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蔓枯病是当前危害瓜类的主要病害,但是蔓枯病菌(Didymella bryoniae)病原学研究还非常落后,关于该菌功能基因的研究报道还较少。为了建立聚乙二醇(polyethylene glycol,PEG)介导的甜瓜蔓枯病菌原生质体遗传转化体系,本研究利用带有潮霉素B磷酸转移酶(hph)基因的质粒pSGate1为载体,通过PEG(分子量3350)介导的融合法转化蔓枯病菌株ZJDB32。将病菌分生孢子于PDB培养液(200g/L马铃薯煎汁,20g/L葡萄糖)内震荡培养20h,然后收集产生的幼嫩菌丝,在10mg/mL lysing enzyme+5mg/mL driselase酶液内28℃酶解3h,每克湿菌丝能产生3.8×107个原生质体。PEG介导融合转化pSGate1至D.bryoniae原生质体,每毫克ZJDB32的DNA转化效率最高能得到1230个转化子。结果表明,20个代表性的ZJDB32的转化子继代4次后其潮霉素抗性、生长速率、产孢量和致病性与野生型都没有明显变异,这些转化子可以进一步用于相关功能研究。因此,该转化体系的建立为甜瓜蔓枯病菌功能基因的深入研究提供了基础资料。
Viburnum is the main disease currently threatening melons, but the etiology of Didymella bryoniae is still very backward. There are few reports on the functional genes of this strain. In order to establish the genetic transformation system of protoplast of meliloti (Glycyrrhiza uralensis) mediated by polyethylene glycol (PEG), the plasmid pSGate1 with hygromycin B phosphotransferase (hph) Molecular weight 3350) mediated fusion method to transform the Bacillus strain ZJDB32. The conidia of the bacteria were cultured in PDB broth (200g / L potato dextrin, 20g / L glucose) for 20h with shaking, then the young mycelium was collected and incubated in 10mg / mL lysing enzyme + 5mg / mL driselase solution 28 ℃ digestion 3h, each gram of wet mycelium can produce 3.8 × 107 protoplasts. PEG-mediated fusion transformation of pSGate1 to D. bryoniae protoplasts resulted in a DNA conversion efficiency of up to 1230 transformants per mg of ZJDB32. The results showed that the 20 representative ZJDB32 transformants showed no significant variation in hygromycin resistance, growth rate, sporulation and pathogenicity compared with the wild type after 4 generations of subculture, and these transformants could be further used for correlation Functional research. Therefore, the establishment of this transformation system provided the basic information for the further study of the functional genes of G. meliloti.