玉蜀黍赤霉异核体的遗传变异及抗药性进化规律研究

In recent years the incidence and severity of Fusarium head blight (FHB) on wheat, infected by Gibberella zeae (Schwein.) Petch, anamorph Fusarium graminearum Schwabe, has increased. At present, however, the inheritance and allele expression of heterokaryon in G. zeae and other homothallic fungi is unclear. In this study, heterokaryon will be obtained by hyphal fusion, crossed two strains which histone tagged with GFP and RFP, respectively. The dynamic state of heterogeneous nuclei in hyphal routine transferring and the nuclear distribution in conidia, ascus and ascospore will be studied by observation the characteristics of nuclei fluorescence. The results will indicate the stability of heterogeneous nuclei of homothallic fungus in vegetative growth and the inheritance of heterokaryon in asexual and sexual reproduction and parasexual cycle. Meanwhile, two strains of G. zeae which β2-tubulin tagged with GFP and RFP, respectively, will be crossed under laboratory conditions and heterokaryon will be obtained by hyphal fusion. The genetic expression of allele in heterokaryon of this homothallic fungus will be determined by observation the characteristics of microtubules fluorescence. By the same thinking and system of research, the mechanisms of carbendazim resistance in heterokaryon obtained by hyphal fusion, crossed two strains of G. zeae, wild type strain and field carbendazim-resistant strain which β2-tubulin tagged with GFP and RFP, respectively. These results will provide a theoretical foundation to study the pathogenicity variation and fungicide-resistance inheritance and evolution of homothallic phytopathogenic fungi and the genetic breeding of beneficial fungi.

玉蜀黍赤霉引起的赤霉病是小麦上的重要病害。该菌为同宗配合真菌，通过菌丝融合产生异核体的遗传变异、等位基因的表达及抗药性进化等方面目前仍不清楚。本研究用GFP、RFP分别标记该菌MBC敏感和抗性菌株的H2B-His和H2A.Z-His，荧光观察两菌株产生的异核体内异质核在连续转代、分生孢子及子囊和子囊孢子中的分布及杀菌剂处理下的变化，从而阐明异核体内异质核的稳定性、抗药性进化、在繁殖（无性、有性及准性）过程中的的遗传变异规律。同时用GFP、RFP分别标记敏感和抗性菌株的β2-tubulin，观察两菌株产生的异核体内微管的荧光表型，阐明异核体中等位基因的表达规律，明确抗性与敏感菌株菌丝融合产生异核体对药剂表现为抗性的机理。以上研究结果将阐明玉蜀黍赤霉异核体的遗传变异及其等位基因的表达规律，为研究同宗配合病原真菌的致病性变异、抗药性遗传进化、有益真菌的遗传育种等方面奠定理论基础。

玉蜀黍赤霉菌引起的赤霉病是危害麦类作物的重要病害，化学防治是目前控制该病害的主要措施，但抗药性的产生是该措施的主要缺陷。玉蜀黍赤霉为同宗配合真菌，在自然条件下极易通过菌丝融合产生异核体，但通过异核体导致致病性变异、抗药性进化等遗传变异及等位基因表达等方面的规律目前仍不清晰。本研究利用绿色荧光蛋白GFP、红色荧光蛋白RFP分别标记玉蜀黍赤霉多菌灵抗性和敏感菌株的组蛋白H2A.Z、H2B及β2-微管蛋白，分别将荧光标记菌株通过原生质体融合获得异核体。通过测定荧光标记菌株及其异核体菌株的生物学特性，结果表明荧光标记菌株的菌丝生长速率、菌丝及菌落形态、分生孢子形态及无性产孢能力等方面与出发菌株无显著差异，而异核体菌株的菌丝生长能力和无性产孢能力强于亲本菌株；通过显微观察异核体菌株的荧光表型，结果表明玉蜀黍赤霉异核体菌株中等位基因可以同时表达，但多菌灵抗性菌株和敏感菌株原生质体融合获得的异核体菌株中多菌灵抗性细胞核出现频率远高于敏感细胞核，说明多菌灵抗性异核体中β2-微管蛋白基因多菌灵抗性突变能抑制野生型等位基因的表达，多菌灵抗性异核体中抗性等位基因在药剂胁迫下的其抗性比例增加；通过测定异核体菌株连续转代后的抗药性表型，结果表明玉蜀黍赤霉多菌灵抗性异核体中异质核能稳定遗传；通过不同抗生素及多菌灵组合筛选大量的异核体菌株的分生孢子，结果未能获得遗传重组的后代，从而说明玉蜀黍赤霉多菌灵抗性异核体通过无性繁殖发生变异（准性生殖）的概率较低。以上研究结果初步阐明玉蜀黍赤霉异核体的遗传变异及其等位基因的表达规律，为研究同宗配合植物病原真菌的致病性变异、抗药性遗传进化、有益真菌的遗传育种等方面奠定了一定的理论基础。