摘要
以青杄花粉和针叶为材料,将青杄全长cDNA与Gateway供体载体pDONR222重组,构建了其非剪切型全长cDNA原始文库,利用基因组DNA饱和杂交技术对原始cDNA文库进行均一化处理,构建青杄的均一化全长cDNA文库。文库的总库容量为1.1×106CFU/mL,平均插入片段长度大于1.0 kb,重组率大于95%。定量RT-PCR检测表明,青杄高丰度表达基因EF1-α在均一化cDNA文库中的表达量下降了约41倍。接着对文库中随机的5 144个克隆进行了测序,获得高质量的有效EST(expressedsequence tag)序列为5 144条,经拼接共获得单一基因(unigene)为2 717个,其中包括片段重叠群(contig)628个和单一EST序列(singlet)2 089个。NCBI同源比对分析表明,其中1 887个序列unigenes获得分子功能注释,这些EST涉及细胞生长、信号转导、转录、抗逆、能量代谢等功能。这些数据有助于对青杄的相关功能蛋白及分子机制开展进一步的研究。
The primary full-length cDNA library was constructed with the pollen and needles of Picea wilsonii using recombination of the full-length cDNAs with the Gateway donor vector of pDONR222. The plasmids of the primary cDNA library were normalized through self-subtraction with the Picea wilsonii a genome DNA, and the normalized full-length cDNA library was constructed using the subtracted plasmids. The normalized full-length cDNA library had a total CFU of 1.1 × 10^6 CFU/mL. The average size of inserted cDNAs was 1.0 kb with a recombination of about 100%. Quantity RT-PCR results demonstrated that normalization produced about 41 folds average reduction of a high abundant gene EFI-a. A high-quality normalized full-length cDNA library was successfully constructed, and 5 144 of random clones in this cDNA library were then sequenced. A total of 5 144 ESTs （ expressed sequence tag ） were attained, and could be assembled into 2 717 unigenes, including 628 contigs and 2 089 singlets. These genes were found to be involved in the multiple biological process including cell development, signal transport, transcription, stress tolerance response and energy metabolism. The ESTs presented here provide a valuable resource for further investigation on functions and molecular mechanisms of similar proteins in Picea wilsonii.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第6期71-76,共6页
Biotechnology Bulletin
基金
转基因生物新品种培育科技重大专项(2011ZX08009-003)
关键词
青杄
均一化
CDNA文库
EST序列分析
Picea wilsonii Normalized cDNA library Expressed sequenced tags analysis
