苹果树腐烂病菌染色质重塑因子Vmles4的功能研究

染色质重塑因子INO80是一类由多亚基构成的遗传学调控因子，调控多种DNA代谢，在基因的表达中发挥着重要的调控功能。但其在苹果树腐烂病菌（Valsa mali）中是否存在及其生物学功能并不清楚，为此，本研究从病菌基因组中分析鉴定到INO80的一个亚基基因Vmles4，利用qRT-PCR技术分析了Vmles4在病菌侵染过程中的表达模式，利用Double-joint PCR技术和PEG介导的原生质体转化方法进行了基因敲除，然后对3个突变体的营养生长及致病力进行测定和分析，并对病菌的非核糖体肽合成酶基因VmNRPS12及VmNRPS14在突变体中的表达进行定量分析。结果表明：Vmles4在侵染初期的表达显著上调，接种后6 h上调表达6.2倍。敲除突变体的生长速率平均降低28%且菌丝生长稀疏，在富士苹果品种（Malus domestic cv. Fuji）叶片和枝条上的致病力分别降低到22.5%和27.5%，VmNRPS12及VmNRPS14基因在Vmles4突变体侵染苹果枝条24 h后的表达量分别下调86.5%和50%；综上所述，Vmles4正调控腐烂病菌的营养生长、致病力以及次级代谢合成酶基因VmNRPS12和VmNRPS14的表达。

Functional characterization of a chromatin remodeling factor Vmles4 in Valsa mali

Chromatin remodeling factor INO80 is a kind of genetic regulatory factors composed of multiple subunits that regulate multiple DNA metabolism and play an important regulatory role in gene expression. But, it is not clear whether it exists in Valsa mali and its biological functions. Therefore, a subunit gene Vmles4 of INO80 was analyzed from the fungal genome in this study. The expression patterns of Vmles4 was analyzed by qRT-PCR technology during the infection process. Using Double\|joint PCR techniques and PEG mediated protoplast transformation methods for gene knock out. Then, the vegetative growth and pathogenicity of 3 mutants were measured and analyzed. The expressions of non\|ribosomal peptide synthase genes VmNRPS12 and VmNRPS14 in mutants were analyzed quantitatively. The results showed that the expression of Vmles4 was up\|regulated significantly in the early stage of infection, and 6.2 times in 6h after inoculation. Growth rate of knockout mutants decreased by 28% on average and mycelium growth was sparse, and the pathogenicity in leaves and twigs of Fuji apple varieties (Malus domestic cv.Fuji) decreased to 22.5% and 27.5%, respectively. The expression of VmNRPS12 and VmNRPS14 genes was down\|regulated by 86.5% and 50%, respectively after 24 h of infection of twigs by Vmles4 mutant. In conclusion, Vmles4 is regulating the vegetative growth, pathogenicity, and the expression of secondary metabolic synthase gene VmNRPS12 and VmNRPS14 of Valsa mali.