苹果树腐烂病菌丝氨酸蛋白酶基因VM1G_08720的功能分析

Functional Analysis of Serine Protease Gene VM1G_08720 in Valsa mali

苹果是我国种植面积最广的温带水果之一,由苹果黑腐皮壳菌(Valsa mali)引起的苹果树腐烂病(Valsa canker of apple)是危害我国苹果树最为严重的病害之一。明确病原菌生长发育、响应环境胁迫及致病作用,解析病原菌的适应性及致病分子机制,有利于提出病害新的防控措施。本研究应用Double-joint PCR以及gap-repair方法获得基因VM1G_08720的缺失突变体和回补菌株。利用十字交叉法和伤口接种等方法,研究了基因VM1G_08720对病菌生长发育和致病性的影响。通过在PDA培养基中分别添加400μg/mL的荧光增白剂、200μg/mL的刚果红、0.01%的SDS三种细胞壁干扰剂和0.5M的NaCl、0.5M山梨醇两种渗透胁迫因子,研究了该基因对病菌细胞壁完整性的维持及渗透胁迫的影响。通过设置不同pH的培养基,研究了基因VM1G_08720在不同pH下对病菌生长发育的影响。研究结果显示,与野生型sdau11-175相比,基因VM1G_08720缺失突变体在菌落颜色、菌丝致密度、生长速率无显著差异,分生孢子器产生数量减少;在含有200μg/mL刚果红、0.01%SDS的培养基上,抑制率显著降低,在含有0.5 M NaCl的培养基上的抑制率显著增加,而在含有400μg/mL荧光增白剂和0.5 M山梨醇的培养基上,无显著差异;在pH为4-10时,生长速率显著降低,而在pH为11-12时,无显著性差异;在苹果果实和苹果枝条上的病斑大小无显著性差异,回补菌株均恢复到野生型水平。表明基因VM1G_08720在病菌分生孢子器的产生、酸碱度的响应、维持细胞壁完整性以及渗透胁迫方面发挥重要作用。

Apple is one of the most widely planted temperate fruits in China.Valsa canker of apple caused by Valsa mali is one of the most serious diseases of apple tree in China.Therefore,strengthen the scientific prevention and control of Valsa mali,and clarify the adaptability and pathogenic effect of pathogens to the environment is the key problem for diease.In this paper,Double-joint PCR and gap-repair methods were used to obtain deletion mutants and complement strains of the gene VM1G_08720.The effect of the gene VM1G_08720 on nutritional growth and pathogenicity was investigated used the cross-over method and the wound inoculation.The role on the maintenance of cell wall integrity and the regulation of osmotic stress of the gene VM1G_08720 were investigated by adding 400μg/mL calcofluor white,200μg/mL Congo red,0.01% SDS,and 0.5 M NaCl,0.5 M sorbitol.The effect of the gene VM1G_08720 on the hyphal growth of the pathogen under different pH was investigated by setting the medium for different gradient.The results showed that the gene VM1G_08720 deletion mutant was no significant differences in colony color,mycelium density,growth rate compared with wild-type sdau11-175.The numbers of pycnidial production were reduced.The inhibition rate was significantly reduced on medium containing 200μg/mL of congo red and 0.01% SDS,and the inhibition increased significantly on medium containing 0.5 M NaCl,and there was no significant difference in the culture medium containing 400μg/mL calcofluor white and 0.5 M sorbitol.Growth rate was significantly reduced at pH 4-10,with no significant difference at pH 11-12.There was no significant difference in diseased spot sizes between mutant and wild strain on apple fruit and apple branches.All the complement strains were recovered to wild-type levels.It indicates that the gene VM1G_08720 plays an important role in pycnidial production,pH response,maintenance of cell wall integrity,and osmotic stress.