盾壳霉生长和产孢相关基因CmTERT的功能研究
发布时间：2018-09-04 13:51
【摘要】：盾壳霉是植物病原真菌核盘菌的重寄生菌,在菌核病的生物防治中具有举足轻重的作用。在前期,实验室通过农杆菌介导的转化(ATMT)获得了盾壳霉的T-DNA插入突变体库。通过筛选发现突变体ZS-1TN4685表型异常。通过克隆T-DNA插入位点的侧翼序列,并将其在盾壳霉基因组中进行比对,发现破坏了基因Cm_03805。本研究以ZS-1TN4685为研究材料,并对基因Cm_03805的功能进行了初步研究,研究结果如下:1.与野生型ZS-1相比,突变体ZS-1TN4685菌落形态不规则,生长速率下降。在生长后期,菌落生长停止,且不能长满整个培养皿。在显微镜下观察发现菌丝尖端弯曲、分枝增多。此外,突变体产孢量下降,但孢子寄生菌核的能力无显著差别。2.将基因Cm_03805在NCBI上进行比对发现该基因含有端粒酶RNA结合结构域和端粒酶逆转录酶特异性结构域,故将该基因命名为CmTERT。克隆该基因发现其全长为4155 bp,含有10个外显子,9个内含子,编码1168个氨基酸。3.通过本地blast比对,发现基因CmTERT在盾壳霉基因组中为单拷贝。进化分析发现该蛋白与番茄匍柄霉的端粒酶逆转录酶具有较高的同源性。对CmTERT的表达动态分析发现该基因在分生孢子器形成起始期表达量高。4.根据同源重组的原理,对CmTERT进行了基因敲除,获得了4株该基因的敲除转化子。与野生型ZS-1相比,基因CmTERT的敲除突变体生长速率降低,菌丝尖端弯曲、分枝增多,生物学产量降低,菌丝ROS含量升高,产孢量下降,但孢子寄生菌核的能力无明显变化。这些特征与突变体ZS-1TN4685基本一致。以上研究结果表明,基因CmTERT参与调控盾壳霉的生长和产孢量,但不影响孢子的寄生能力,这对于深入理解盾壳霉的生长发育具有重要意义。
[Abstract]:As a hyperparasite of Sclerotinia sclerotiorum, it plays an important role in the biological control of Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). In the early stage, the T-DNA insertion mutant library was obtained by Agrobacterium tumefaciens mediated transformation of (ATMT). The mutant ZS-1TN4685 phenotype was found to be abnormal by screening. By cloning the flanking sequence of the insertion site of T-DNA, and comparing it with the genome of C. capsidii, it was found that the gene Cm_03805. was destroyed. In this study, ZS-1TN4685 was used as the research material and the function of gene Cm_03805 was preliminarily studied. The results are as follows: 1. Compared with wild type ZS-1, the mutant ZS-1TN4685 colony was irregular in shape and decreased in growth rate. At the late stage of growth, colony growth stopped and the whole dish could not grow. The mycelium tip was curved and branched under microscope. In addition, the spore production of the mutant decreased, but there was no significant difference in the ability of spore parasite. The gene Cm_03805 was compared on NCBI. It was found that the gene contained telomerase RNA binding domain and telomerase reverse transcriptase specific domain, so the gene was named CmTERT.. The total length of the gene was 4155 bp, with 10 exons and 9 introns, encoding 1168 amino acids. The gene CmTERT was found to be a single copy in the genome of Trichoderma auratus by local blast comparison. Evolutionary analysis showed that the protein had high homology with the telomerase reverse transcriptase of Tomato. Dynamic analysis of the expression of CmTERT showed that the gene was highly expressed in the initial stage of conidial organ formation. According to the principle of homologous recombination, CmTERT gene knockout was carried out, and four knockout transformants were obtained. Compared with wild-type ZS-1, knockout mutants of gene CmTERT decreased in growth rate, mycelium tip bent, branching increased, biological yield decreased, hyphal ROS content increased and sporulation decreased, but the ability of spore parasite sclerotia did not change significantly. These characteristics are basically consistent with the mutant ZS-1TN4685. These results suggest that the gene CmTERT is involved in the regulation of the growth and sporulation of C. capsidii, but does not affect the parasitic ability of the spores, which is of great significance for the further understanding of the growth and development of the fungus.
【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】：S476
本文编号：2222292
[Abstract]:As a hyperparasite of Sclerotinia sclerotiorum, it plays an important role in the biological control of Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). In the early stage, the T-DNA insertion mutant library was obtained by Agrobacterium tumefaciens mediated transformation of (ATMT). The mutant ZS-1TN4685 phenotype was found to be abnormal by screening. By cloning the flanking sequence of the insertion site of T-DNA, and comparing it with the genome of C. capsidii, it was found that the gene Cm_03805. was destroyed. In this study, ZS-1TN4685 was used as the research material and the function of gene Cm_03805 was preliminarily studied. The results are as follows: 1. Compared with wild type ZS-1, the mutant ZS-1TN4685 colony was irregular in shape and decreased in growth rate. At the late stage of growth, colony growth stopped and the whole dish could not grow. The mycelium tip was curved and branched under microscope. In addition, the spore production of the mutant decreased, but there was no significant difference in the ability of spore parasite. The gene Cm_03805 was compared on NCBI. It was found that the gene contained telomerase RNA binding domain and telomerase reverse transcriptase specific domain, so the gene was named CmTERT.. The total length of the gene was 4155 bp, with 10 exons and 9 introns, encoding 1168 amino acids. The gene CmTERT was found to be a single copy in the genome of Trichoderma auratus by local blast comparison. Evolutionary analysis showed that the protein had high homology with the telomerase reverse transcriptase of Tomato. Dynamic analysis of the expression of CmTERT showed that the gene was highly expressed in the initial stage of conidial organ formation. According to the principle of homologous recombination, CmTERT gene knockout was carried out, and four knockout transformants were obtained. Compared with wild-type ZS-1, knockout mutants of gene CmTERT decreased in growth rate, mycelium tip bent, branching increased, biological yield decreased, hyphal ROS content increased and sporulation decreased, but the ability of spore parasite sclerotia did not change significantly. These characteristics are basically consistent with the mutant ZS-1TN4685. These results suggest that the gene CmTERT is involved in the regulation of the growth and sporulation of C. capsidii, but does not affect the parasitic ability of the spores, which is of great significance for the further understanding of the growth and development of the fungus.
【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】：S476
【参考文献】
相关硕士学位论文 前4条
1 郝艳飞;生防菌盾壳霉LaeA基因的克隆与功能研究[D];华中农业大学;2015年
2 王祺;枯草芽孢杆菌S-16抑制核盘菌形成活性物质的鉴定及其相关功能基因的克隆[D];内蒙古农业大学;2014年
3 王萍萍;盾壳霉产孢相关基因的克隆及腺苷脱氨酶(CMADA)基因的初步研究[D];华中农业大学;2010年
4 张雄;生防菌盾壳霉双相发酵工艺的优化及放大[D];华中农业大学;2010年
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