文献类型： 外文期刊

作者： Liu, Qin ¹ ; Dang, Hui-Jie ¹ ; Wu, Yuan-Hang ¹ ; Li, Min ¹ ; Chen, Yin-Hua ¹ ; Niu, Xiao-Lei ¹ ; Li, Kai-Mian ² ; Luo, Li ¹ ;

作者机构： 1.Hainan Univ, Inst Trop Agr & Forestry, Hainan Key Lab Sustainable Utilizat Trop Bioresou, Haikou 570228, Hainan, Peoples R China

2.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Haikou 570228, Hainan, Peoples R China

关键词： PCR product; Cloning; T-vector; Blunt-end ligation; High efficiency

期刊名称：BMC BIOTECHNOLOGY （ 影响因子：2.563； 五年影响因子：3.292 ）

ISSN： 1472-6750

年卷期： 2018 年 18 卷

页码：

收录情况： SCI

摘要： Background: With the rapid development of sequencing technologies, increasing amount of genomic information has been accumulated. To clone genes for further functional studies in large scale, a cheap, fast and efficient cloning vector is desired. Results: A bifunctional vector pXST has been constructed. The pXST vector harbors a Xcml-ccdB-Xcml cassette and restriction site Smal. Digestion the vector with Xcml generates a single thymidine Cr) overhang at 3' end which facilitates TA cloning, and Smal gives blunt end that enables the blunt-end ligation. Multiple products with various sizes were amplified from cassava genome by PCR and each PCR fragment was separately cloned into a pXST using TA cloning and blunt-end ligation methods. In general, the TA cloning gave higher transformation efficiency than blunt-end ligation for inserts with all different sizes, and the transformation efficiency significantly decreased with increasing size of inserts. The highest transformation efficiency (8.6 x 10(6) transformants/mu g) was achieved when cloning 517 bp DNA fragment using TA cloning. No significant difference observed in the positive cloning efficiency between two ligation methods and the positive cloning efficiency could reach as high as 100% especially for small inserts (e.g. 517 and 957 base pairs). Conclusions: We describe a simple and general method to construct a novel pXST vector. We confirm the feasibility of using pXST vector to clone PCR products amplified from cassava genome with both TA cloning and blunt-end ligation methods. The pXST plasmid has several advantages over many currently available vectors in that (1) it possesses Xcml-ccdB-Xcml cassette and restriction site Smal, enabling both TA cloning and blunt-end ligation. (2) it allows direct selection of positive recombinant plasmids in Escherichia coli through disruption of the ccdB gene. (3) it improves positive cloning efficiency by introducing the ccdB gene, reducing the possibility of self-ligation from insufficient digested plasmids. (4) it could be used by high performance and cost-effective cloning methods. Therefore, this dual function vector would offer flexible alternatives for gene cloning experiments to researchers.