稻瘟菌糖原合成酶激酶MoGSK3功能探究

【目的】观测稻瘟菌（Magnaporthe oryzae）糖原合成酶激酶基因MoGSK3敲除突变体表型，明确MoGSK3在稻瘟菌中的潜在生物学功能，为挖掘防治稻瘟菌新型药剂的潜在靶标提供参考。【方法】基于同源重组原理，用split-PCR方法获得稻瘟菌MoGSK3敲除突变体菌株，将MoGSK3基因克隆到pFL2载体上得到MoGSK3-C融合载体，并将其通过PEG介导的原生质体转化法导入MoGSK3突变体中得到回补菌株。培养观察野生型菌株Guy11、突变体菌株G3-9及回补菌株GC-1的菌落形态和生长状况，采用实时荧光定量PCR(qRT-PCR)检测稻瘟菌产孢相关基因的表达量；观察稻瘟菌不同菌株的分生孢子形态，通过附着胞洋葱表皮穿透试验及接种水稻叶片，研究其致病力；通过KI-I-2染色对野生型菌株Guy11及突变体菌株G3-9分生孢子和附着胞中的糖原运输能力进行观测。【结果】稻瘟菌MoGSK3突变体存在多个表型缺陷，与野生型菌株Guy11相比，MoGSK3突变体菌株G3-9菌落直径显著变小，生长缓慢，产孢相关基因表达量下降且分生孢子出现末端伸长畸形的状态。MoGSK3基因缺失还会导致稻瘟菌菌丝末端无法形成正常的分生孢子梗，附着胞无法穿透洋葱表皮形成侵染菌丝，接种划伤水稻叶片也无法形成褐色病斑。对MoGSK3突变体菌株G3-9分生孢子及附着胞进行糖原染色后发现，突变体菌株G3-9在糖原转运能力方面存在明显缺陷。【结论】MoGSK3基因参与稻瘟菌的生长、分生孢子形成及形态建成、侵染、糖原转运等过程，是稻瘟菌重要的毒力因子。

Functions of glycogen synthase kinase MoGsk3 in Magnaporthe oryzea

【Objective】This study observed the phenotype of knockout mutants of glycogen synthase kinase MoGSK3 of Magnaporthe oryzae and clarified potential biological functions of MoGSK3 to provide references for exploring potential targets of novel agents.【Method】Based on the principle of homologous recombination,the MoGSK3 knockout mutant strain was obtained by split-PCR method,and the MoGSK3 gene was cloned into the pFL2 vector to obtain the MoGSK3-C fusion vector,which was then introduced into the MoGSK3 mutant by PEG-mediated protoplast transformation to obtain the replacement strain. The colony morphology and growth status of wild-type strain Guy11 and the mutant strain and replacement strain were observed and the expression of spore-producing related genes of Magnaporthe oryzae was detected using real-time fluorescence quantitative PCR.The conidial morphology was observed using microscopy and its pathogenicity was studied by attachment cell onion epidermal penetration test and inoculation of rice leaves.Glycogen in conidia and appressoria of wild type strain Guy11 and mutant strain were measured by KI-I₂ staining.【Result】The MoGSK3 mutant had several phenotypic defects.Compared with the wild-type strain Guy11,colony diameter of MoGSK3 mutant significantly decreased,growth was slow,expression of sporulation-related genes were reduced and the conidiophore showed an elongated end.The deletion of MoGSK3 gene prevented the formation of normal conidial stalks at the end of mycelium,the formation of infecting mycelium by attachment cell from penetrating onion skin,and the formation of brown spots on inoculated rice leaves.Glycogen staining of MoGSK3 mutant conidia and adherent cells revealed that the mutant strain G3-9 was significantly defective in glycogen transport.【Conclusion】The MoGSK3 gene involved in the processes of nutritional growth, conidia formation,infestation and glycogen transport of rice fungus,and it was an important virulence factor of rice fungus.