根癌农杆菌介导须癣毛癣菌ZafA基因的遗传转化
发布时间：2018-08-16 15:02
【摘要】：须癣毛癣菌是一种重要的人畜共患皮肤癣菌,主要感染人与动物的皮肤角质层及其附属角质化组织。锌是微生物重要的必需微量金属元素之一,是细胞中的第二大过渡金属,对病原微生物的生长、繁殖、致病力具有重要作用。目前已在多种真菌中发现参与调控真菌细胞锌离子稳态的相关基因,其中锌离子反应转录激活因子基因(ZafA)表达蛋白及其同源蛋白已被证明在病原性真菌胞内锌稳态调控和致病力中具有重要作用。通过转录组测序已在须癣毛癣菌中发现ZafA基因,但该基因及其表达的蛋白功能目前仍不清楚。根癌农杆菌介导的遗传转化(ATMT)是目前真菌基因敲除的常用方法之一,已经成功应用于多种丝状真菌的遗传转化。本文利用ATMT法对须癣毛癣菌ZafA基因进行遗传转化,构建ZafA基因转化株,并对须癣毛癣菌ATMT转化体系进行优化,为后续ZafA功能验证奠定基础。试验结果如下:1、将构建完成的含有ZafA基因同源片段及潮霉素抗性标记基因的双元载体pDHt/ZafA-ⅠⅡ::hph转入根癌农杆菌中,PCR验证转化成功后,利用带有双元载体的根癌农杆菌介导转化须癣毛癣菌分生孢子,PCR验证结果证明须癣毛癣菌ZafA基因成功地被hph基因替代实现转化。2、在转化过程中使用不同的固相载体(Hybond N+尼龙膜、PVDF膜、滤纸),不同共培养时间(36 h、48 h、60 h),预培养中是否加入AS,在28℃共培养条件下对转化体系进行优化。发现预培养中加入AS获得的转化株数量显著高于未加AS组,使用Hybond N+尼龙膜、滤纸均可获得转化株,且差异不显著,使用PVDF膜时无转化株出现。在28℃共培养条件下,将共孵育时间延伸至60 h,可显著提高转化效率。综上所述,通过ATMT体系可成功转化须癣毛癣菌ZafA基因,获得须癣毛癣菌ZafA基因转化株。根癌农杆菌在经AS诱导培养后与须癣毛癣菌分生孢子液(1×107CFU/mL)1:1混合,涂布于覆盖尼龙膜或滤纸的共培养基中,28℃共培养60 h可获得较高数量的转化子。
[Abstract]:Trichophyton tubuloides is an important zoonotic dermatophytes, which mainly infects the human and animal skin cuticle and its associated keratinized tissue. Zinc is one of the essential trace metal elements in microorganism and the second transition metal in cells. It plays an important role in the growth, reproduction and pathogenicity of pathogenic microorganisms. Genes involved in regulating the homeostasis of zinc ions in fungal cells have been found in many fungi. Zinc ion-responsive transcriptional activator gene (ZafA) and its homologous proteins have been proved to play an important role in the regulation and pathogenicity of zinc homeostasis in pathogenic fungi. The ZafA gene has been found in Trichophyton mutans by transcriptome sequencing, but the function of the gene and its expressed protein is still unclear. Agrobacterium tumefaciens mediated genetic transformation (ATMT) is one of the commonly used methods of fungal gene knockout and has been successfully applied to the genetic transformation of many filamentous fungi. In this paper, the ZafA gene of Trichophyton tinea was transformed by ATMT method, the transformed strain of ZafA gene was constructed, and the ATMT transformation system of Trichophyton tinea was optimized, which laid a foundation for the subsequent verification of ZafA function. The results are as follows: 1. The constructed binary vector pDHt- ZafA- 鈪，
本文编号：2186337
[Abstract]:Trichophyton tubuloides is an important zoonotic dermatophytes, which mainly infects the human and animal skin cuticle and its associated keratinized tissue. Zinc is one of the essential trace metal elements in microorganism and the second transition metal in cells. It plays an important role in the growth, reproduction and pathogenicity of pathogenic microorganisms. Genes involved in regulating the homeostasis of zinc ions in fungal cells have been found in many fungi. Zinc ion-responsive transcriptional activator gene (ZafA) and its homologous proteins have been proved to play an important role in the regulation and pathogenicity of zinc homeostasis in pathogenic fungi. The ZafA gene has been found in Trichophyton mutans by transcriptome sequencing, but the function of the gene and its expressed protein is still unclear. Agrobacterium tumefaciens mediated genetic transformation (ATMT) is one of the commonly used methods of fungal gene knockout and has been successfully applied to the genetic transformation of many filamentous fungi. In this paper, the ZafA gene of Trichophyton tinea was transformed by ATMT method, the transformed strain of ZafA gene was constructed, and the ATMT transformation system of Trichophyton tinea was optimized, which laid a foundation for the subsequent verification of ZafA function. The results are as follows: 1. The constructed binary vector pDHt- ZafA- 鈪，
本文编号：2186337
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