梨腐烂病菌多聚半乳糖醛酸酶基因功能的初步研究

发布时间：2019-05-27 12:24

【摘要】：梨腐烂病是我国梨树上的一种重要的枝干病害,在北方梨产区发病严重的梨园常造成大量死树或毁园,病菌侵染梨树后导致细胞壁降解而表现树皮腐烂症状,但有关梨腐烂病菌果胶酶基因的研究尚未见报道。本研究根据实验室前期获得的梨腐烂病菌强致病力菌株F-HN-6和弱致病力菌株F-HN-2a-1的转录组数据分析,预测获得1个聚半乳糖醛酸酶相对表达量上调的基因Vamepg,通过基因敲除和转化子致病力比较,对Vamepg基因在梨腐烂病菌侵染梨过程中的功能进行了初步研究,取得的主要研究结果如下:1、通过Real-Time PCR验证Vamepg基因的相对表达量,结果表明梨腐烂病菌强致病力菌株F-HN-6 Vamepg基因的表达量为弱致病力菌株F-HN-2a-1的1.86倍,预测分析发现Vamepg基因编码的外切聚半乳糖醛酸酶具有外分泌功能,能够分泌到细胞膜外。2、采用Double-joint PCR方法构建线性敲除载体,利用PEG介导原生质体遗传转化方法,获得能够在含潮霉素的培养基上生长的转化子。分别采用4对检测引物筛选转化子和Southern Blot鉴定为单拷贝,获得4个缺失Vamepg基因的突变体。并利用基因互补方法获得2个Vamepg基因的互补突变体,说明梨腐烂病菌可以采用线性载体利用PEG介导原型质体转化方法进行基因敲除。3、缺失Vamepg基因的突变体在PDA培养基上生长速度略微降低,分生孢子器大小及形态无差异,但在枝条上的致病力显著降低。Vamepg基因的互补突变体的生长速度和致病力与野生型菌株相比基本相同。体外酶活力测定发现,野生型菌株在培养过程中酶活力不断增强,在10d时酶活力达到最大值;缺失Vamepg基因的突变体菌株体外酶活力一直维持在较低水平,在10d时酶活力未出现最大值。推测缺失Vamepg基因可能导致突变体分泌的外切聚半乳糖醛酸酶含量减少,从而减弱其对梨树枝条的致病力。
[Abstract]:Pear rot disease is an important branch disease on pear trees in China. Pear orchards with serious incidence in northern pear producing areas often cause a large number of dead trees or destroy orchards. After the pathogen infects pear trees, it leads to cell wall degradation and shows bark decay symptoms. However, there is no report on the Pectinase gene of pear rot pathogen. Based on the analysis of transcriptional group data of strong pathogenic strain F-HN-6 and weakly pathogenic strain F-HN-2a-1 obtained in laboratory, a gene Vamepg, with up-regulated relative expression of polygalacturonase was predicted. The function of Vamepg gene in the process of pear infection was studied by comparing the pathogenicity of gene knockout and transformant. The main results were as follows: 1. The relative expression of Vamepg gene was verified by Real-Time PCR. The results showed that the expression of F-HN-6 Vamepg gene was 1.86 times higher than that of weak pathogenicity strain F-HN-2a-1. It was found that the exocrine polygalacturonase encoded by Vamepg gene had exocrine function. 2. The linear knockout vector was constructed by Double-joint PCR method, and the transformant which could grow on hygromycin medium was obtained by PEG mediated protoplast genetic transformation. Four pairs of primers were used to screen transformants and Southern Blot were identified as single copies, and four mutants without Vamepg gene were obtained. Two complementary mutants of Vamepg gene were obtained by gene complementary method, which indicated that pear rot pathogen could be knockout by PEG mediated protoplast transformation by linear vector. The growth rate of mutants deleted with Vamepg gene decreased slightly on PDA medium, but there was no difference in the size and morphology of conidia. However, the pathogenicity of Vamepg gene complementary mutants was significantly lower than that of wild type strains, and the growth rate and pathogenicity of Vamepg gene complementary mutants were basically the same as those of wild type strains. It was found that the enzyme activity of wild strains increased continuously during the culture process, and reached the maximum at 10 days. The enzyme activity of the mutant strain without Vamepg gene remained at a low level in vitro, and there was no maximum enzyme activity at 10 days. It is speculated that the deletion of Vamepg gene may lead to the decrease of extraneous polygalacturonase content secreted by the mutant, thus weakening the pathogenicity of the mutant to pear branches.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S436.612.16

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