β-甘露聚糖酶基因重组整合组成型表达载体的构建  

CONSTRUCTION OF RECOMBINANT CONSTITUTIVE EXPRESSION VECTOR FOR β-MANNANASE GENE

作　　者：包晓兰[1] 王和平[1] 包力高[1] 周平[1] 蔺日胜[1] 赵丽霞[1] 

机构地区：[1]内蒙古农业大学生物工程学院,呼和浩特010010

出　　处：《内蒙古农业大学学报（自然科学版）》2007年第4期137-142,184,共7页Journal of Inner Mongolia Agricultural University(Natural Science Edition)

基　　金：国家自然科学基金项目(39870558)

摘　　要：应用PCR方法从P.pastorisGS115染色体中扩增了GAP启动子(PGAP),大小为500bp左右,以其取代诱导型表达载体pPIC9K上的AOX1启动子(PAOX1),构建了组成型表达载体pGAP;再从野生型酵母菌JSY4染色体中扩增出25S rDNA保守序列,大小为1 700bp左右,以其取代组成型表达pGAP上的组氨酸脱氢酶基因(H IS4)片断,构建了整合组成型表达载体pGAP-rDNA。再双酶切重组质粒pT002M anⅠ得编码β-甘露聚糖酶的ManⅠ基因片断插入pGAP-rDNA的多克隆位点(MCS),获得含有目的基因ManⅠ的重组整合组成型表达载体pGAP-rDNA-ManⅠ。CaC l2法转化感受态大肠杆菌DH5α,对阳性克隆进行质粒PCR鉴定和单、双酶切鉴定。结果表明:成功构建了重组组成型表达载体pGAP-rDNA-ManⅠ,为下一步野生型酵母菌JSY4中β-甘露聚糖酶的组成型表达打下基础。The GAP gene promoter（ PGAP ） about 500 bp was amplified from P. pastoris GS115 and used to replace the AOX1 promoter（ PAOX1 ） on pPIC9K resulting in constitutive expression vector pCAP. The 25S rDNA sequence which is about 1700bp was amplified from wild JSY4 strain used to replace the HISS sequence on the pGAP resulting in integration constitutive expression vector pGAP- rDNA. A 1200bp DNA fragment encoding the β-Mannanase called Man I was cutted from recombinant plasmid pT002-Man I . The Man I was cloned into the MCS of pGAP-rDNA and its recombinant integration constitutive expression vector pGAP - rDNA - Man I was verified by PCR and restriction enzyme digestion. The successful construction of recombinant integration constitutive expression vector on Man I provides a basis for luther work to constitutive express β-Mannanase in wild JSY4 strain.

关 键 词：Β-甘露聚糖酶 组成型表达 GAP启动子 25SrDNA 

分 类 号：Q556.2[生物学—生物化学] Q784