禾谷镰刀菌中<em>FgPDE1</em>基因的敲除及其功能的研究

doi:10.13523/j.cb.20150809

中国生物工程杂志

2015, Vol. 35

Issue (8): 59-67 DOI: 10.13523/j.cb.20150809

研究报告

禾谷镰刀菌中FgPDE1基因的敲除及其功能的研究

常玉梅, 侯占铭

内蒙古师范大学生命科学与技术学院呼和浩特 010020

Research on Gene Knockout and Function of FgPDE1 in Fusarium graminearum

CHANG Yu-mei, HOU Zhan-ming

College of Life Science and Technology, Inner Mongolia Normal University, Hohhot 010022, China

全文: PDF(933 KB) HTML

摘要：

目的:旨在敲除禾谷镰刀菌Fusarium graminearum FgPDE1基因,确定其缺失突变体表型,从而分析该基因的生物学功能。方法:应用Split-marker技术构建含有潮霉素基因敲除盒,通过PEG介导原生质体转化,PCR筛查抗潮霉素转化子以获得缺失突变体ΔFgPDE1,根据突变体表型变化及致病性的检测对FgPDE1基因的功能进行分析。结果:采用Split-marker技术,成功构建了FgPDE1基因敲除盒;PEG介导转化禾谷镰刀菌原生质体后成功获得转化子。经PCR筛查,得到3个PCR确认的敲除突变体;表型观察发现,ΔFgPDE1菌落的外型及菌落生长速度与野生型没有明显差异。孢子侵染西红柿果实实验证明:以西红柿为侵染宿主,相对于野生型,突变体致病性没有明显减弱;但突变体分生孢子产量显著下降。结论:FgPDE1基因可能与禾谷镰刀菌分生孢子的形成有关。

关键词： 禾谷镰刀菌; FgPDE1; 基因敲除; Split-Marker

Abstract:

Objective: The purpose is to knock out FgPDE1 gene in Fusarium graminearum, so as to identify the function of the gene through analysis of the phenotype of the deletion mutants. Methods: The Split-marker strategy is applied to build knockout cassette containing hygromycin phosphotransferase gene and anti-hygromycin transformants are obtained by using PEG-mediated protoplast transformation. The FgPDE1 gene deletion mutants are screened by the absent of the PCR products of the FgPDE1 gene. The function of the gene is analyzed according to the mutant phenotype and pathogenicity detection. Results: The Split-marker knockout cassette is successfully constructed and the transformants are obtained after PEG-mediated transformation of the protoplasts of PH-1 and then, three FgPDE1 gene deletion mutants are obtained through PCR screening. The phenotypic analysis revealed that there is no significant difference between ΔFgPDE1 and wild type in terms of colony phenotype and growth rate. The virulence assay by fruit of tomato infected by conidiospores show that the mutants do not decrease greatly in pathogenicity. However, microscopic observation show that the conidiospore amount of ΔFgPDE1 reduce significantly. Conclusions: FgPDE1 gene might be related to conidia formation of the Fusarium graminearum.

Key words: Fusarium graminearum FgPDE1 Gene knockout Split-marker

收稿日期: 2015-03-10 出版日期: 2015-08-25

ZTFLH:

Q789

基金资助:

国家自然科学基金资助项目(31160280)

通讯作者: 侯占铭 E-mail: 670645337@qq.com;hou42@wisc.edu

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引用本文:

常玉梅, 侯占铭. 禾谷镰刀菌中FgPDE1基因的敲除及其功能的研究[J]. 中国生物工程杂志, 2015, 35(8): 59-67.

CHANG Yu-mei, HOU Zhan-ming . Research on Gene Knockout and Function of FgPDE1 in Fusarium graminearum. China Biotechnology, 2015, 35(8): 59-67.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150809 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I8/59

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