Mabinlin基因分离与克隆中T－载体技术的应用

APPLICATION OF T-VECTOR TECHNIOUE IN ISOLATION AND CLONING OF MABINLIN GENE

从采自云南屏边的马槟榔（ＣａｐｐａｒｉｓｍａｓａｉｋａｉＬｅｖｌ）叶片中分离提纯到ＤＮＡ，依据植物超甜蛋白Ｍａｂｉｎｌｉｎ的氨基酸序列，回译出Ｍａｂｉｎｌｉｎ的核苷酸序列，用简并密码子优先使用的原则和计算机ＤＮＡ系统分析，设计基因的上、下游引物，通过快速扩增得到Ｍａｂｉｎｌｉｎ基因的ＰＣＲ产物。用平末端内切酶ＥｃｏＲＶ和Ｓｍａｌ分别消化质粒Ｂｌｕｅｓｅｒｉｐｔ和Ｐｖｚ－１在标准缓冲液条件下，使用２ｍｍｏｌ／ＬｄＴＴＰ和Ｔａｑ多聚酶，７０℃温育２ｈ，反应中缺乏ｄＴＴＰ以外任何其它核苷酸均可导致线状载体３’端单－Ｔ的增加，此Ｔ－载体可用于Ｍａｂｉｎｌｉｎ基因的分离与克隆。其克隆效率比将ＰＣＲ扩增产物克隆到平末端载体上的效率高出１００倍以上。

The DNA which contains Mabinlin a super sweet-tasting protein gene,was isolated and purified from the leaves of Capparis masaikai Levl.in Pingbian County,Yunnan Province,and the PCR products of Mabinlin gene were obtained through PCR amplification Bluescript andPvz-1 plasmid were digested with blunt-ended restriction endonuclease EcoRV and Smal separate-lv and incubated with Taq polyrnerase(1 unitl/μg plasmid/20μl volume)using standard bufferconditions(50 mmol/LKCI,10 mmol/L Tris pH 8.3,1.5 mmol/L。 MgCl2 and 200μg/mL 。 BSA)in the presence of 2 mmol/L dTTP for 2 hours at 70, The。bsence of any other nucleotides inthe reaction resuIted in an addition of a single thymidine at the 3’end of each fragment, and T-Vector constructed in this way can be used for isolation and cloning of Mabinlin gene。Our studyshowed this procedure gave at least one hundred fold cloning efficiency as compared to cloning thePCR product into a blunt-ended cut vector。