兴安落叶松纤维素合酶基因的克隆及特性分析

发布时间：2018-05-22 12:46

本文选题：兴安落叶松 + CesA　；参考：《内蒙古大学》2017年硕士论文

【摘要】：兴安落叶松(Larix gmelinii)是中国东北地区荒山造林和森林更新的主要树种,因其材质优良,所以也为建筑、木材加工,木纤维工业等提供原料,具有巨大的经济价值。纤维素合酶(Celllμlose Synthase)是纤维素生物合成途径中的一个关键酶,它的表达丰度可以直接影响到纤维素的合成,进而对木材材质实现有效调控。因此研究兴安落叶松CesA基因的表达调控机理和功能特性意义非凡。本研究利用兼并PCR和RACE-PCR技术分离了兴安落叶松纤维素合酶基因(命名为LgCesA3),并对该基因的功能和表达特性进行了分析。LgCesA3基因的cDNA全长为4220bp,其中包含3297bp的开放阅读框(ORF)、531bp的5,-UTR和392bp的3,-UTR,编码1099个氨基酸。LgCesA3基因的DNA全长为7255bp,包含14个外显子和13个内含子。亚细胞定位结果显示LgCesA3::GFP融合蛋白信号在细胞膜周围被检测到,因此推测LgCesA3定位于细胞膜上。为了调查LgCesA3的表达调控机理,利用基因组步移的技术克隆了 1180bp的LgCesA3启动子序列。序列分析显示,该基因的启动子区域包含水杨酸、茉莉酸甲酯等植物激素应答原件,光诱导元件以及干旱、机械压力等非生物胁迫相关的应答元件。实时荧光定量PCR检测结果显示,LgCesA3在兴安落叶松的根、茎、叶中均稳定表达,茎中的表达量要远远高于根和叶。同时LgCesA3基因的表达受外源激素茉莉酸甲酯和水杨酸的诱导,但在15%PEG和GA3处理条件下,该基因表现出先升高后下降的表达趋势。为了深入研究LgCesA3的功能和异源表达特性,构建了 pBI101-LgCesA3双元表达载体,并通过农瘤杆菌介导的花序浸染法获得了转基因拟南芥。RT-PCR和酶活性检测结果证实了 LgCesA3在转基因拟南芥中的稳定表达。超表达LgCesA3基因拟南芥的表型分析结果显示LgCes43基因的过量表.达促进了转基因拟南芥植株中纤维素的积累。以上研究结果表明,可通过对纤维素合成路径中的关键酶CesA进行遗传修饰调控纤维素的生物合成,进而有目的地改良材质。该研究成果亦为深入研究兴安落叶松ZgCesA3基因的表达调控机制以及利用该基因进行木本植物的材质改良奠定了基础。
[Abstract]:Larix gmelinii (Larix gmelinii) is the main tree species for afforestation and forest regeneration in northeast China. Because of its excellent material quality, Larix gmelinii also provides raw materials for construction, wood processing and wood fiber industry, which is of great economic value. Cellulosic synthase (Celll 渭 lose Synthase) is a key enzyme in cellulose biosynthesis pathway. Therefore, it is of great significance to study the expression and regulation mechanism and functional characteristics of CesA gene in Larix gmelinii. In this study, the cellulose synthase gene of LgCesA3 (LgCesA3) gene was isolated by devolving PCR and RACE-PCR techniques. The function and expression characteristics of LgCesA3 gene were analyzed. The cDNA length of LgCesA3 gene was 4220 BP, including the open reading frame of 3297bp. The DNA encoding 1099 amino acids, LgCesA3, was 7255bp. it contained 14 exons and 13 introns. The subcellular localization results showed that the LgCesA3::GFP fusion protein signal was detected around the cell membrane, so we speculated that LgCesA3 was located on the cell membrane. In order to investigate the expression regulation mechanism of LgCesA3, the LgCesA3 promoter sequence of 1180bp was cloned by genomic step technique. Sequence analysis showed that the promoter region of the gene contained plant hormone responses such as salicylic acid methyl jasmonate photoinduced elements and abiotic stress response elements such as drought and mechanical pressure. The results of real-time fluorescence quantitative PCR showed that LgCesA3 was stably expressed in roots, stems and leaves of Larix gmelinii, and the expression of LgCesA3 in stems was much higher than that in roots and leaves. At the same time, the expression of LgCesA3 gene was induced by exogenous hormones methyl jasmonate and salicylic acid, but under the condition of 15%PEG and GA3 treatment, the expression of LgCesA3 gene increased first and then decreased. In order to study the function and heterologous expression characteristics of LgCesA3, a double expression vector of pBI101-LgCesA3 was constructed. The stable expression of LgCesA3 in transgenic Arabidopsis thaliana was confirmed by Agrobacterium tumefaciens mediated inflorescence soaking method. Phenotypic analysis of overexpression of LgCesA3 gene in Arabidopsis thaliana showed that LgCes43 gene was overrated. Da promoted the accumulation of cellulose in transgenic Arabidopsis thaliana plants. The results show that the cellulosic biosynthesis can be controlled by genetic modification of the key enzyme CesA in the cellulose synthesis pathway, and then the material can be modified purposefully. The results also laid a foundation for the further study of the regulation mechanism of ZgCesA3 gene expression and the material improvement of woody plants by using this gene.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S791.222;Q943.2

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