CAJ | 학술논문

用RDP试剂提取三丁基锡暴露诱导的杂色鲍肝胰腺总RNA,经Oligotex纯化得到 mRNA;应用SMART技术合成双链cDNA,双链特异核酸酶(DSN)进行双链cDNA的均一化,构建了杂色鲍三丁基锡暴露诱导下的均一化cDNA文库.原始文库的库容为4.3× 10~6 CFU/ml,重组率为 97.9%.从文库中随机挑选了3 288个克隆进行测序,得到3 048个高质量EST序列,其中有 370条 Contigs,2 103条 Singlets,Unigenes共 2 473条,冗余率为18.86%.以上结果说明该文库质量较好,为进一步筛选相关功能基因打下基础;较低的冗余率说明该文库值得继续使用大规模ESTs测序的方法寻找相关功能基因,并为进一步使用基因芯片技术研究相关功能基因的表达谱提供便利.
용RDP시제제취삼정기석폭로유도적잡색포간이선총RNA,경Oligotex순화득도 mRNA;응용SMART기술합성쌍련cDNA,쌍련특이핵산매(DSN)진행쌍련cDNA적균일화,구건료잡색포삼정기석폭로유도하적균일화cDNA문고.원시문고적고용위4.3× 10~6 CFU/ml,중조솔위 97.9%.종문고중수궤도선료3 288개극륭진행측서,득도3 048개고질량EST서렬,기중유 370조 Contigs,2 103조 Singlets,Unigenes공 2 473조,용여솔위18.86%.이상결과설명해문고질량교호,위진일보사선상관공능기인타하기출;교저적용여솔설명해문고치득계속사용대규모ESTs측서적방법심조상관공능기인,병위진일보사용기인심편기술연구상관공능기인적표체보제공편리.
A normalized cDNA library from hepatopancreas of abalone Haliotis diversicolor supertexta exposure to Tributyltin was constructed. Total RNA were prepared using RDP reagent. Oligotex (QIAGEN) was used to separate the mRNA from total RNA. The first strand cDNA was synthesized by transcription of mRNA with the SMART technique. The LD-PCR was performed using a modified SMART primer as the primer set,and the first -strand cDNA as the template to synthesize double strand cDNA. Double strand cDNA was normalized using Duplex-Specific Nuclease ( DSN ) . The containing capacity of the unamplified cDNA library was 4. 3 × 10~6 CFU/ ml with a recombinant rate of 97. 9% . A total of 3 288 clones were random selected to be sequenced, and 3 048 high quality ESTs were generated. After processing,a total of 2 473 unigenes comprising 370 contigs and 2 103 singlets were obtained. The redundancy was 18. 86% . These results indicate that the normalized cDNA library is suitable to be used for further cloning and analysis of genes related to tributyltin exposure.