【作者】 田志坚；

【导师】 张学文；

【作者基本信息】 湖南农业大学 ， 细胞生物学， 2006， 硕士

【摘要】 苎麻(Boehmeria nivea)是一种我国传统的重要纤维作物，其麻皮纤维是主要收获物。当今一些模式植物纤维素合成分子生物学研究取得了良好的进展，为苎麻纤维素合成的分子生物学研究提供了丰富的参考。 本研究以苎麻为材料，提取其总RNA并反转录成cDNA，通过设计一对简并引物扩增出纤维素合成酶基因中间片段，然后通过RACE技术分别获得基因3′端包含多聚A尾的序列和5′端大部分序列(尚缺失包括起始密码子在内的约450bp序列)。将三段序列分别测序并拼接成一长3276bp的cDNA序列，其读码框可翻译成一段包含938个氨基酸的蛋白质。经BLAST及蛋白质结构分析，证实我们得到的cDNA序列即是苎麻纤维素合成酶基因序列，按照纤维素合成酶基因命名规则将其命名为BnCesA1，并将此序列提交GenBank，登陆号为：DQ077190。 为了进一步了解BnCesA1基因在苎麻组织中的表达情况，揭示苎麻纤维素合成及其基因表达调控机制，以BnCesA1基因3′端非保守区域为检测目的基因，苎麻18S rRNA为内参基因，应用RT-PCR技术对苎麻纤维素合成酶基因的表达进行半定量，建立一个针对BnCesA1的特异、稳定的RT-PCR半定量检测体系。结果显示在苎麻根、茎、叶、芽中都可检测到BnCesA1基因的表达，其表达模式为：茎＞叶＞芽＞根，相对表达量依次为：0.791，0.381，0.319，0.183；推导BnCesA1蛋白与模式植物拟南芥(Arabidopsis thaliana)AtCesA1蛋白具有很高的一致性和同源性(87／93％)；功能上可能参与苎麻细胞初生和次生壁的形成。 通过PCR将BnCesA1基因中编码纤维素合成酶催化和底物结合结构域的cDNA序列从T载体上释放，反向插入到植物表达载体pWM101中，将其置于35S启动子下游，并通过PCR和酶切实验验证，获得了在植物中组成型表达反义BnCesA1核心区段的表达载体。以农杆菌介导将其转化模式植物—更多还原

【Abstract】 Ramie is one of the most important economic crops in China, its cortex fibers are the main product that to be harvested. Recent researches on molecular mechanism of cellulose biosynthesis in some model plants have made great progress and providing perfect alternative information for understanding the molecular mechanism of cellulose biosynthesis in ramie.In this research, ramie was used as material, the ramie cellulose synthase gene cDNA most sequence was cloned by combination the techniques of degenerate PCR and RACE. There are only 450bp of the 5’very end of the expected cDNA still under pursuing. The cloned cDNA for cellulose synthase is designated as BnCesA1. Sequencing analysis showed the cDNA sequence be cloned is 3276 bp with a partial ORF from neuclotide 1 to 2814. Bioinformatic analysis ascertained this cDNA sequence is the ramie cellulose synthase gene and was submitted to GenBank. Its accession number is DQ077190.In order to investigate the expression model and regulation mechanism of BnCesA1 in different tissues of ramie, the nonconservative sequence at 3’terminal of BnCesA1 was amplified by semi-quantitative RT-PCR respectively at the same time. Taking 18S rRNA gene as an inner control, the integrate optic density (IOD) of BnCesA1 and 18S rRNA gene bands were detected with gel analysis software and defined the ratio of IOD as relative expressive quantity. The results indicated that BnCesA1 expressing both in leaf, stem, root and bud. The expression level from high to low is stem, leaf, bud and root, the relative content is: 0.791, 0.381, 0.319, 0.183 respectively. The expression data presented shows that BnCesA1 is highly expressed in developing stem undergoing significant secondary cell-wall biosynthesis as well as更多还原