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新疆棕色棉色素合成相关基因的克隆及表达特性研究
梁明炜
学位类型硕士
导师李晓波
2011-05-25
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业有机化学
关键词Race 序列分析 查尔酮合成酶 类黄酮3’-羟化酶 类黄酮3’ 5’-羟基化酶
摘要根据葡萄的类黄酮3’-羟化酶(F3’H)全长cDNA序列blast所得棉花的EST序列设计引物,以开花后16 d(16 DPA)的新彩棉5号(XC-5)纤维为材料,利用RACE和RT-PCR技术分离得到了2个类黄酮3’-羟化酶基因cDNA序列,此2个序列编码区完全相同,仅在3’UTR存在片段长短的差异,推测可能是基因转录后加工方式不同所造成。克隆所获得的棉花F3’H基因编码区全长1533 bp,编码510个氨基酸,氨基酸序列分析预测表明,该基因所编码蛋白含有一个跨膜结构域,是一种分泌蛋白,定位于内质网上,并含有一段与细胞色素P450功能区相匹配的保守功能域;序列比对结果表明,棉花F3’H基因与其他多个物种的F3’H基因在氨基酸序列上有很高的同源性;聚类分析结果表明,棉花F3’H蛋白与双子叶植物大豆的F3’H亲缘关系较为接近,而与单子叶植物高粱等作物则较远。 应用半定量RT-PCR方法检测花色苷合成途径中查尔酮合成酶基因(GhCHS)、类黄酮3’-羟基化酶基因(GhF3’H)、类黄酮3’5’-羟基化酶基因(GhF3’5’H)在不同色泽棕色棉品种中的时空表达特性,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基因主要在胚珠1 DPA中有一定表达,在纤维和花瓣中表达较低。这些试验结果表明,棕色棉纤维色素形成与花色苷的合成可能存在相关性。
其他摘要Two full-length cDNA sequences encoding flavonoid 3’-hydroxylase gene (F3’H) were cloned from brown-colored cotton (Gossypium hirsutum L.) cultivar “XC-5” fibers of 16 days post anthesis (DPA) by rapid amplification of cDNA ends (RACE) and RT-PCR technique using specific primers based on the EST sequences from the blast results with the full-length cDNA of F3’H in Vitis vinifera and were named as GhF3’H-1 and GhF3’H-2 (GenBank accession: HM598123, HM598124). The two cDNA sequences contain an identical opening reading frame (ORF) of 1533bp in length, encoding a protein of 510 amino acids. There are few differences between the two sequences except for the sequence length of the 3’ untranslational region (UTR), which may be resulted from the difference of post-transcriptional processing. The bioinformatics analyses showed that: the F3’H protein, holding with a membrane spaning domain,is located on endoplasmic reticulum and belongs to secretory proteins; in addition, it contains a conservative functional domain which matches the cytochrome P450 binding domain; the GhF3’H gene shows the highly homogeneity at amino acid level in comparison to the F3’H genes of several other higher plant species; the phylogenetic analysis of F3’H genes from different species reveals that the F3'H has closer relationship with the F3’H from Glycine max than from other plant species. The temporal and spatial expression patterns of chalcone synthase gene (GhCHS), flavonoid 3’,5’-hydroxylase gene (GhF3’5’H) and flavonoid 3’-hydroxylase gene (GhF3’H) in several brown cotton cultivars (lines) were examined by the Semi-quantitative RT-PCR analysis with the specific primers. The expression analysis indicated that the expressed levels of GhCHS and GhF3’5’H gene in white and brown-fiber cotton cultivars during development were relatively high. But the GhF3’H gene was mainly expressed in the ovule of 1 day post anthesis, and were almost not expressed in the fibres and petals. All the results suggested that the production of pigments in brown cotton fibers is related to the biosynthesis pathway of anthocyanins.
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4438
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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梁明炜. 新疆棕色棉色素合成相关基因的克隆及表达特性研究[D]. 北京. 中国科学院研究生院,2011.
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