大豆疫霉菌原生质体制备及再生菌株的生物学性状

Protoplast preparation of Phytophthora sojae and biological characterization of regenerated cells

为了建立聚乙二醇(PEG)介导的大豆疫霉菌的遗传转化系统,对大豆疫霉菌原生质体的制备条件及再生菌株的生物学性状进行了研究。结果表明:D riselase以及D riselase和Lysing酶的混合液均能有效地裂解菌株Pmg 2-3的细胞壁并获得原生质体;其中混合酶液的裂解效果优于D riselase单一酶液,当混合酶液中两个酶的浓度均为15 mg.mL-1时,在30℃消化3 h可产生4×106mL-1的原生质体。所制备的原生质体经细胞核染料4,6-d iam id ino-2-phenyl-indole(DAPI)染色后发现,大型均一的原生质体内含有细胞核;原生质体在再生培养基上再生率达1.0%,再生菌株在利马豆培养基上菌落形态呈白色、圆形、毛毡状,其生长速率、致病性均与亲本菌株保持一致。

To establish transformation system of Phytophthora sojae, we prepared protoplasts of the well-studied strain Pmg 2-3 of the oomycete and investigated biological characters of regenerated strains. Protoplasts of 4 ×10^6 mL^- 1 of osmotica were produced by digesting the cell wall for 3 h at 30℃ with 15 mg·mL^- 1 solution of Driselase and Lysing enzyme, which provided better effect than Driselase used singly. Nuclei of the generated protoplasts were observed by fluorescence microscope. Nuclei were found in the fully developed and uniform protoplasts. Regeneration was made at a rate of 1.0% in the medium applied with 1 mol· L^-1mannitol. Regenerated strains that formed gray, orbicular, and fehy colonies grew on the medium and showed pathogenicity to the host plant similarly as did their parent.