摘要
目的克隆贵阳腐霉、Pr2蛋白酶的cDNA片段,改造贵阳腐霉基因。方法用几丁质诱导培养基培养贵阳腐霉24 h,然后抽提菌丝的总RNA,反转录合成cDNA第1链,根据Pr2蛋白酶的氨基酸保守序列设计合成简并引物,以cDNA第1链为模板,采用降落PCR技术扩增,将扩增的产物纯化、连入pGEM-T载体,转化大肠杆菌DH5α,送阳性克隆子测序。结果所获片段中,1个215 bp的cDNA片段与trypsin样蛋白酶家族基因有较高的相似性,其中与亚洲玉米螟的类胰蛋白酶基因相似性最大(52.8%)。结论这一DNA片段可能是贵阳腐霉Pr2蛋白酶的1条cDNA片段,更进一步的工作是设法获得全长cDNA片段,并加以证实。
Objective To clone a cDNA fragment of Pr2, Pythium guiyangense, and to transform Pythium guiyangense. Methods Mycelia of the fungus were cultured in 1% chitin suspension for 24 hours, and then the total RNA were isolated, the first strand cDNA were synthesized and amplified cDNA with a pair of degenerated primers basing on the highly conservative region of Pr2, the purifying the amplification product were inserted into the pGEM-T vector,and the E. coli DHSct were transformed, cloned and sequenced. Results One of the obtained fragments, a cDNA of 215 bp, showed high homology to the trypsin-like, especially to the Ostrinia fumacalis（52.8% ）. Conclusion The fragment might be a part of a novel Pr2 cDNA of Pythium guiyangense, and the further work should be done to identify the full length of Pr2 cDNA and prove it.
出处
《广西医学》
CAS
2011年第12期1554-1556,共3页
Guangxi Medical Journal
基金
广西教育科学研究基金资助项目(200810MS026)
柳州医学高等专科学校硕士研究生科研启动项目(2007Y01)
