双亲灭活原生质体电诱导融合法选育高漆酶活性菌株

Screening for Strain with High Laccase Activity by Protoplast Electrofusion of Inactivated Parental Strains

以杏鲍菇和平菇为亲本菌株,采用双亲灭活原生质体融合技术选育高漆酶活性且遗传稳定的融合菌株。结果表明:杏鲍菇原生质体采用紫外灭活,在30 W紫外灯、照射距离30 cm条件下处理20 min,灭活率达100%。平菇原生质体采用热灭活,65℃处理30 min,灭活率达100%。两菌株按1∶1混合,施加1 MHz、250 V/cm的成串脉冲电压,5.5 kV/cm的融合脉冲电压的电场诱导下使其电融合,原生质体成对率达到70%,融合率为3.2×10-3。从147株融合菌株中筛选出1株高漆酶活性菌株F49,其漆酶和TTC-脱氢酶酶活比双亲分别提高20.6%和25.3%,遗传稳定性分别达到92.0%和92.7%以上。从菌落特征、菌丝形态、菌丝生长量、拮抗试验等方面对融合子进行鉴定,证实为具有双亲性状的融合株。

With Pleurous eryngii and Pleurotus ostreatus as parents, the strain with high laccase activity and genetic stability was screened by protoplast elecrofusion of the inactivated parental strains in this paper. With UV method, Pleurous eryngii was handled at 30 W,irradiation distance of 30 cm for 20 minutes, and the lethality rate was 100% ; with the method of heat inactivation, Pleurotus ostreatus was handled at 65℃ for 30 minutes, and the lethality rate was also 100%. The strains of Pleurous eryngii and Pleurotus ostreatus were mixed by 1 ： 1 and electrofused in the alternating electric field with frequency of 1 MHz, intensity of 250 V/cm and fusion pulse intensity of 5.5 kV/cm. The pairing rate of protoplasts arrived at 70% and the fusion rate was 3.2 × 10 ^-3. One strain with characters of both parents was picked out from 147 fusion strains and the enzyme activity of laccase and TTC - dehydrogenase increased by 20.6% and 25.3% respectively than its parents while their heredity stability arrived at 92.0% and 92.7%. The fusant was identified based on colony character, morphological state, growth rate and antagonistic effect, then the screened strain was confirmed with the characters of parents.