天然棕色棉纤维葡萄糖苷转移酶基因gh3gt的克隆与功能研究

石河子大学 硕士学位论文 天然棕色棉纤维葡萄糖苷转移酶基因Gh3GT的克隆与功能研究 姓名：李明月 申请学位级别：硕士 专业：作物遗传育种 指导教师：孙杰 20100401 天然棕色棉葡萄糖苷转移酶基因Gh3GT的克隆与功能分析 I 摘 要 棉花是世界上重要的经济作物 ,棉纤维是人类日常生活的必需品和纺织工业的重要原 料。彩色棉(colored cotton)又称有色棉 ,无需加工和染色可直接织成具有天然色彩纺织物, 是绿色环保产品,因此这种棉花又被称为 “生态棉” 。但是天然彩色棉目前存在的共同问题 是:产量不高 ,纤维长度短， 颜色偏少且色素遗传不稳定， 所以培育天然彩色棉新品种 (系) 难度较大。因此，研究棉纤维色素发育的分子机理就显得极为紧迫。但迄今为止，对于棉纤 维色素发育的分子机理还知之甚少，克隆到的彩色棉纤维中特异表达的色素基因也为数不 多， 还有很多纤维特异或优先表达的色素基因有待克隆和研究， 这些工作不仅有助于深入了 解棉纤维色素发育的分子机理， 同时也可以为基因工程改良彩色棉纤维品质和色泽品质提供 目的基因和理论基础。 本研究通过抑制性差减杂交方法从棕色棉纤维中克隆了一个600bp的序列。 通过3 RACE 和 5RACE 技术克隆到了基因的全长 cDNA 序列，为 1663bp，其开放阅读框全长 1350bp，编 码含 450 个氨基酸残基的蛋白序列。 蛋白序列与糖苷转移酶基因的蛋白序列进行比对， 此序 列 C-端具有糖苷转移酶的所有保守氨基酸功能序列 PSPG 功能域以及 N-端的功能基团。 通过 BLAST 比对，根据同源性和保守结构域将该基因命名为Gh3GT（GeneBank NO.eu921265） 。半 定量 RT-PCR 分析表明：该基因在棕色棉纤维中的表达量明显高于其他组织及其近等基因系 白色棉品种， 且其在纤维中的表达规律与棕色棉的色素形成过程一致。 该基因被定位于叶绿 体中。设计全长引物，得到了Gh3GT的全长 cDNA。构建了Gh3GT的植物表达载体，经蘸花 转化法侵染了野生型拟南芥，转化了烟草 BY-2 悬浮细胞，经烟草叶盘转化法转化了野生型 烟草。通过 Kan 的筛选以及提取拟南芥基因组 DNA，用 PCR 的方法证实得到了转基因拟南芥 的阳性植株。观察发现，转基因拟南芥没有得到预想的花色的变化，但其生长速度比野生型 明显缓慢， 生育期延长至三个月左右， 且耐旱能力明显增强， 植株大小及花色没有明显变化， 茎秆变扭曲；而转基因 BY-2 悬浮细胞出现褐化，细胞紧密，不易摇开。推测Gh3GT可能与 植物的生长发育以及逆境胁迫响应和防御有关，但Gh3GT的具体作用机制目前还不清楚,还 有待进一步研究。 关键词：关键词：棕色棉；糖苷转移酶；RACE；载体构建；遗传转化 天然棕色棉葡萄糖苷转移酶基因Gh3GT的克隆与功能分析 II Abstract Cotton is an important cash crop all over the world, and cotton fiber is necessary in daily life and indispensable as industrial material. Natural colored cotton can be directly made into colored texture without processing and dyeing, which is green, environmental protection product, so this kind of cotton is also called eco-cotton. But natural colored cotton share the same problem: low productivity, short fiber length, color shortage and unstable pigment, make it difficult to create new cultivars (lines). So it is urgent to study the molecular mechanism of cotton fiber development .But till now, it is still very little known, and genes specially expressed in colored cotton fiber cloned are not many enough, there are still lots of genes specially or preferentially expressed in cotton fiber need to be cloned and studied, and this work will help understand the molecular mechanism of cotton fiber development, and lay a solid foundation for colored cotton fiber quality and color quality improvement. A 600bp sequence was cloned from fiber of Gossypium hirsutum through SSH (suppression subtractive hybridization). The whole cDNA sequence was cloned through 3RACE and 5RACE, and the length is 1663bp, with its ORF of 1350bp, encodes a protein contains 450 residues. The protein sequence was compared with the members of glucosyltransferase family, and the result suggested that the sequence contains N-terminal functional groups and the conserved functional amino acid sequence (PSPG motif) in C-terminal, which is shared by flavonoid 3-O-glucosyltrans -ferases. The results of nucleic acid and protein sequences analysis suggested that the gene cloned is a member of glucosyltransferase family, and it was named as Gh3GT. Semi-quantitative RT-PCR analysis revealed that the expression of Gh3GT is much higher in brown cotton fiber than in other tissues and in white cotton fiber, and the gene expression coincides with pigment formation in brown cotton fiber. The gene was located in chloroplast. The full-length cDNA of Gh3GT was obtained by using full-length primers. Sense expression vector was constructed and transformed into arabidopsis, BY-2 cells, and through Kan selection, genome DNA extraction and PCR idebtification, the positive transgenic arabidopsis were obtained. The results were found that transgenic arabidopsis grew slowly, and the growth period was extended to about 3 months, the plant type, plant size and flower color had no obvious change. And transgenic BY-2 cells grew slower 天然棕色棉葡萄糖苷转移酶基因Gh3GT的克隆与功能分析 III compared to the wild type. It presumed that Gh3GT may have close connection with plant development and response to reversity and defence, but the molecular mechanism is still not clear, and need to be further studied. Key words： natural brown cotton； glucosyltransferase； RACE； vector construction； genetic transformation 天然棕色棉葡萄糖苷转移酶基因Gh3GT的克隆与功能分析 VI 缩写词中英文对照表 List of abbreviation 缩写缩写 Abbreviation 英文名称英文名称 English Name 中文名称中文名称 Chinese Name Amp Ampicillin 氨苄青霉素 AS Acetosyringone 乙酰丁香酮 6-BA 6-benzylaminopurine 6-苄基腺嘌呤 bp Base pair 碱基对 cDNA Complementary DNA 互补 DNA CTAB Cetyltriethylammonium Bromide 十六烷基三甲基溴化胺 ddH2O Distilled and deionized water 重蒸水 DEPC Diethyl Pyrocarbonate 焦碳酸二乙酯 Ecoli Escherichia coli 大肠杆菌 EDTA Ethylendiaminotetra acetic acid 乙二胺四乙酸 IPTG Isopropylthio-D-galactoside 异丙基-D-硫代半乳糖 Kan Kanamycin 卡那霉素 MS Murashinge在生产应用中，彩色棉常出现产量低、纤维短、强度差、 衣分低、色素物质不稳定等问题，且通过常规育种方法解决问题有很大难度，因 此，通过生物工程技术来解决问题就显得非常必要和紧迫。目前，国内外对天然 彩色棉研究主要集中在产量和纤维品质的提高及改进,缺乏对彩色棉品质形成机 理的认识，已成为限制彩色棉产业进一步发展的重要因素。已知彩色棉纤维颜色 形成主要是纤维细胞中腔色素物质的沉积所致,受到遗传因子得控制。虽然目前 已经明确彩色棉是由于纤维中腔里沉积了某种色素物质所致, 但对色素物质及 其形成分子机制知之甚少， 因此利用基因工程方法改良彩色棉纤维品质和色泽品 质的