番茄qRT-PCR内参基因的筛选

Selection of tomato reference genes for qRT-PCR

以高温、低温、盐胁迫、TYLCV接种处理和对照的番茄幼苗为植物材料,通过实时荧光定量RT-PCR(Real-time fluorescence quantitative PCR,qRT-PCR)方法分析了常用的8个内参基因肌动蛋白基因(ACT)、甘油醛-3-磷酸脱氢酶基因(GAPDH)、泛素基因(UBI)、18S核糖体RNA基因(18S)、转录延伸因子基因(EF-1a)、β-微管蛋白基因(TUB)、表达蛋白基因(EXP)和接合素蛋白复合物基因(CAC)在不同样本中的表达情况。利用ge Norm和Norm Finder软件分析筛选表达稳定性较高的内参基因。结果表明,在不同胁迫处理条件下,UBI和ACT的稳定性较高;在同样的胁迫处理中,8个内参基因的表达稳定性不同。综合而言,虽然UBI和ACT的稳定性同样较高,但在TYLCV接种处理试验中ACT的稳定性相对较差,而TUB和GAPDH表达较稳定,故TUB和GAPDH可在TYLCV接种处理试验中作为番茄qRT-PCR的内参基因。

In this study, the tomato seedlings treated under high and low temperature, salt stress, TYLCV whiteflies vaccination and the relative controls were used as plant materials, and the expression levels of eight commonly used reference genes including actin(ACT), glyceral-dehyde-3-phosphate dehydrogenase(GAPDH), ubiquitin(UBI), 18S ribosomal RNA(18S), transcription elongation factors(EF-1a), β-1arip(TUB), expressed protein(EXP) and clathrin adaptor complexes(CAC)in different samples were analyzed through the qRT-PCR.The genes with stable expression levels were identified using geNorm and NormFinder softwares.The results showed that under different conditions of stress treatments, the stability of UBI and ACT were higher,while at the same stress treatment, the stability of expression of the eight reference genes were inconsistent.On the whole, the stability of UBI and ACT were equally high.In the TYLCV vaccination, the stability of ACT was relatively poor, so stably expressed TUB and GAPDH could be used as tomato reference genes for qRT-PCR.