产植酸酶黑曲霉菌株S8的基因扩增与鉴定

Amplification and Identification of Phytase phyA Gene of Aspergillus niger S8

本文主要报道了用 3种不同方法从S8黑曲霉菌丝体中提取基因组DNA ,最适方法是经改进的Vitagene试剂盒法。PCR扩增效果最好的是设计时没有去掉phyA基因内含子的引物 ,获得的特异性PCR产物长约 1.5kb。同时对PCR反应体系进行了优化。对扩增出的目的片段用 3种不同方法进行纯化回收 ,结果是PCRFragmentRe coveryKit法回收效果最好 ,其产量可达到约 10ng/ μL。根据已报道的植酸酶phyA基因序列酶切位点 ,对回收的目的片段进行酶切鉴定 ,该基因能被BamHⅠ、SalⅠ酶切 ,不能被HindⅢ、XbalⅠ、KPnⅠ酶切 ,与已知的phyA基因酶切图谱基本一致 。

The thesis mainly reported that in the three methods used to extract genome DNA from S8 Aspergillus niger mycelium, the best was modified Vitagine Kit. In the two pairs of primers selected by software and one pair of primer reported as control, the best primer was not got rid of the intron of phyA gene and brought KPnⅠand XbalⅠenzyme digestion site,and the length of the PCR product was about 1.5 kb. In the meantime, the system of PCR was optimized. The best result of purifing and recovering the objective fragment was PCR Fragment Recovery Kit in the three varied methods, and the output could attain 10 ng/μL. According to the reports about the enzyme digestion site of the phyA gene, the objective fragment could be digested by BamHⅠ、SalⅠ and not by HindⅢ、XbaⅠ、KPnⅠ. This result was consistent with the known enzyme digestion.