摘要
目的:为筛选和克隆大乳头水螅发育调控相关基因的全长cDNA,构建大乳头水螅RACE cDNA文库。方法:提取大乳头水螅总RNA后从其中分离mRNA,运用SMART技术构建RACE cDNA文库。为鉴定所构建文库的质量,根据GenBank中大乳头水螅actin基因cDNA序列设计5'RACE和3'RACE的引物及用于扩增actin基因编码区全长序列的引物。结果:琼脂糖凝胶电泳结果表明,RACE cDNA文库中全长cDNA的长度集中在500-2 000 bp之间。5'RACE、3'RACE PCR及扩增actin基因编码区全长序列时均以本文构建的大乳头水螅RACE cDNA文库为模板,这3个PCR反应均能扩增出产物,产物大小与目标片段预计大小相似。PCR产物分别经T/A克隆及测序后证明为大乳头水螅actin基因cDNA的相应序列。结论:RACE cDNA文库的成功构建为通过RACE方法获得大乳头水螅功能基因cDNA全长序列奠定了基础。
Objective： To construct RACE cDNA library of Hydra magnipapillata.Methods： Total RNA was isolated from Hydra magnipapillata,and purified mRNA from total RNA was used to construct RACE cDNA library with the SMART cDNA library con-struction kit.In order to identify the cDNA library,the polymerase chain reaction（PCR） primers for 5＇ RACE,3＇ RACE and the full-length cDNAs of actin gene were designed based on the pupative cDNA sequence of actin from GenBank.Results： Agarose gel elec-trophoresis showed that the lengths of full-length cDNAs in this library were pooled mainly between 500 and 2 000 base pairs.By RACE PCR,amplified products were obtained with all the gene-specific primers and adaptor primers.Conclusion： The quality of the RACE cD-NA library was high and appropriate for cloning the full-length cDNAs of functional genes in Hydra magnipapillata.
出处
《现代生物医学进展》
CAS
2013年第4期647-650,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(31171415)
安徽省高等学校省级自然科学研究重点项目(KJ2011A130)
安徽师范大学优秀创新团队建设计划(2010-2011)
