花生种仁特异表达基因的初步筛选

Primary Screening of Differentially Expressed Genes in the Kernel of Peanut

应用抑制消减杂交技术,以花生种仁cDNA作为消减杂交的试验方(tester),以花生果皮cDNA作为驱动方(driver)进行消减杂交。经过两轮抑制PCR后,将第2次PCR产物与pGEM-TEasy载体相连,电击转化大肠杆菌,成功构建种仁特异表达cDNA文库。所长出菌落中91.2%为白色克隆,采用PCR法对阳性克隆进行鉴定,发现其中单一扩增条带的克隆约占86.5%,片段大小集中分布于200～800 bp之间。用花生种仁cDNA探针和果皮cDNA探针分别对文库高密度杂交点阵膜进行反向Northern杂交检测。根据杂交结果,初步从文库中挑取出254个差异点。花生种仁特异表达基因的初步筛选,为了解花生产量、品质形成相关的重要功能基因,也为将来应用植物基因工程手段改良花生产量和品质奠定基础。

To screen the specific genes in the kernel of peanut, a differentially expressed eDNA library was constructed by using suppression subtractive hybridization （SSH）. For subtractive hybridization, cDNA from the kernel of peanut was used as the tester, and cDNA from the pod of peanut was used as the driver. After two times of subtractive hybridization and two times of nested PCR, the products of the second PCR amplification were inserted into pGEM -T Easy vectors to transform the E. coli, a differentially expressed cDNA library of peanut kernel was successfully constructed. 91.2% of the colonies were white. The positive recombinants were confirmed by PCR, 86.5% of them showed single band with eDNA fragments ranging from 200 bp to 800 bp. The PCR products were picked to make array membranes, after checking the array by reverse northern blotting with two probes, the results demonstrated that 254 dots showed visual difference. The primary screening results of differentially expressed genes in peanut kernel can be used to study the important genes related to the quality and yield, also can be further used to improve the yield and quality of peanut by plant genetic engineering in future.