Selection Of Candidate Reference Genes For Gene Expression Studies By Quantitative Real-time PCR In Lonicera Japonica Thunb.

Selection and validation of candidate reference genes is necessary when real-time quantitative PCR is used to study gene expression.Lonicera japonica Thunb.as a commonly used traditional Chinese medicine,the application has a long history,in addition to the medicinal and edible,and the good ecological plants.At present,there are few studies on the gene expression of honeysuckle,and no internal reference genes suitable for honeysuckle real-time quantitative PCR are screened.In this study,10 traditional genes(Lonja.ACT11,Lonja.ACT2/7,Lonja.G6PD,Lonja.GAPDH,Lonja.MTP,Lonja.TUA,Lonja.TUB,Lonja.UBQ10,Lonja.EF1A,Lonja.UBC)and 11 new genes(Lonja.23040,Lonja.2392,Lonja.27738,Lonja.32161,Lonja.36969,Lonja.44744,Lonja.46545,Lonja.49672,Lonja.48053,Lonja.53402,Lonja.66846)were selectedfrom the data of low temperature stress of honeysuckle leaves,they were analyzed in Lonicera japonica Thunb.leaves subjected to low temperature stress and high temperature stress,in root,stem,leaf and flower.Results were comprehensive analysis by geNorm and NormFinder.The results are as follows:(1)It is recommended to use Lonja.48053,Lonja.GAPDH and to standardize transcripts in leaf of temperature stress.Lonja.49802 and Lonja.23040 were used in combination under low temperature stress.Lonja.48053 and Lonja.23040 were the best combination under high temperature stress.(2)A comprehensive study of honeysuckle when the different organs to Lonja.23932,Lonja.36369 combination of standardization.Only study the vegetative organs can also use Lonja.23932,Lonja.36369 combination.Recommended Lonja.36369,Lonja.ACT2/7 combination in the root;Lonja.36369,Lonja.27738 combination in the stem;Lonja.23932,Lonja.23040 combination in the leaf;Lonja.27738,Lonja.23932 combination in the flower.(3)It is suggested that Lonja.36369 and Lonja.27738should be used as the internal reference gene to improve the accuracy of quantitative real-time PCR.In addition,Lonja.49672 and Lonja.ACT 2/7 were used as the internal reference gene,and Lonja.39828 and Lonja.56446 were used as the target gene to study the gene expression.Quantitative real-time PCR results and transcriptome data analysis results are consistent,indicating that the study of the internal reference gene screening accuracy is high.In this study,we first selected the candidate reference gene of Lonicera japonica Thumb.by quantitative real-time PCR,and laid the foundation for the molecular research of honeysuckle gene expression.