大丽轮枝菌VdCPMO基因克隆与功能研究

发布时间：2018-04-05 08:26

本文选题：环戊酮1　切入点：2-单加氧酶　出处：《西北农林科技大学》2017年硕士论文

【摘要】：大丽轮枝菌是一种分布广泛的土传病原真菌,能够在660多种植物上引起黄萎病,造成巨大的经济损失。微菌核是大丽轮枝菌特殊的休眠结构,可以在土壤中长期存活,是病害的主要初侵染来源。微菌核的萌发是大丽轮枝菌成功侵染寄主的先决条件,但迄今为止,尚未见微菌核萌发相关基因的研究报道。本研究在前期微菌核萌发表达谱的基础上,选择上调倍数最高的环戊酮1,2-单加氧酶基因(VDAG_03943),进行克隆与功能研究。主要取得了如下结果:1.基于已公布的大丽轮枝菌Vd Ls.17的基因组序列,克隆得到VDAG_03943序列,该基因全长1929bp,c DNA序列全长1668bp,编码555个氨基酸组成的蛋白,命名为Vd CPMO。利用一步法的载体构建方法成功得到了Vd CPMO的敲除质粒p OSCAR-CPMO。2.通过农杆菌介导的遗传转化方法,从野生型菌株JY中敲除了Vd CPMO,经过50mg/L的潮霉素抗性筛选,得到了26株候选的敲除转化子菌株。分别使用潮霉素基因检测引物Hyg-F和Hyg-R、目的基因检测引物CPMO-F和CPMO-R进行PCR验证,确认得到了敲除突变体菌株ΔCPMO-1和ΔCPMO-2。利用PEG介导的原生质体转化方法进行功能互补后,得到了14株互补转化子菌株,经过G418抗性筛选、表型测定和PCR扩增检测后,确认得到3株互补突变体菌株,将其中1株互补突变体菌株并命名为ΔCPMO-C进行后续试验。3.敲除突变体菌株ΔCPMO-1、ΔCPMO-2,互补突变体菌株ΔCPMO-C和野生型菌株JY的菌落生长情况、微菌核萌发率、芽管平均长度的测定结果表明,敲除突变体菌株ΔCPMO-1和ΔCPMO-2的菌落生长减慢,微菌核萌发率显著降低,芽管平均长度明显较短,而互补突变体菌株ΔCPMO-C与野生型菌株JY间无显著性差异;对棉花和烟草植株致病力测定结果表明,敲除突变体菌株ΔCPMO-1和ΔCPMO-2的微菌核丧失了对棉花的致病力,同时对烟草的致病力有所下降,而互补突变体菌株ΔCPMO-C和野生型菌株JY间的致病力无差异。
[Abstract]:Verticillium dahliensis is a widely distributed soil-borne pathogenic fungus, which can cause verticillium wilt in 660 species of plants and cause huge economic losses.Microsclerotia is a special dormant structure of Rhizoctonia dahliensis, which can survive in soil for a long time, and is the main primary infection source of the disease.The germination of microsclerotia is a prerequisite for the successful infection of Rhizoctonia dahliensis, but up to now, no studies on the genes related to microsclerotia germination have been reported.Based on the expression profile of microsclerotia germination in the early stage, the VDAG03943 gene of cyclopentanone 1 + 2-monooxygenase gene was selected to study the cloning and function of cyclopentanone 1 + 2 -monooxygenase gene (VDAG03943).The main results are as follows: 1.Based on the published genome sequence of Vd Ls.17, the VDAG_03943 sequence was cloned. The total length of the gene was 1668 BP, encoding 555 amino acids, which was named Vd CPMO.The whole length of the gene was 1 929 BP, and the total length of the gene was 1668 BP.The knockout plasmid pOSCAR-CPMO.2of VD CPMO was successfully obtained by one-step vector construction method.By means of Agrobacterium tumefaciens mediated genetic transformation, 26 candidate knockout transformants were obtained from wild-type strain JY by screening for hygromycin resistance of 50mg/L.Hygromycin gene detection primer Hyg-F and Hyg-Rand objective gene detection primer CPMO-F and CPMO-R were used for PCR validation. The knockout mutants 螖 CPMO-1 and 螖 CPMO-2 were identified.After functional complementation by PEG mediated protoplast transformation, 14 complementary transformants were obtained. After G418 resistance screening, phenotype determination and PCR amplification, 3 complementary mutants were identified.One of the complementary mutants was named 螖 CPMO-C for follow-up test. 3.The colony growth of mutant 螖 CPMO-1, 螖 CPMO-2, complementary mutants 螖 CPMO-C and wild-type strain JY, the germinating rate of microsclerotia and the average length of bud tube were measured. The results showed that the colony growth of 螖 CPMO-1 and 螖 CPMO-2 decreased.The germinating rate of microsclerotia decreased significantly, the average length of bud tube was significantly shorter, but there was no significant difference between the complementary mutants 螖 CPMO-C and wild type strain JY, and the pathogenicity of cotton and tobacco plants was determined.The microsclerotia of the knockout mutants 螖 CPMO-1 and 螖 CPMO-2 lost their pathogenicity to cotton and decreased their pathogenicity to tobacco, but the pathogenicity of complementary mutants 螖 CPMO-C and wild-type strain JY was not different.
【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

【相似文献】