西兰花SUR1基因的克隆与功能分析

发布时间：2018-08-24 13:11

【摘要】：西兰花(Brassica oleracea var.italica)又名花椰菜、绿菜花,是十字花科(Brassicace ae)芸薹属(Brassica)植物中的一种重要作物,在我国被广泛种植。西兰花原产于意大利,19世纪末20世纪初传到我国,被誉为“蔬菜皇冠”,其花蕾和茎叶均具有丰富的营养物质,富含蛋白质、胡萝卜素、维生素及各种矿物质。但西兰花真正引起科学家们兴趣的是其卓越的抗癌功效,研究表明经常食用西兰花可有效降低人们罹患多种不同肿瘤疾病如胰腺癌、卵巢癌、乳腺癌、直肠癌及胃癌等的风险,存在于西兰花中的具有抗癌活性的物质是一种异硫氰酸盐,异硫氰酸盐是次生代谢物质芥子油苷水解后的产物,因此研究西兰花中芥子油苷的代谢意义重大。本研究以西兰花为实验材料,参考前期工作中西兰花转录组测序结果,通过RACE方法克隆了西兰花芥子油苷合成途径的关键酶基因SUR1的mRNA全长,并将其转入模式植物拟南芥中,通过过量表达BoSUR1的转基因拟南芥的主要表型验证了Bo SUR1的功能,并将其命名为Bo SUR1。为深入探讨西兰花芥子油苷的代谢及利用基因工程方法培育具有较高芥子油苷含量和抗癌活性的西兰花品种提供了理论参考和基因资源。本论文的主要研究结果:(1)克隆获得了西兰花Bo SUR1基因mRNA全长序列。Bo SUR1基因CDS序列全长1371bp,编码蛋白质含456个氨基酸残基,分子量50.45KD,。BLAST结果表明:该基因与芜菁亚种(EF611275.1、EF611274.1、EF611276.1、EF611273.1)、甘蓝型油菜(EF611272.1)、卷心菜(EF611271.1)、白菜(FJ376052.1)、小白菜(EF611278.1)具有98%的同源性,与芝麻菜亚种(JX946194.1)具有94%同源性,与山嵛菜(XM_006408931.1)具有92%同源性,与拟南芥具有88%的同源性。(2)获得过量表达Bo SUR1基因的转基因拟南芥,为验证及深入研究Bo SUR1的功能奠定了物质基础。(3)对35S::Bo SUR1转基因拟南芥进行表型分析结果显示,脂肪族芥子油苷含量升高显著,吲哚族芥子油苷含量升高不显著,验证了Bo SUR1基因可参与脂肪族芥子油苷和吲哚族芥子油苷的合成。本研究的意义:西兰花中Bo SUR1基因的克隆及功能的验证,为深入探讨西兰花中芥子油苷的合成代谢及培育具有较高含量芥子油苷的西兰花新品种奠定了基础。
[Abstract]:Broccoli (Brassica oleracea var.italica), also known as cauliflower, is an important crop in (Brassica) of (Brassicace ae) Brassica, which is widely planted in China. Broccoli originated from Italy at the end of the 19th century and spread to China at the beginning of the 20th century. It is called "vegetable crown". Its flower buds and stems and leaves are rich in nutrients, protein, carotene, vitamins and various minerals. But broccoli is of real interest to scientists because of its remarkable anticancer properties. Studies have shown that regular consumption of broccoli can reduce the risk of various cancer diseases such as pancreatic cancer, ovarian cancer, breast cancer, rectal cancer and stomach cancer. The substance with anticancer activity in broccoli is a kind of isothiocyanate. Isothiocyanate is the product of the hydrolysis of the secondary metabolite mustard oil glucoside, so it is of great significance to study the metabolism of mustard oil glycoside in broccoli. In this study, using broccoli as experimental material and referring to the sequencing results of broccoli transcriptome in previous work, the mRNA length of the key enzyme gene SUR1 of broccoli mustard glucoside synthesis pathway was cloned by RACE method and transferred into Arabidopsis thaliana, a model plant. The main phenotypes of transgenic Arabidopsis thaliana (Arabidopsis thaliana), which overexpressed BoSUR1, proved the function of Bo SUR1 and named it Bo SUR1.. This study provides a theoretical reference and genetic resource for the further study of the metabolism of mustard oil in broccoli and the cultivation of broccoli varieties with higher mustard oil content and anticancer activity by genetic engineering method. The main results of this thesis are as follows: (1) the mRNA full-length sequence of broccoli Bo SUR1 gene. Bo SUR1 gene CDS sequence is 1371bp. the encoding protein contains 456 amino acid residues. The results of molecular weight 50.45 KDX. Blast showed that the gene had 98% homology with EF611275.1,EF611274.1,EF611276.1,EF611273.1, EF611272.1, EF611271.1, FJ376052.1, EF611278.1, 94% homology with JX946194.1 and 92% with XM_006408931.1. (2) transgenic Arabidopsis thaliana with overexpression of Bo SUR1 gene was obtained, which laid a material foundation for the verification and further study of the function of Bo SUR1. (3) the phenotype analysis of 35S::Bo SUR1 transgenic Arabidopsis thaliana showed that, The content of aliphatic mustard oil increased significantly, but the content of indoline mustard oil did not. It was proved that Bo SUR1 gene could be involved in the synthesis of aliphatic mustard and indoline mustard oil. The significance of this study: cloning and functional verification of Bo SUR1 gene in broccoli laid a foundation for further study on the synthesis and metabolism of mustard oil in broccoli and the breeding of new varieties with higher content of mustard oil glucoside.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S635.3;Q943.2

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