基于无缝克隆方法构建毛木耳多功能纤维素酶基因农杆菌转化载体

Construction of Plasmid Contributing to Agrobacterium-mediated Transformation of Multi-Functional Cellulase Gene in Auricularia Polytricha Using Seamless Cloning

【目的】构建毛木耳农杆菌介导转化双元表达载体,为毛木耳后续多功能纤维素酶基因Mfc转化研究奠定基础。【方法】通过EcoR I酶切p PK2质粒并回收大片段,应用实验设计引物分别对扩增毛木耳Gpd启动子、Mfcsg基因、intron、Hpt基因,通过无缝克隆方法,将p PK2质粒大片段与表达序列同源组装,分别构建农杆菌转化载体pAKMFC、pAKIMFC。【结果】测序验证发现各个元件都已在改造后的载体中进行了正确组装,符合实验预期。【结论】本实验通过毛木耳Gpd启动子、Mfcsg、Hpt潮霉素B抗性基因和终止子组装,构建形成了能够应用于毛木耳农杆菌介导转化的多功能纤维素酶基因双元表达质粒p AKMFC和p AKIMFC,为多功能纤维素酶Mfcsg基因转化增强毛木耳纤维素降解能力奠定了基础。

[ Objective ] The expression binatT vector was constructed to provide a basis for the study of Agrobacterium-mediated transformation of nmlti-functional cellulase gene （Mfc） in Auricularia polytricha. [ Method] After digesting with EcoR I, the large backbone of pPK2 was purified. A. polytricha Gpd promoter, Mjcsg gene, intron, Hpt gene were amplified, by seamless cloning, the large backbone of pPK2 with expression sequence was homologily assembled to construct the vectors of pAKMFC, pAKIMFC. [ Result] Sequencing results validated the components of Gpd pronmter, Mfcsg, intron and Hpt in line with expectations. [ Conclusion] By seamless cloning ofA. polytricha Gpd promoter, Mfcsg gene, intron and Hpt gene, the expression binary vectors of pAKMFC, pAKIMFC were constructed to pave the way for subsequent transgenic study of Mjcsg gene in A. polytricha.