大丽轮枝菌VdLac基因的克隆与功能分析

发布时间：2018-06-03 21:39

本文选题：大丽轮枝菌 + 漆酶　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：大丽轮枝菌是一种典型的土传病原真菌,寄主范围广泛。微菌核是大丽轮枝菌的主要休眠结构,抗逆性强,可在没有寄主的情况下存活14年,这与其体内含有丰富的黑色素密切相关。黑色素是一种由酚类或吲哚类化合物组成的带有负电荷的疏水性高分子聚合物,能够保护真菌免受环境和寄主的影响。漆酶是黑色素合成通路中的关键酶,在真菌生长发育的过程中发挥着重要的作用。本文通过农杆菌介导的遗传转化和PEG介导的原生质体转化方法对大丽轮枝菌中的漆酶基因VdLac进行了敲除和互补,研究漆酶基因在大丽轮枝菌中的主要功能。结果如下:1.根据NCBI上已公布的VdLs.17的基因组序列,克隆得到漆酶基因VdLac序列,VdLac基因全长1978 bp,c DNA序列全长1713 bp,编码570个氨基酸。2.利用农杆菌介导的遗传转化方法(ATMT)对VdLac进行敲除,并通过潮霉素抗性筛选和PCR检测确定阳性敲除突变体,获得了4个敲除突变体。3.通过PEG介导的原生质体转化方法将互补质粒随机插入到敲除突变体中,经G418抗性筛选和PCR检测确定了1株阳性互补突变体。4.通过对敲除和互补突变体进行一系列的生物学功能测定,分析漆酶基因的功能。结果表明,VdLac基因缺失突变体对硝化压力更敏感,其在CM培养基上仍可产生漆酶,但产生漆酶的能力有所降低;VdLac基因不影响菌株的营养生长、产孢能力、致病力以及黑色素合成。
[Abstract]:Verticillium dahliensis is a typical soil-borne pathogenic fungus with a wide range of hosts. Microsclerotia is the main dormant structure of Rhizoctonia dahliensis, which has strong resistance to stress and can survive for 14 years without host, which is closely related to the rich melanin in its body. Melanin is a negatively charged hydrophobic polymer composed of phenols or indole compounds, which can protect fungi from environmental and host effects. Laccase is a key enzyme in melanin synthesis pathway and plays an important role in the growth and development of fungi. The laccase gene VdLac was knockout and complementary by Agrobacterium tumefaciens-mediated genetic transformation and PEG mediated protoplast transformation, and the main function of laccase gene was studied. The result is as follows: 1. According to the genomic sequence of VdLs.17 published on NCBI, the VdLac sequence of laccase gene was cloned. The total length of 1978 BP DNA sequence of laccase gene was 1713 BP, encoding 570 amino acids. 2. VdLac knockout was carried out by Agrobacterium tumefaciens mediated genetic transformation method. Four knockout mutants were obtained by hygromycin resistance screening and PCR detection. The complementary plasmids were randomly inserted into the knockout mutants by PEG mediated protoplast transformation. A positive mutants. 4 was identified by G418 resistance screening and PCR detection. The function of laccase gene was analyzed by a series of biological functions of knockout and complementary mutants. The results showed that the mutant of VdLac gene was more sensitive to nitrification pressure, and laccase was still produced in CM medium, but the ability of laccase production could not affect the vegetative growth, sporulation ability, pathogenicity and melanin synthesis of the strain.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.44

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