利用改进的Oligo-capping法构建手掌参全长cDNA文库

Construction of a Full-length cDNA Library of Gymnadenia conopsea by Modified Oligo-capping

Oligo-capping法是构建全长cDNA文库的重要方法之一.以名贵中药手掌参(Gymnadenia conopseaR.Br.)的幼芽组织为材料,通过减少mRNA的用量,用pUC18作载体,设计特异引物对第1链cDNA进行PCR扩增,按片段大小分级回收cDNA,形成了一种以Oligo-capping法为基础,构建珍稀材料全长cDNA文库的快速、简单的技术体系.利用该方法,首次成功地构建了手掌参的全长cDNA文库.经综合评价,文库容量达到了8×105个/μg cDNA,全长cDNA比例达到68%,重组率超过96%,说明本实验的改进非常成功.该文库的建成为手掌参的遗传资源保护和功能基因发掘等理论与应用研究奠定了基础.

Oligo-capping is one of the important methods in the construction of full-length cDNA libraries. A specific primer was designed by employing the plumelets of Gymnadenia conopsea as the material,pUC18 as the vector and reducing the mRNA dosage,and then used to amplify cDNA in the first order by PCR and cDNA fragments was extracted according to their length classification. In this way,a quick,simple and convenient technical system of constructing a full-length cDNA library of precious and endangered plants, which was based on Oligo capping, with the system a full-length cDNA library of Gymnadenia conopsea was successfully developed for the first time. Comprehensive evaluations of the library indicated that its capacity reached 8×10^5 independent clones,its full-length cDNA proportion amounted to as high as 68% and it recombination rate surpassed 96%, proving that the modifications done in the study to Oligo capping were very successful. The construction of the library had laid a foundation for the conservation of genetic materials and the theoretical and applied exploitation of functional genes in Gymnadenia conopsea.