农杆菌介导凝乳酶基因转化黑曲霉载体的构建

Construction of Chymosin Gene Transformation to Aspergillus niger by Agrobacterium-mediated

目的:研究针对黑曲霉中高表达的糖化酶基因(glaA)位点,构建含有潮霉素抗性的农杆菌介导的牛凝乳酶基因转化黑曲霉基因置换载体,并在潮霉素基因两端设计了正向重复序列,使在后续的研究中消除抗性基因成为可能。方法:将glaA上游(Gla5)和下游(Gla3)片段作为同源臂,通过重叠延伸PCR技术连接在Cym基因两端构成GlaA5-Cym-GlaA3(CYM)片段,并在潮霉素基因下游引入与Cym基因下游方向相同的Gla3,通过中间载体获得GlaA5-Cym-GlaA3-hph-Gla3结构。结果:将上述结构克隆至在T-DNA区内只含有多克隆位点的Ti质粒载体pSZA,经过酶切鉴定,成功获得载体pSZH-CYM。

Objective：GlaA can be highly expressed in Asperogillus niger,this study therefore constructed the Asperogillus gene vector transformed by chymosin gene which contained hygromycin resistance and was mediated by Agrobacterium.In order to eliminate resistance gene possibly,the directed repeats were designed for hygromycin gene.Method：Taken upstream gla5 and downstream gla3 as homologous arm,the segment of GlaA5-Cym-GlaA3（CYM） was constructed by overlap extension PCR.Subsequently,structure of GlaA5-Cym-GlaA3-hph-Gla3 was obtained by mediated vector which Gla3 that equals to Cym gene was introduced to hygromycin downstream gene.Result：After the identification of enzyme cleavage,the vector pSZH-CYM was successfully gained by cloning GlaA5-Cym-GlaA3-hph-Gla3 to Ti plasmid vector pSZA which only contained multiple cloning sites in T-DNA region.