茶树炭疽菌CfHTF5基因的功能初探

摘要茶树炭疽病是影响茶叶产量和品质的重要原因之一，进而使茶产业发展受到抑制，茶树炭疽菌的生物学性状影响其对茶树的侵染能力、繁殖能力及抗逆性，研究生物学特性用于防治茶树炭疽病有重要意义。在茶树炭疽菌的同源异形盒家族中，挑选其中一个CHTF15基因进行操作，探讨其功能。通过PCR扩增，融合PCR等技术成功获得用于CfHTF 15基因敲除的同源片段AG、GB。进一步通过原生质体转化、筛选等步骤获得敲除CHTF15基因的缺失突变体。以野生型为参照，通过对比并分析其表型，可以观察到茶树炭疽菌在生长发育形态以及产孢过程中的变化，分析CHT℉I5基因的生物学功能。结果表明，CHT℉I5基因的缺失对茶树炭疽菌的形态、生长速度、孢子形态、孢子萌发状况、附着胞形成、致病性均无显著影响，初步判断CfHTF 15基因对茶树炭疽菌的生长发育不是必需的。关键词：茶叶：茶树炭疽菌：同源异型盒基因AbstractTea anthrax is one of the important reasons affecting tea yield and quality,whichin turn inhibits the development of tea industry,and the biological characteristics ofanthrax affect its infection,reproductive ability and stress resistance to tea plants,andthe study of biological characteristics is of great significance for the prevention andtreatment of anthrax.In the Homeobox family of Colletotrichum fructicola,one of theCHTF/5 genes was selected to explore its function.Homologous fragments AG andGB for CHTF/5 gene knockout were successfully obtained by PCR amplificationand fusion PCR.Furthermore,the deletion mutant of knockout CH/TF/5 gene wasobtained by protoplast transformation and screening.Taking the wild type as areference,by comparing and analyzing its phenotype,the changes of the growth anddevelopment morphology and spore-production of C.fructicola can be observed,andthe biological function of CHTF/5 gene can be analyzed.The results showed that thedeletion of CHTF/5 gene had no significant effect on the morphology,growth rate,spore morphology,spore germination,appressorium formation and pathogenicity of C.fructicola,and it was preliminarily judged that CfHTF/5 gene was not necessary forthe growth and development of C.fructicola.Keywords:Tea;Tea anthrax;Homeobox目录1引言…1.1茶.21.2茶树炭疽病1.2.1茶树炭疽病的介绍1.2.2茶树炭疽菌研究现状1.2.3茶树炭疽病的防治现状21.3果生炭疽菌介绍及其侵染过程21.4 Homeobox基因.31.5本研究的目的和意义42材料与方法……42.1试验使用的菌株、质粒42.2供试植物.…42.3实验试剂.…42.4培养基配制52.5仪器.62.6茶树炭疽菌基因组DNA的提取.72.7DNA分子琼脂糖电泳72.8PCR反应.82.9PCR产物胶回收92.10融合PCR.92.11茶树炭疽菌原生质体的制备与转化112.12阳性转化子的筛选及验证.112.13茶树炭疽菌的表型观察122.13.1菌丝生长速率测定122.13.2产孢情况观察…132.13.3孢子形态观察.132.13.4孢子萌发及附着胞形态观察133结果与分析.….133.1 CfHTFI.5基因的敲除.133.1.1CHTF15基因同源重组片段AG,GB的获得133.1.2重组基因阳性克隆子的筛选结果153.2CTF15基因缺失突变体的表型分析163.2.1CHT℉15基因缺失突变体对茶树炭疽菌营养生长的影响.163.2.2CHT℉15基因缺失突变体对茶树炭疽菌产孢活力的影响.173.2.3 CfHTFI.5基因缺失突变体对茶树炭疽菌孢子形态、孢子萌发状况、附着胞形态的影响.17