大豆疫霉多聚半乳糖醛酸酶pspg1基因毕赤酵母表达载体的构建

[目的]构建大豆疫霉多聚半乳糖醛酸酶pspg1基因毕赤酵母表达载体。[方法]利用PCR方法从大豆疫霉菌cDNA中扩增多聚半乳糖醛酸酶pspg1基因。用限制性内切酶EcoRI和Not1分别酶切此基因和毕赤酵母表达载体pPIC9K，然后用T4连接酶将目的基因克隆到pPIC9K载体中构建此载体。[结果]扩增的大豆疫霉菌pspgl基因成熟肽片段大小为1250bp。用基因特异性g}物扩增阳性重组子，可得到大小为1200bp的片段。而用载体特异性引物扩增阳性重组子，可得到大小为1200和1700bp两条带，多出来的400bp恰好符合载体部分的大小。将此特异性片段PCR扩增回收，序列测定发现大豆疫霉pspg1基因已经成功地插入到表达载体中。[结论]该研究为进一步研究大豆疫霉菌pspg1基因在毕赤酵母中高效表达奠定了基础。

Construction of Expression Vector of Polygalacturonase Gene pspg1 from Phytophthora sojae

[Objective] The aim was to construct Pichia expression vector of polygalacturonase gene pspg1 from Phytophthora sojae.[Method] The polygalacturonase gene pspg1 was amplified from P.sojae cDNA by PCR, then the polygalacturonase gene pspg1 and expression vector pPIC9K were cut by restriction enzymes of EcoRⅠand NotⅠ, resp.and then the target gene fragment was cloned into the Pichia expression vector pPIC9K through T4 ligase to construct the vector.[Result] The length of mature peptide fragment of polygalacturonase gene pspg1 from P.sojae was 1 250 bp.Using gene specific primers amplifying positive recombinant, a fragment of 1 200 bp was obtained.While vector specific primers amplifying positive recombinant, 2 fragments of 1 200 and 1 700 bp were obtained, and the surplus 400 bp just accorded with the length of vector fragment.The specific fragment was amplified by PCR, then recovered and then sequenced.The sequencing result showed that the pspg1 gene from P.sojae was inserted into expression vector successfully.[Conclusion] The research laid the foundation for further study on the high effective expression of polygalacturonase gene pspg1 in Pichia.