Abstract: [Objective] Choosing a suitable reference gene is an effective method to improve the accuracy and to reduce the experimental error of quantitative real-time PCR (qPCR). It has become an important technique to select stable expression of genes as novel reference genes using gene expression data in recent years. The purpose of our study is to select the novel reference genes which are expressed stably under salt and drought stresses in poplar using multiple microarray data. Our study will enrich the reference genes in poplar, and provide more channels for exploring stable and desirable reference genes.[Method] The gene microarray data of Populus trichocarpa in different growth periods under different stress treatments were collected from public gene chip database. The data were normalized to rank the stability of the expression of genes under different experimental conditions. Based on the functional annotation information of the genes, six novel reference genes were selected. In order to further verify the stability of gene expression, we have chosen the leaves of Populus deltoides ‘Nanlin95’ assessed by NaCl, PEG for analysis. Six traditional reference genes(PtUKN1, PtUBQ, Actin, EF1α, 18S rRNA, TUA8)and six novel reference genes were assessed by qPCR at six points (0, 1, 4, 8, 12, 24 h). Using the reference gene analyzing programs of geNorm, NormFinder and BestKeepe, the experimental data were counted and ranked in order to compare the expression stability of the 12 genes and accordingly select the suitable novel reference genes.[Result] In this study, six stable expression genes (PtRG1, PtRG2, PtRG3, PtRG4, PtRG5, PtRG6) were selected from the gene chip database. Comparisons of the stability between these six new reference genes and the six traditional reference genes displayed that, in the geNorm program analysis, the stability of PtRG2 and PtRG3 was better under salt stress and the stability of PtRG3 and PtRG5 was better under the drought stress; in NormFinder program analysis, the stability of TUA8 and PtRG1 was better under salt stress and the stability of PtRG1 and PtRG2 was better under the drought stress; in BestKeeper program analysis, the stability of PtRG1 and PtRG5 was better under salt stress and the stability of PtRG3 and PtRG5 was better under the drought stress. To further verify the stability of these gene expression, PtVQ6, PtVQ13 and PtVQ37 expressed highly in the poplar VQ gene family under NaCl and PEG treatment were selected as the target gene to conduct qPCR again and found the results were consistent with the previous study, suggesting PtRG1, PtRG3 and PtRG5 were stable.[Conclusion] PtRG1, PtRG3 and PtRG5 identified in our study are suitable as the reference genes in poplar under salt and drought stresses, which would contribute to a more accurate analysis for the expression of the resistance genes in qPCR.

Key words: poplar, quantitative real-time PCR, reference genes, salt stress, drought stress