中国南瓜内参基因18S rRNA的克隆及其引物开发

发布时间：2018-05-25 21:58

本文选题：中国南瓜 + S　；参考：《西北农林科技大学学报(自然科学版)》2017年03期

【摘要】：【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。
[Abstract]:[objective] to clone and obtain 18s rRNA from Chinese pumpkin, and to design a suitable fluorescent quantitative PCR internal reference. [methods] the genomic DNA of Chinese pumpkin 'Miamoto' was used as a template to clone the 18SrRNA gene sequence of Chinese pumpkin by PCR. Then the fluorescent quantitative PCR primers were designed with Primer Premier software to detect the expression stability of the internal reference gene in different tissues, growth stages and abiotic stress conditions of Chinese pumpkin. [results] the 18SrRNA gene sequence of Chinese pumpkin was cloned for the first time. Its length is 1 857 BP GenBank accession number is KM979454, the homology of this gene with watermelon, squash and other melon vegetable 18SrRNA gene is more than 90%, based on the full-length 18SrRNA sequence of Chinese pumpkin internal reference gene, a pair of fluorescent quantitative PCR primers were designed. The first cDNA strand of total RNA reverse transcription in Chinese pumpkin pulp was used as template for PCR amplification. The amplified fragment size was 132bp. the amplification efficiency was high and the specificity of 18s rRNA gene was strong in different tissues of Chinese pumpkin. [conclusion] the Chinese pumpkin 18SrRNA gene was obtained by cloning, which is suitable for the study of Chinese pumpkin gene expression as an internal reference gene.
【作者单位】：福建省农业科学院作物研究所;福建省农业科学院蔬菜研究中心;福建省蔬菜工程技术研究中心;福建省种植业技术推广总站;
【基金】：福建省属公益类科研院所基本科研专项(2015R1026-3) 福建省农业科学院创新团队PI项目(2016PI-40) 福建省自然科学基金项目(2015J01118)
【分类号】：S642.1

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