管花肉苁蓉实时荧光定量PCR分析中内参基因的选择和验证

Selection and validation of appropriate reference genes for qRT-PCR analysis in Cistanche tubulosa

本实验基于管花肉苁蓉的转录组数据,选择了12个常用候选内参基因,研究其在管花肉苁蓉不同组织中以及不同非生物胁迫下的稳定性。利用geNorm、NormFinder和BestKeeper三种常用软件计算候选内参基因的稳定性,并用RefFinder对其综合稳定性进行评价。实验结果表明,在愈伤组织中,EF-1α和ACT是干旱胁迫下内参基因的最佳组合;RPL和PP2A是盐处理下最稳定的两个内参基因;重金属胁迫下,H2A和F-box是最合适的内参基因,而ACT和F-box是冷处理和水杨酸处理下最稳定的内参基因;茉莉酸甲酯处理样本中最合适的内参基因是α-TUB2、F-box和GAPDH。种子中,EF-1α与F-box是脱落酸处理下的最佳内参基因;EF-1α和H2A结合分别是赤霉素处理下最合适的内参基因;而ACT和DNAj是氟啶酮处理下最稳定的内参基因。此外,DNAj和F-box被认为是最适合管花肉苁蓉中各组织的内参基因组合。本研究还对愈伤组织中G6PDH1和G6PDH2在不同非生物胁迫条件下的相对表达水平进行了研究,证明内参基因的选择对实时荧光定量PCR结果精确度有很大影响。

In our study,the expression stability of 12 candidate reference genes across Cistanche tubulosa(Orobanchaceae)tissue types,calli with drought,salt,heavy metal,cold,salicylic acid(SA)and methyl jasmonate(MeJA)treatments,and seeds with abscisic acid(ABA),gibberellin(GA3)and fluridone treatments were calculated,respectively.After calculation of PCR efficiencies,the expression stability of 12 candidates was calculated by three commonly used software(geNorm,NormFinder,and BestKeeper),and comprehensive stability rankings were merged by RefFinder.The optimal reference genes selected for gene expression analysis in calli were EF-1α(Elongation factor-1 alpha)and ACT(Actin)for drought treatment,RPL(Ribosomal protein)and PP2 A(Protein phosphatase 2 A)for salt treatment,H2A(Histone H2A)and F-box(F-box family protein)for heavy metal treatment,ACT and F-box(F-box family protein)for cold and SA treatment,andα-TUB2(alpha-tubulin 2),F-box and GAPDH for MeJA treatment.The relatively stable genes for seed as follows:EF-1αand F-box for ABA treatment,EF-1αand H2A for GA3 treatment,and ACT and DNAj(Chaperone protein dnaJ)for fluridone treatment.Moreover,our results indicated that DNAj and F-box were the most appropriate reference genes across different tissues.Furthermore,the relative expression levels of the glucose 6-phosphate dehydrogenase(G6 PDH)1 and 2 under different abiotic stresses were conducted to confirm the validity of the reference genes.