【摘要】：須癬毛癬菌是一種重要的人畜共患皮膚癬菌,主要感染人與動物的皮膚角質(zhì)層及其附屬角質(zhì)化組織。鋅是微生物重要的必需微量金屬元素之一,是細(xì)胞中的第二大過渡金屬,對病原微生物的生長、繁殖、致病力具有重要作用。目前已在多種真菌中發(fā)現(xiàn)參與調(diào)控真菌細(xì)胞鋅離子穩(wěn)態(tài)的相關(guān)基因,其中鋅離子反應(yīng)轉(zhuǎn)錄激活因子基因(ZafA)表達蛋白及其同源蛋白已被證明在病原性真菌胞內(nèi)鋅穩(wěn)態(tài)調(diào)控和致病力中具有重要作用。通過轉(zhuǎn)錄組測序已在須癬毛癬菌中發(fā)現(xiàn)ZafA基因,但該基因及其表達的蛋白功能目前仍不清楚。根癌農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化(ATMT)是目前真菌基因敲除的常用方法之一,已經(jīng)成功應(yīng)用于多種絲狀真菌的遺傳轉(zhuǎn)化。本文利用ATMT法對須癬毛癬菌ZafA基因進行遺傳轉(zhuǎn)化,構(gòu)建ZafA基因轉(zhuǎn)化株,并對須癬毛癬菌ATMT轉(zhuǎn)化體系進行優(yōu)化,為后續(xù)ZafA功能驗證奠定基礎(chǔ)。試驗結(jié)果如下:1、將構(gòu)建完成的含有ZafA基因同源片段及潮霉素抗性標(biāo)記基因的雙元載體pDHt/ZafA-ⅠⅡ::hph轉(zhuǎn)入根癌農(nóng)桿菌中,PCR驗證轉(zhuǎn)化成功后,利用帶有雙元載體的根癌農(nóng)桿菌介導(dǎo)轉(zhuǎn)化須癬毛癬菌分生孢子,PCR驗證結(jié)果證明須癬毛癬菌ZafA基因成功地被hph基因替代實現(xiàn)轉(zhuǎn)化。2、在轉(zhuǎn)化過程中使用不同的固相載體(Hybond N+尼龍膜、PVDF膜、濾紙),不同共培養(yǎng)時間(36 h、48 h、60 h),預(yù)培養(yǎng)中是否加入AS,在28℃共培養(yǎng)條件下對轉(zhuǎn)化體系進行優(yōu)化。發(fā)現(xiàn)預(yù)培養(yǎng)中加入AS獲得的轉(zhuǎn)化株數(shù)量顯著高于未加AS組,使用Hybond N+尼龍膜、濾紙均可獲得轉(zhuǎn)化株,且差異不顯著,使用PVDF膜時無轉(zhuǎn)化株出現(xiàn)。在28℃共培養(yǎng)條件下,將共孵育時間延伸至60 h,可顯著提高轉(zhuǎn)化效率。綜上所述,通過ATMT體系可成功轉(zhuǎn)化須癬毛癬菌ZafA基因,獲得須癬毛癬菌ZafA基因轉(zhuǎn)化株。根癌農(nóng)桿菌在經(jīng)AS誘導(dǎo)培養(yǎng)后與須癬毛癬菌分生孢子液(1×107CFU/mL)1:1混合,涂布于覆蓋尼龍膜或濾紙的共培養(yǎng)基中,28℃共培養(yǎng)60 h可獲得較高數(shù)量的轉(zhuǎn)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，

本文編號：2186337