本文選題：突變體 + 過表達　； 參考：《華中農業(yè)大學》2017年碩士論文

【摘要】：菜籽油是我國的傳統(tǒng)食用油,因其較高的營養(yǎng)價值而得到國民的青睞。但是,目前我國每年進口的菜籽油占需求的30%以上。提高油菜的含油量,推廣高含油量品種是提高我國菜籽油自給率的有效手段。一般地提高含油量的辦法是通過改變脂肪酸合成路徑中某些基因的表達來促進脂肪酸合成,從而達到增加油脂積累的目的,如何降低TAG在作物中合成后的分解對于油脂的積累也顯得十分重要的。GDSL型酶是一類廣泛存在于動植物中的水解酶,可以水解硫基酯、芳香酯、磷脂和氨基酸等多種類型的底物。本研究主要在擬南芥中鑒定了AT1G71250(GDSLlipase like,GLIPL)和AT5G15720(GDSL-motif lipase 7,GLIP7)及油菜同源基因(BnGLIPL,BnGLIP7)對含油量的影響,結果如下:1)在油菜中克隆到GLIPL以及GLIP7的同源基因的CDS全長和完整啟動子。2)在幾種蕓薹屬植物中比對同源基因的DNA和氨基酸序列,發(fā)現(xiàn)GLIPL和GLIP7在蕓薹屬不同植物中的保守性非常高,序列相似度達90%,且都存在4個保守的結構域,也預示兩個基因具有相似的功能。3)對擬南芥根、莖、葉、花蕾、角果做qRT-PCR,發(fā)現(xiàn)GLIPL在擬南芥各器官中都有不同程度的表達,在開花后4天的角果中表達量顯著提高;對GUS陽性苗染色發(fā)現(xiàn):BnGLIPL在萌發(fā)的種子、花器官以及發(fā)育的種子中表達量比較高;BnGLIP1在幼苗、花序以及幼嫩角果中有明顯的表達。4)在擬南芥中得到GLIPL的純合突變體;構建GLIPL以及GLIP7油菜同源基因的含雙35s啟動子的CDS過表達載體并得到陽性單株。5)通過檢測過表達株系、純合T-DNA插入突變體以及野生型的種子含油量發(fā)現(xiàn):BnGLIPL比對照含油量下降9.54%,達到極顯著水平;突變體與對照相比含油量上升7.63%,達到極顯著水平;BnGLIP7的過表達株系含油量比對照低4.66%,差異顯著。
[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.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S565.4

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相關期刊論文 前10條

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本文編號：1967585