中国南瓜内参基因18S rRNA的克隆及其引物开发

Cloning of 18S rRNA gene from Cucurbita moschata and development of its primers

【目的】克隆获得中国南瓜18SrRNA,并设计合适的荧光定量PCR内参,为开展中国南瓜重要功能基因的表达模式和调控机制研究奠定基础。【方法】以中国南瓜‘密本’品种的基因组DNA为模板,通过PCR方法克隆中国南瓜18SrRNA基因序列,再利用Primer Premier软件设计荧光定量PCR引物,对该内参基因在中国南瓜不同组织、生长阶段及非生物胁迫条件下的表达稳定性进行检测。【结果】首次克隆得到中国南瓜18SrRNA基因序列,其长度为1 857bp,GenBank登录号为KM979454,该基因与西瓜、西葫芦等瓜类蔬菜18SrRNA基因同源性大都在90%以上;以中国南瓜内参基因18SrRNA核苷酸全长序列为基础设计1对荧光定量PCR引物,以中国南瓜果肉总RNA逆转录的cDNA第一链为模板进行PCR扩增,该引物扩增的片段大小为132bp,扩增效率高,特异性强;18SrRNA基因在中国南瓜不同组织、生长发育阶段及非生物胁迫条件下均能稳定表达。【结论】克隆获得中国南瓜18SrRNA基因,该基因适合在中国南瓜基因表达研究中作为内参基因。

【Objective】The study cloned 18 SrRNA gene of Cucurbita moschata and designed suitable qRT-PCR primers for analyzing expression patterns and regulation mechanisms of important critical genes.【Method】Sequence of 18 SrRNA gene was cloned by PCR using the genomic DNA of Cucurbitamoschata cultivar‘Miben＇as template.A pair of qRT-PCR primers were then designed by Primer Premier software.Furthermore,the stability of 18 SrRNA gene was determined in Cucurbita moschata in different tissues,growth stages and abiotic stress conditions.【Result】The 18 SrRNA gene of Cucurbita moschata wascloned firstly（GenBank accession number：KM979454）.It was 1 857 bp long and shared more than 90%homology with 18 SrRNA genes from melons vegetables such as watermelon and squash.According to the deduced 18 SrRNA gene sequence,apair of qRT-PCR primers were then designed.The fragment of 132 bp was successfully amplified by the primers using the first-strand cDNA as template,which was reverse-transcribed from the total RNA of Cucurbita moschata.The primers had high specificity and amplification efficiency.RT-PCR indicated that the 18 SrRNA gene was stably expressed under different growth stages and abiotic stresses.【Conclusion】The 18 SrRNA gene was suitable as a reference gene for analyzing gene expression patterns in Cucurbita moschata.