摘要
本文通过构建金黄地鼠神经管cDNA文库,筛选了RPL30基因,并探讨了其与神经管发育的关系。提取胚胎(E)8.5d的金黄地鼠神经管总RNA,用SMART技术构建神经管cDNA的噬菌体表达文库。利用分部分排除技术(SSS法),结合PCR法逐级筛选cDNA文库。将得到的RPL30阳性噬菌斑进行质粒转化、酶切鉴定及测序分析,进而回收、纯化RPL30cDNA片段并制备探针。用Northern杂交技术检测神经管发育过程中RPL30mRNA的表达状况。结果显示,构建的神经管cDNA文库容量为1.5×106pfu/ml,重组率达到99%,平均插入片段长度大于1kb。经过筛选得到含RPL30cDNA的阳性克隆。序列测定及同源性分析发现,该cDNA片段全长535bp,其中含有完整的开放阅读框。Northern杂交结果显示,在E8d^E12d各时间段,神经管中RPL30mRNA的表达均处于较高水平,且随着时间的增长,表达量递增,于E12d达到高峰。本文结果表明,我们用SSS技术结合PCR法从神经管cDNA文库中克隆到了RPL30基因的全长cDNA序列,该基因可能与神经管发育密切相关。
To construct a cDNA library frpont neural tube of golden hamster and sereen RPL30 gene, and to discuss the relationship between RPL30 gene and the development of the neural tube. Total RNAs were isolated from neural tube using TRIZOL reagent, the neural tube eDNA library was constructed by SMART method, Using subsection screening （SSS） method to screen the cDNA library by PCR step by step. positive plaques were then converted to plasmids, enzyme identified, and sequence analyzed. The level of RPL30 mRNA in the neural tube at different development stages was detected by Northern blotting. The results showed thai the titer of the neural tube cDNA library was 1.5×10^6pfu/ml, the recombirnation rate was 99%, the average insert was larger than 1kb. The positive clone containing RPL30 cDNA was obtained after screeniug, and the nucleotide sequence of the cDNA insert was determined and analyzed with BLAST. The length of the cDNA insert was 535bp, comtaining full open reading frame. Northern hlut showed that the level of RPL30 mRNA was higher in the neural tube from E8 d to E12 d, increased gradually during neurulation and increased significantly at E12 d group. The full-length of RPL30 cDNA was cloned from this neural tube cDNA lihrary using SSS method combined with PCR method, and RPL30 gene was associated closely with the development of the neural tube.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
2006年第1期33-37,共5页
Chinese Journal of Neuroanatomy
基金
国家自然科学基金(No.30270701)
卫生部优秀青年科技人才专项基金(No.1999BA2CCA1)资助项目
