棕色棉DFR基因的克隆与生物信息学分析

生物技术通报 ›› 2014, Vol. 0 ›› Issue (5): 88-95.

棕色棉DFR基因的克隆与生物信息学分析

肖向文^1，2 朱奇朗¹ 刘海峰² 王俊铎³ 罗城² 曾闻¹ 梁亚军³ 龚兆龙³ 李晓波¹

(1. 中国科学院新疆理化技术研究所干旱区植物资源化学重点实验室，乌鲁木齐 830011；2. 中国彩棉(集团)股份有限公司，乌鲁木齐 830016；3. 新疆农业科学院经济作物研究所，乌鲁木齐 830091)

收稿日期:2013-11-27 出版日期:2014-05-23 发布日期:2014-05-24
作者简介:肖向文，博士，研究方向：植物基因工程；E-mail：xiaoxw@ms.xjb.ac.cn
基金资助:
中国博士后科学基金项目(2012M521827)，兵团博士资金项目(2013BB005)，高品质彩色棉花种质资源创制(2013AB003)，转基因特色专用棉新品种培育(2011ZX08005-005)

Cloning and Bioinformatics Analysis of DFR Gene from Brown Cotton(Gossypium hirsutum L.)Fiber

Xiao Xiangwen^1，2 Zhu Qilang¹ Liu Haifeng² Wang Junduo³ Luo Cheng² Zeng Wen¹ Liang Yajun³ Gong Zhaolong³ Li Xiaobo¹

(1. Key Laboratory of Chemistry of Plant Resources in Arid Regions，Xinjiang Technical Institute of Physics and Chemistry，Chinese Academy of Sciences，Urumqi 830011；2. China Colored-Cotton(group)Co.，Ltd.，Urumqi 830016；3. Economic Crop Research Institute，Xinjiang Academy of Agricultural Sciences，Urumqi 830091)

Received:2013-11-27 Published:2014-05-23 Online:2014-05-24

摘要/Abstract

摘要： 花色素苷是影响花色的主要色素，二氢黄酮醇4-还原酶(DFR)基因是花色素苷生物合成途径的关键酶基因。通过同源克隆策略，以新彩棉6号(XC-6)纤维的RNA以及DNA为模板克隆得到GhDFR基因的CDS全长编码序列及带有内含子的基因组序列，并进行了生物信息学分析。序列分析结果显示，该基因含有6个外显子，5个内含子结构，其cDNA包含一个1 020 bp的开放阅读框，编码355个氨基酸，其氨基酸序列包含具有高度保守性的NADP(H)的结合位点以及底物特异性结合位点。推定的GhDFR蛋白质分子量为39.65 kD，等电点为5.67。该蛋白氨基酸序列同毛果杨、葡萄、天竺葵等物种DFR蛋白显示出较高同源性，而系统进化分析结果表明其与天竺葵、芍药DFR亲缘关系较近。氨基酸序列分析预测表明，GhDFR基因所编码的蛋白不具备信号肽区段，无明显跨膜区域，不属于分泌蛋白，可能为亲水性蛋白，定位于细胞质的可能性最高，其主要二级结构元件为α-螺旋和无规则卷曲。GhDFR属于NADB-Rossmann superfamily。

关键词: 二氢黄酮-4-还原酶(DFR), 天然彩色棉, 基因克隆, 花色素苷

Abstract: The main pigment that affects flower colors is the anthocyanin, and dihydroflavonol 4-reductase(DFR)is a key enzyme involved in anthocyanin biosynthesis pathway. A full-length gene from brown cotton(Cultivar Xincaimian 6)fiber at the 16 day post anthesis(16 DPA)encoding dihydroflavonol 4-reductase(GhDFR)was isolated, through homology cloning techniques. A full length coding sequence(CDS)and genomic sequence including introns were obtained. Bioinformatics approach was used to analyze and predict the function and structure domain of GhDFR. According to sequence analysis, the genomic sequence of GhDFR contained six exons and five introns. The full-length CDS of the GhDFR ortholog consisted of a 1 500 bp open reading frame(ORF)encoding a polypeptide composed of 355 amino acid residues. The molecular weight of deduced GhDFR polypeptide was 39.65 kD, with an isoelectric point(pI)of 5.67. The deduced amino acid sequence of the GhDFR ortholog had a highly conserved NADP(H)-binding site and substrate specificity site. The deduced DFR amino acid sequence showed high homology with DFRs from other plants, such as Populus trichocarpa, Vitis vinifera and Pelargonium zonale. Phylogenetic analysis showed the closest relationship with DFRs of Pelargonium zonale and Paeonia lactiflora.

Key words: Dihydroflavonol 4-reductase(DFR) Naturally, Colored Cotton, Gene Clone, Anthocyanin

肖向文, 朱奇朗, 刘海峰, 王俊铎, 罗城, 曾闻, 梁亚军, 龚兆龙, 李晓波. 棕色棉DFR基因的克隆与生物信息学分析[J]. 生物技术通报, 2014, 0(5): 88-95.

Xiao Xiangwen, Zhu Qilang, Liu Haifeng, Wang Junduo, Luo Cheng, Zeng Wen, Liang Yajun, Gong Zhaolong, Li Xiaobo. Cloning and Bioinformatics Analysis of DFR Gene from Brown Cotton(Gossypium hirsutum L.)Fiber[J]. Biotechnology Bulletin, 2014, 0(5): 88-95.

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