蛋白-O-岩藻糖基转移酶1基因CfPOFUT1在果生炭疽菌中的功能分析

Functions of CfPOFUT1 in Colletotrichum fructicola

摘要:

【目的】蛋白-O-岩藻糖基转移酶1 (protein O-fucosyltransferase 1，POFUT1)是催化蛋白质O-岩藻糖基化的关键酶，在动物和人体内被证明调控一系列的生理病理过程，然而POFUT1基因在果生炭疽菌乃至真菌中还未见报道。本研究旨在克隆果生炭疽菌中CfPOFUT1基因，并分析其生物学功能。【方法】利用RT-PCR技术扩增CfPOFUT1的基因并进行生物信息学分析，构建了CfPOFUT1基因的沉默和过表达载体，通过PEG介导法将载体导入原生质体中获得CfPOFUT1基因的沉默和过表达突变体。测定了野生型菌株、CfPOFUT1沉默菌株和过表达菌株在PDA上的菌丝生长、分生孢子产生、萌发与附着胞形成、胁迫应答和致病力、杀菌剂敏感性等生物学表型。【结果】与野生型菌株相比，基因过表达突变体产孢量显著增加，致病力增强，对嘧菌酯敏感性降低，但对多菌灵和咪鲜胺敏感性增强。基因沉默突变体产孢量减少，细胞壁完整性、内质网应激敏感性提高，致病力减弱，对嘧菌酯敏感性提高，但对多菌灵和咪鲜胺敏感性降低。【结论】CfPOFUT1基因参与调控果生炭疽菌分生孢子产量，细胞壁完整性、内质网对应激和药剂敏感性，并对其致病性也具有一定的影响。

Abstract:

[Objective] Protein O-fucosyltransferase 1(POFUT1),a key enzyme catalyzing protein O-fucosylation,has been verified to regulate a series of physiological and pathological processes in animals and humans.However,POFUT1 gene in Colletotrichum fructicola or even fungi has not been reported.This study aims to clone the CfPOFUT1 gene from C.fructicola and analyze its biological functions.[Methods] RT-PCR was employed to amplify the CfPOFUT1 gene and then the bioinformation of the gene was analyzed.We constructed the CfPOFUT1 silencing and overexpression vectors,which were then transformed into the protoplasts with the PEG-mediated method to yield the CfPOFUT1-silenced and-overexpressing mutants.The biological phenotypes of the wild-type LY5-1,and the CfPOFUT1-silenced and-overexpressing mutants were determined,such as the mycelia growth,conidial production,conidial germination,appressorium formation,stress response,pathogenicity,and fungicide sensitivity.[Results]CfPOFUT1-overexpressing mutant showed higher conidial production,stronger pathogenicity,lower sensitivity to azoxystrobin,but higher sensitivity to carbendazim and prochloraz than the wild-type LY5-1.CfPOFUT1-silenced mutant demonstrated lower conidial production,intact cell wall,higher sensitivity to endoplasmic reticulum (ER) stress and azoxystrobin,lower pathogenicity,and lower sensitivity to carbendazim and prochloraz.[Conclusion] CfPOFUT1 is involved in conidial production,cell wall integrity,ER stress,and fungicide sensitivity of C.fructicola,affecting the pathogenicity.