绿色巴夫藻cDNA文库的构建及EST序列分析  

cDNA library construction and EST analysis of Pavlova viridis

作　　者：牛艳[1] 孔文涛[1] 杨婧[1] 孔健[1] 

机构地区：[1]山东大学微生物技术国家重点实验室,山东济南250100

出　　处：《中国水产科学》2006年第6期902-907,共6页Journal of Fishery Sciences of China

基　　金：国家自然科学基金资助项目(30370035)

摘　　要：将绿色巴夫藻（Pavlova viridis）培养液逐种加入氨苄青霉素、硫酸庆大霉素、硫酸阿米卡星3种抗生素处理，经检验后无杂菌污染。以该藻种为实验材料，以λTriplEx2为载体，利用SMART技术构建其cDNA文库。经测定文库滴度为6×10^6pfu／mL，重组率为98％。利用PCR方法检测cDNA文库插入DNA片段的大小，其长度为0．5～2kb，表明该文库达到建库要求，可用来筛选低丰度mRNA的基因克隆。对cDNA文库中的阳性克隆进行随机PCR扩增，挑选插入DNA片段较大的克隆，进行5’末端单向序列测定，测序结果与NCBI数据库进行BLAST比对，获得了200个有研究价值的EST序列和cDNA克隆，为进一步研究和开发绿色巴夫藻的功能基因奠定了基础。Pavlova viridis is rich in polyunsaturated fatty acids （PUFAs）, which represents an attractive production system for PUFAs such as EPA and DHA, and has been considered to be a useful source for cloning fatty acid desaturase genes. In this study, total RNA was extracted from Pavlova virids cells in the middle of the exponential phase using acid guanidinium thiocyanate. Poly ＋ A RNA was isolated and purified by Oligotex Suspension. The mRNA template was reversely transcriptionized to a single strand eDNA with modified Oligo（dT） primer using SMART（Switching Mechanism At 5 ＇end of RNA Transcript） technology. Synthesized dscDNA was ligated with vector ）,Triplex2. The recombined vectors were packaged in vitro. The titer of the unamplified constructed eDNA library was 6 ×10 ^6 pfu/mL and recombination rate was 98 %. The length of the inserted DNA fragment was between 500 bp and 1.5 kb. All of these suggest that the library can be suitable for screening low abundant mRNA for cDNA clones. The library was subsequently amplified. The presence of insert was checked by PCR using universal primers and clones containing cDNA longer than 500 bp were selected for sequencing. 200 ESTs were obtained by sequencing from the 5＇ end of the cDNA clones. Then the ESTs were compared with sequences in the GenBank data of NCBI using Blast. Partial sequence of some functional genes was identified, including atpI and atpH genes for subunit of ATPase complex, rRNA gene, carboxypeptidase A, cytochrome b, chloroplast photosystem II 12 kD extrinsic protein and so on. The results provide the basis for further study of Pavlova viridis gene expression. Further studies on those genes and large scale sequencing of the library may be helpful to elucidate and understand the function of those proteins at molecular level.

关 键 词：绿色巴夫藻 cDNA文库SMART技术 序列表达标签 多不饱和脂肪酸 

分 类 号：Q949.26[生物学—植物学]