摘要：

通过对尖孢镰刀菌亚麻专化型Folcdc15基因敲除及敲除突变体互补试验,探究该基因在尖孢镰刀菌亚麻专化型中的功能。对Folcdc15基因进行DNA测序后,使用Split-Marker技术,构建含有潮霉素抗性基因(hph)缺失盒。通过PEG介导法转入野生型原生质体,在含有潮霉素的选择培养基上进行转化子筛选,通过PCR正负筛选确定敲除突变体。利用pZWH1载体构建含有 Folcdc15的互补载体pZLY1,将其转入敲除突变体中进行互补测验。与野生型hm相比,敲除突变体ko1菌落生长速率下降了73%,菌丝形态发生了显著改变,菌丝变短且分叉变多,分生孢子隔膜数量增多长度增加,分生孢子数量显著减少。亚麻侵染试验表明,ko1的致病力显著降低。通过互补测验发现,回复突变株ca1恢复了Folcdc15基因缺失带来的分生孢子产量及形态、菌落生长和形态及致病力的缺陷。Folcdc15参与调控尖孢镰刀菌亚麻专化型的分生孢子发生及菌丝生长。

关键词: 尖孢镰刀菌亚麻专化型, 亚麻枯萎病, Split-Marker, Folcdc15, 基因功能, 致病性

Abstract:

To identify the Folcdc15 gene and its function in Fusarium oxysporum f.sp.lini through disruption of the gene and complementation test.After DNA sequencing of Folcdc15 gene,Split-Marker strategy was used to construct a deletion cassette containing hygromycin resistance gene(hph),which was transformed into protoplast of wild-type hm by PEG mediated method.Transformants were collected in the medium containing hygromycin and Folcdc15 deleted mutants were screened through PCR using positive and negative primers.The complementary vector pZLY1 containing Folcdc15 was constructed by using pZWH1 vector and transformed into the knockout mutant for complementation test.Compared with wild-type hm,the growth rate of deletion mutant ko1 decreased by 73%,and the mycelia morphology changed significantly and became shorter and more bifurcated.The number of conidia decreased significantly,and the septum and length of conidia increased.The infection experiment in flax showed that the virulence of deletion mutant ko1 was significantly reduced.The complementation test revealed that the revertant ca1 recovered phenotype of wild-type hm from the defects in conidiation,morphology of conidium,growth rate of colony,morphology of colony and pathogenicity caused by the Folcdc15 gene deletion.Folcdc15 is involved in the regulation of conidiogenesis and hyphal growth in F.oxysporum f.sp.lini.

Key words: Fusarium oxysporum f.sp.lini, Flax blight, Split-Marker, Folcdc15, Gene function, Pathogenicity