甘蔗鞭孢堆黑粉菌原生质体转化DNA双片段的基因敲除方法的建立

A gene knockout method based on protoplast transformation with two PCR fragments in Sporisorium scitamineum

由甘蔗鞭孢堆黑粉菌Sporisoriumscitamineum引起的甘蔗黑穗病是甘蔗生产的主要病害,严重影响了甘蔗的产量和品质,然而其分子致病机制报道很少,极大地影响了有效防控技术的开发。为了提高该病菌基因功能的研究效率,本研究以甘蔗鞭孢堆黑粉菌有性配合两个关键基因SsGPA3和SsPRF1为目标,建立了基于DNA双片段的甘蔗鞭孢堆黑粉菌原生质体转化基因敲除技术体系。结果表明甘蔗鞭孢堆黑粉菌单倍体细胞生长期在OD600约为0.7,28℃下10mg/mL细胞壁裂解酶作用30min获得的原生质体产率最高,再生率最优;使用线性DNA双片段转化获得阳性转化子的成功率达90%以上,显著高于线性DNA单片段和质粒。本方法的建立将极大地提高甘蔗鞭孢堆黑粉菌的基因敲除效率,同时也为其他真菌的基因敲除提供借鉴。

Sugarcane smut caused by Sporisorium scitamineum results in a great loss of yield as well as quality of sugarcane.The molecular mechanism of its pathogenesis remains largely elusive,hindering from the development of new method of disease control.Here,a concise method for gene knockout in Sporisorium scitamineum,based on the protoplast transformation with a pair of two PCR fragments was developed.Employing two key genes SsGPA3 and SsPRF1 as working model,the feasibility and efficiency of this method were evaluated.The results unraveled that when the haploid cells at exponential growth stage(OD600 about 0.7)were treated with 10 mg/mL cell wall lyase for 30 min at 28℃,a maximium of protoplast yield and the highest regeneration rate were achieved.Co-transformation of a pair of two linearized DNA fragments appeared more efficient than the transformation of one linearized DNA single fragment or plasimid.Successful establishment of this gene knockout strategy provides an important tool to investigate the gene function of Sporisorium scitamineum,and it could also be applied to knockout specific genes in other fungal pathogens.