应用抑制消减杂交技术 构建花生果皮差异表达文库

为构建花生果皮差异表达基因消减文库，分离花生果皮差异表达cDNA片段，本研究采用抑制消减杂交技术，以花生种仁cDNA为Driver，果皮cDNA为Tester，进行两次消减杂交和两次抑制性PCR，将第二次PCR产物与pGEM-T easy 载体相连，电击转化大肠杆菌，成功构建果皮差异表达cDNA文库。所长出菌落中89.2%为白色克隆。采用PCR法对重组子进行鉴定，发现其中单一扩增条带的克隆约占83.5%， 片段大小集中分布于250～750bp之间。

Construction of cDNA library from peanut pod by using suppression subtractive hybridization

To isolate peanut pod specially expressed genes,a cDNA library was constructed by using suppression subtractive hybridization(SSH).For subtractive hybridization,cDNA from the kernel of peanut was used as Driver,and cDNA from the pod of peanut was used as Tester.After two times of subtractive hybridization and two times of nested PCR,the products of last PCR amplification were inserted into pGEM-T Easy vectors and be transformed into the E.coli,a cDNA library of peanut pod was successfully constructed.89.2% of the colonies were white.The positive recombinants were confirmed by PCR method,83.5% of them showed single band.Those cDNA in the library can be used to isolate differentially expressed genes and promoters of peanut pod,also can be further studied for the purpose of understanding the molecular mechanism in the specially expression of pod genes.