尖孢镰刀菌亚麻专化型FolSwe1基因功能分析  

Functional Analysis of FolSwe1 Gene in Fusarium oxysporum f.sp.lini

作　　者：冯欣茹 侯占铭[1] FENG Xinru;HOU Zhanming(College of Life Science and Technology,Inner Mongolia Normal University,Hohhot 010022,China)

机构地区：[1]内蒙古师范大学生命科学与技术学院,呼和浩特010022

出　　处：《内蒙古大学学报（自然科学版）》2024年第4期376-387,共12页Journal of Inner Mongolia University：Natural Science Edition

基　　金：国家自然科学基金项目(31160280);内蒙古自然科学基金项目(2015MS0311)。

摘　　要：本文旨在对尖孢镰刀菌亚麻专化型FolSwe1基因进行功能分析。利用同源比对,通过PCR对FolSwe1基因及其侧翼序列进行扩增和测序,使用Split-Marker技术构建该基因缺失突变盒,使用聚乙二醇(PEG)介导转化野生型原生质体,获得了FolSwe1基因缺失突变株,对突变体表型和致病力进行测定。构建荧光载体pZESH1(pZWH1+EGFP+FolSwe1)对FolSwe1蛋白进行亚细胞定位。结果显示,FolSwe1基因编码区为3219 bp,含有一个内含子,长度为54 bp,所测序列包括编码区上游1704 bp,编码区下游为1694 bp。表型观察发现,敲除突变体的菌丝比野生型短小且密实,菌落生长速度较慢,特别显著的变化是缺失突变体不再产生孢子,致病力也明显下降。亚细胞定位显示FolSwe1蛋白在培养时间较长菌丝的细胞膜中有荧光信号。综上所述,FolSwe1基因主要调控尖孢镰刀菌亚麻专化型的孢子发生、菌丝营养生长和对亚麻的致病性。To identify the function of the FolSwe1 gene in Fusarium oxysporum f.sp.lini,the coding region of the gene and its flanking sequence were amplified and sequenced, and a gene deletion cassette was constructed by using Split-Maker strategy.The FolSwe1 gene deletion mutant strains were obtained by transforming wild-type protoplasts mediated by polyethylene glycol(PEG).The phenotype and pathogenicity of the deletion mutant were assayed through conventional biological observation and measurement.A fluorescent vector pZESH1(pZWH1+EGFP+FolSwe1) was constructed for subcellular localization of the FolSwe1 protein.The sequencing results showed that the coding region of FolSwe1 gene is 3219 bp, containing one intron with length of 54 bp, and the obtained flanking sequence contained upstream 1704 bp and downstream 1694 bp of the coding region.Phenotypic observation revealed that the hyphae of the deletion mutant were shorter and grew denser compared with the wild-type, and the colony of deletion mutant was significantly smaller than that of the wild type.The most remarkable change was that the deletion mutant did not produce spores anymore, indicating that the gene was closely related to the conidiogenesis of the fungus.The infection assay to the seedling of the flax indicated that the pathogenicity of the deletion mutant decreased significantly.The subcellular localization showed that the fluorescence signals mainly appeared in the cell membrane.In summary, the FolSwe1 gene is mainly responsible for the conidiogenesis, vegetative growth and pathogenicity of the fungus.

关 键 词：尖孢镰刀菌亚麻专化型 FolSwe1基因 Split-Marker技术 基因敲除 

分 类 号：Q933[生物学—微生物学] Q78