甘薯黑斑病菌木聚糖酶xylanase 1基因敲除体系的建立

Construction of gene knockout system for C.fimbriata xylanase 1 gene

由甘薯长喙壳菌(Ceratocystis fimbriata Ellis.et Halsted)引起的黑斑病是严重危害甘薯的病害之一,但目前甘薯黑斑病菌致病的分子机制尚不明确.本研究以徐州农科院甘薯病虫害防控研究室田间分离的黑斑病菌为材料,以广泛存在于植物病原真菌中的木聚糖酶基因xylanase 1为例,参考其他常见植物病原真菌的基因敲除方法,优化原生质体制备步骤、基因融合片段合成方法及PEG介导的转化过程,结果发现,黑斑病菌菌丝体在质量分数为2%的纤维素酶(Cellulase)、2%的溶壁酶(Lysing Enzyme)、2%的崩溃酶(Driselase)的混合酶液中震荡酶解4h,即可获得转化所需的高质量原生质体.通过2轮融合PCR合成Xyl-Up::HYG::Xyl-Down片段,借助改良的PEG转化技术,成功地敲除黑斑病菌中的木聚糖酶基因xylanase 1.该敲除体系的建立,为深入研究甘薯黑斑病菌分子致病机制提供了技术保证.

Sweetpotato black spot caused by Ceratocystis fimbriata Ellis.et Halsted is one of the sweetpotato serious diseases.However,the molecular mechanism of this disease is still unclear.In this study,xylanase 1,a xylanase gene widely distributed in plant pathogenic fungi,was taken as an example,and the protoplast preparation steps,gene fusion fragment synthesis method and PEG transformation process were optimized according to common gene knockout methods of other plant pathogenic fungi.The results showed that the high quality protoplasts required for transformation could be obtained by splitting the mycelium with in the mixed enzyme solution the mass fraction of 2%Cellulase,2%Lysing Enzyme and 2%Driselase for 4 h.Furthermore,Xyl-Up::HYG::Xyl-Down fragment was synthesized by two rounds of fusion PCR assay.Totally,xylanase gene was successfully knocked out by improved PEG transformation technology.The establishment of this knockout system could provide a technical guarantee for further study on the molecular pathogenesis of sweetpotato black spot.