequences(GenBank accession: DT545210, CO071403 and BG447485)from the blast results with the full-length cDNA of F3'H in Vitis vinifera that had already known and were named as GhF3'H-1 and GhF3'H-2 (GenBank accession: HM598123, HM598124). The lengths of the two cDNA sequences were 1 761 and 1 892 bp, respectively. They contained an identical opening reading frame (ORF) of 1 533 bp, encoding a protein of 510 amino acids. There were few differences between the two sequences except for the sequence length of the 3' untranslational region (UTR). The temporal and spatial expression patterns of chalcone synthase (CHS), flavonoid-3’，5'-hydroxylase as (F3'5'H) and flavonoid 3'-hydroxylase (F3'H) genes were examined by the semi-quantitative RT-PCR analysis with the specific primers. The expression analysis indicated that the expressed levels of CHS and F3'5'H gene in white and brown-fiber cotton cultivars during development were relatively high. But the F3'H gene was mainly express in the ovule of 1 day post anthesis, and were almost not expressed in the fibers and petals. All the results suggest that the production of pigments in brown cotton fibers is related to the biosynthesis pathway of anthocyanins, the principle of pigment formation in colored cotton has been disclosed, and this is of help to improve the colors of naturally colored cotton.