丝瓜18S rRNA基因克隆及其作为内参基因的应用

Cloning of 18S rRNA Gene From Luffa cylindrical and Its Application as an Internal Standard

选择合适的内参基因是提高实时荧光定量PCR分析(q RT-PCR)准确性的重要条件。18S r RNA基因表达范围广、表达量恒定,常作为内参基因应用于实时荧光定量PCR中。为了获得丝瓜18S r RNA基因,并设计合适的荧光定量PCR内参引物,解决丝瓜实时荧光定量PCR检测中无内参基因的现状,通过PCR和序列测定,首次克隆到了丝瓜的18S r RNA基因序列,其长度为1 862 bp,Gen Bank登录号为KM656452。在此基础上设计1对荧光定量PCR引物,该引物特异性强,扩增效率高,在丝瓜各生长发育阶段及各种非生物胁迫条件下均能稳定表达,适合在丝瓜基因表达研究中作为内参基因。该研究结果可为开展丝瓜重要功能基因的表达模式和调控机制的研究奠定基础。

The selection of a suitable reference gene is a critica1 condition for improving the precision of gene expression analyzing by quantitative real time PCR（ q RT-PCR）. The 18 S r RNA gene which has a broad and constant expression was always used as reference gene for q RT-PCR. The study was to obtain the 18 S r RNA gene of luffa and design the q RTPCR primers. Through PCR and sequencing,the 18 S r RNA gene of luffa was cloned firstly. It was 1 862 bp long and the Gen Bank accession number was KM656452. A pair of q RT-PCR primer was designed based on the 18 S r RNA gene sequence showed,high specificity and amplification efficiency. The RT-PCR indicated that the 18 S r RNA gene was stable expression under abiotic stress and in different growth stages,so it was suitable as a reference gene for the analysis of gene expression patterns in luffa. The present study has provided an important reference for analysis the expression of critical genes in luffa.