一种可用于外源基因功能鉴定的真菌分泌表达载体74HSP-EGFP的构建及验证  

Construction and Verification of a Fungal Secretory Expression Vector 74HSP-EGFP for Functional Identification of Exogenous Genes

作　　者：王丹[1] 彭存智[1] 常丽丽[1] 郑杏梅 徐兵强 仝征[1] Wang Dan;Peng Cunzhi;Chang Lili;Zheng Xingmei;Xu Bingqiang;Tong Zheng(Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences,Haikou,571101;Haikou Experimental Station(Institute of Tropical Fruit Tree Research),Chinese Academy of Tropical Agricultural Sciences,Haikou,571101)

机构地区：[1]中国热带农业科学院热带生物技术研究所,海口571101 [2]中国热带农业科学院海口实验站(热带果树研究所),海口571101

出　　处：《基因组学与应用生物学》2021年第1期230-236,共7页Genomics and Applied Biology

基　　金：中国热带农业科学院基本科研业务费专项资金项目(1630052017006,1630052017009)资助。

摘　　要：传统的植物遗传转化体系能够鉴定植物基因的功能,但该过程烦琐、耗时长,且存在一定的局限性。本研究构建具有蛋白分泌信号肽和荧光蛋白EGFP的真菌分泌载体74HSP-EGFP,通过尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense, Foc)携带侵染巴西蕉(Musa nana)植株对载体上的外源基因功能进行快速鉴定。利用蛋白质组学技术鉴定获得来源于香蕉枯萎病致病菌Foc的主要分泌蛋白Secreted in xylem 1(SIX1d) N端分泌信号肽。基于pCT74载体、SIX1d分泌信号肽序列以及供外源基因插入的多克隆位点序列构建真菌分泌表达载体74HSP,将EGFP基因通过多克隆位点插入到该载体中获得分泌载体74HSP-EGFP。转化Foc后发现,Foc分泌载体74HSP-EGFP转化菌株在PDA培养基和KK培养基中生长4 d后,菌体中无绿色荧光信号,培养基中检测到绿色荧光信号。转化菌株侵染巴西蕉后,在植株根部组织切片中检测到绿色荧光信号。研究结果表明分泌载体74HSP-EGFP能够利用病原菌将携带的外源蛋白表达并分泌到被侵染的植物体内,实现对外源基因功能的快速分析。The function of plant genes can be identified by traditional plant transgenic technology, but the transgenic process is cumbersome, time-consuming and has some limitations. 74HSP-EGFP was constructed as the secretion vector of fungal pathogens with protein secretion signal peptide and EGFP, and the function of the target gene was quickly identified by using pathogens as carriers. Secretory in xylem 1(SIX1d), a major secretory protein of Fusarium oxysporum f. sp. cubense(Foc), was identified by proteomics. Bioinformatics analysis showed that the N-terminal of SIX1d had a secreted signal peptide(SP). We successfully constructed the sequences of SP and multiple clone sites(MCS) into pCT74 skeleton to obtain 74HSP, and put an EGFP gene into the 74HSP from MCS(74HSP-EGFP) for function test. Transforming Foc found that after 74HSP-EGFP transformed strain grew in PDA medium and KK medium for 4 days, there was no green fluorescence signal in the cells, but strong green fluorescence signal in the culture medium, indicating that EGFP could be secreted completely into the extracellular by using this vector. After infection of Brazilian banana, green fluorescent signals were detected in tissue sections of banana roots. The results showed that 74HSP-EGFP, a fungal secretory vector, can express and secrete the carried foreign proteinsinto the infected plants to achieve rapid analysis of the function of foreign genes.

关 键 词：分泌表达载体 真菌 功能验证 

分 类 号：Q78[生物学—分子生物学]