油菜BnGLIPL和BnGLIP7对种子含油量的影响

发布时间：2018-06-02 05:28

本文选题：突变体 + 过表达　；参考：《华中农业大学》2017年硕士论文

【摘要】：菜籽油是我国的传统食用油,因其较高的营养价值而得到国民的青睐。但是,目前我国每年进口的菜籽油占需求的30%以上。提高油菜的含油量,推广高含油量品种是提高我国菜籽油自给率的有效手段。一般地提高含油量的办法是通过改变脂肪酸合成路径中某些基因的表达来促进脂肪酸合成,从而达到增加油脂积累的目的,如何降低TAG在作物中合成后的分解对于油脂的积累也显得十分重要的。GDSL型酶是一类广泛存在于动植物中的水解酶,可以水解硫基酯、芳香酯、磷脂和氨基酸等多种类型的底物。本研究主要在拟南芥中鉴定了AT1G71250(GDSLlipase like,GLIPL)和AT5G15720(GDSL-motif lipase 7,GLIP7)及油菜同源基因(BnGLIPL,BnGLIP7)对含油量的影响,结果如下:1)在油菜中克隆到GLIPL以及GLIP7的同源基因的CDS全长和完整启动子。2)在几种芸薹属植物中比对同源基因的DNA和氨基酸序列,发现GLIPL和GLIP7在芸薹属不同植物中的保守性非常高,序列相似度达90%,且都存在4个保守的结构域,也预示两个基因具有相似的功能。3)对拟南芥根、茎、叶、花蕾、角果做qRT-PCR,发现GLIPL在拟南芥各器官中都有不同程度的表达,在开花后4天的角果中表达量显著提高;对GUS阳性苗染色发现:BnGLIPL在萌发的种子、花器官以及发育的种子中表达量比较高;BnGLIP1在幼苗、花序以及幼嫩角果中有明显的表达。4)在拟南芥中得到GLIPL的纯合突变体;构建GLIPL以及GLIP7油菜同源基因的含双35s启动子的CDS过表达载体并得到阳性单株。5)通过检测过表达株系、纯合T-DNA插入突变体以及野生型的种子含油量发现:BnGLIPL比对照含油量下降9.54%,达到极显著水平;突变体与对照相比含油量上升7.63%,达到极显著水平;BnGLIP7的过表达株系含油量比对照低4.66%,差异显著。
[Abstract]:Rapeseed oil is the traditional edible oil in China. However, at present, the rapeseed oil imported each year accounts for more than 30% of the demand. It is an effective means to improve the self-sufficiency rate of rapeseed oil in China by increasing the oil content of rapeseed and popularizing the varieties with high oil content. The general way to increase oil content is to promote fatty acid synthesis by changing the expression of certain genes in the fatty acid synthesis pathway, thereby increasing oil accumulation. How to reduce the decomposition of TAG in crops after synthesis is also very important for the accumulation of oil. GDSL-type enzyme is a kind of hydrolase that widely exists in animals and plants. It can hydrolyze many kinds of substrates, such as thioester, aromatic ester, phospholipids and amino acids. In this study, the effects of AT1G71250(GDSLlipase like GLIPL) and AT5G15720(GDSL-motif lipase 7 (GLIP7) and Brassica napus homologous gene BnGLIP7) on oil content were identified in Arabidopsis thaliana. The results were as follows: (1) the CDS length and complete promoter of GLIPL and GLIP7 homologous genes were cloned in Brassica napus and compared the DNA and amino acid sequences of homologous genes in several Brassica plants. It was found that GLIPL and GLIP7 were highly conserved in different plants of Brassica, with a sequence similarity of 90 and four conserved domains, indicating that the two genes had similar functions in Arabidopsis roots, stems, leaves and buds. It was found that GLIPL was expressed in all organs of Arabidopsis thaliana to varying degrees, and increased significantly in pods 4 days after anthesis, and GUS positive seedling staining showed that: BnGLIPL was expressed in germinated seeds. A homozygous mutant of GLIPL was obtained in Arabidopsis thaliana with higher expression of BnGLIP1 in floral organs and developing seeds than in seedlings, inflorescences and young pods. To construct CDS overexpression vector containing double 35s promoter of GLIPL and GLIP7 homologous gene and obtain positive single plant. The oil content of seed inserted by homozygous T-DNA and wild-type seeds was 9.54% lower than that of the control, and reached a very significant level. Compared with the control, the oil content of the mutant increased 7.63%, and reached the extremely significant level. The oil content of the overexpression strain of BnGLIP7 was 4.66% lower than that of the control, and the difference was significant.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4

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