尖孢镰刀菌亚麻专化型FolFTL1基因敲除及功能分析

Knockout and Function Analysis of FolFTL1 Gene in Fusarium oxysporum f.sp.lini

为鉴定尖孢镰刀菌FolFTL1基因的功能,本研究采用Split Marker技术,构建了含有潮霉素抗性基因(hph)的基因缺失盒。通过聚乙二醇(PEG)介导法转化野生型hm原生质体,获得了FolFTL1基因缺失突变体ΔFolFTL1。与野生型hm相比,尽管ΔFolFTL1分生孢子形态和大小没有不同,但产量显著减少,各菌株平均孢子产量分别为hm:2.45×10^(7)个/mL、ecFolFTL1:1.63×10^(7)个/m L、ΔFolFTL1:0.08×10^(7)个/mL,表明FolFTL1基因缺失影响了孢子发生。形态学观察发现,敲除突变体的菌落比野生型更致密,并且生长速率显著降低,各菌落生长速率分别为hm:1.78 cm/d、ecFolFTL1:1.75 cm/d、ΔFolFTL1:0.88 cm/d。亚麻幼苗侵染实验显示,敲除突变体侵染的幼苗生长正常,而野生型和外插入突变体侵染的幼苗严重枯萎,表明FolFTL1基因的缺失降低了尖孢镰刀菌的致病力。外插入突变体是为了进一步证明FolFTL1基因功能的改变是由基因敲除导致。本研究结果显示FolFTL1基因是尖孢镰刀菌亚麻专化型分生孢子发生、菌丝营养生长和植物侵染相关基因。

To identify the function of FolFTL1 gene in Fusarium oxysporum f.sp.lini,the deletion cassette containing hygromycin resistance gene(hph)was constructed by using split marker strategy and the deletion mutant of FolFTL1,ΔFolFTL1,was obtained through transformation of the deletion cassette into protoplasts of wild type hm mediated by polyethylene glycol(PEG).Compared with the wild type,the conidia produced by FolFTL1 deletion,ΔFolFTL1,was significantly reduced,although the morphology and size of the conidium were not different from the wild type hm.The average conidia yield of each strain was 0.08×10^(7)sporolium/mL,1.63×10^(7)sporolium/mL,2.45×10^(7)sporolium/mL forΔFolFTL1,ecFolFTL1,hm respectively.Indicating that the deletion FolFTL1 gene obviously affected on its conidiation.The morphological observation found that the colony of the deletion mutant was denser than that of the wild type and the growth rate of the deletion mutant was significantly reduced.The growth rate of each colony was 0.88 cm/d,1.75 cm/d,1.78 cm/d forΔFolFTL1,ecFolFTL1,hm respectively.The infection experiment showed that the seedlings infected by the deletion mutants grew normal while the seedlings infected by the wild type and ectopic transformants withered seriously,suggesting that virulence of the deletion FolFTL1 gene reduced the virulence of the fungus.The purpose of the ectopic transformants is to further prove that the functional change of FolFTL1 gene is caused by gene knockout.The results of this research demonstrated that FolFTL1gene played a pivotal role in conidiogenesis,vegetative growth and pathogenicity of the F.oxysporum f.sp.lini.