棘孢木霉嗜铁素sidA基因敲除及表型分析

Deletion and phenotyping of a siderophore biosynthetic gene sidA in Trichoderma asperellum

明确棘孢木霉菌Trichoderma asperellum嗜铁素合成的关键基因,能够为进一步探索嗜铁素在生防和促生中的作用奠定基础。本研究在对sidA基因进行定位、结构分析和RT-PCR检测的基础上,利用double-jointPCR技术构建基因敲除载体,经聚乙二醇(PEG)介导原生质体转化、潮霉素初筛、PCR和southern blot验证获得突变株,并对其表型进行分析,获得2株性能稳定的敲除突变体?sidA1和?sidA2。与野生型相比,?sidA1和?sidA2的嗜铁素产量在5d时分别下降了38.67%和36.65%;孢子萌发率在12h时分别下降了45.33%和47.47%;产孢量在10d时分别下降了33.01%和41.02%,且突变株在受NaCl、KCl、SDS等胁迫时的抗性较野生型降低。表明sidA基因的缺失降低了嗜铁素产量,抑制了菌体的生长以及对胁迫因子的抗性,sidA基因是影响棘孢木霉嗜铁素产生的关键基因之一。

The key genes involved in siderophore biosynthesis of Trichoderma asperellum,serving the functions of biocontrol and growth promotion of the fungus were identified.The knockout vector was constructed using double-joint PCR on the basis of localization of sidA gene,structural analysis and RT-PCR detection.The protoplasts were transformed by using polyethylene glycol(PEG)and the transformants were selected by using hygromycin and verified by PCR and southern blot.Two knockout mutants,?sidA1 and?sidA2,were obtained and their phenotypes were analyzed.Compared with the wild type,the siderophore yield of?sidA1 and?sidA2 decreased by 38.67%and 36.65%respectively in 5 d after inoculation.The germination rate of conidia decreased by 45.33%and 47.47%in 12 h and the conidial production also decreased by 33.01%and 41.02%in ten days.The mutants showed lower resistance to NaCl,KCl and SDS than the wild type.Deletion of sidA results in reduction of siderophore production and mycelial growth as well as stress resistance,suggesting that sidA might be one of the key genes for siderophore biosynthesis in T.asperellum.