Identification Of Reference Genes And Transcriptional Expression Of NcSUT4 In Neolamarckia Cadamba

Neolamarckia cadamba belongs to the family of Rubiaceae.Based on the features of erect trunks and growing fast,it has a broad prospect in the fields of medical,construction,food etc.Reverse transcription quantitative real-time PC R is a sensitive technique for quantifying gene expression,its success depends on the stability of the reference gene(s)used for data normalization.With the whole genome sequencing and functional gene research of Neolamarckia cadamba,there is a lack of an effective and stable method for quantifying the transcription of functional gene.This study selected,amplified and cloned the reference gene(s)and NcSUT4,NcNCED3 by transcriptome data.Analyzed the reference genes of stable expression in 66 samples and the transcription expression characteristics of NcSUT4.The main research results are as followed:1.Selected,amplified and cloned c DNA and gDN A sequences of 15 candidate reference genes from Neolamarckia cadamba transcriptome data,including: ACT1,ACT11,TUA2,TUA4,TUA5,GAPDH,RPS25,EF-1-?,Rubisco,RPL10 A,MDH,F-BOX,SAMDC,NcSUT4 and NcNCED3.Analyzed with BLAST,the cDNA sequence of each gene was consistent with the sequence in the transcriptome.The intron of each gene was obtained by comparative analysis,which was used as a reference for the design of primers in quantitative analysis later.2.The expression stability in 66 Neolamarckia cadamba samples were evaluated and analyzed using three algorithms,geNorm,NormFinder and BestKeeper.The results showed that SAMDC was the most stable reference genes in all samples,but showed the difference between the different experimental conditions.To verify the stability of reference genes based on NcSUT4 and NcNCED3,this study showed that the stability of reference genes and combinations,is more reasonable than unstable genes for data normalization.It certified that these stable reference genes selected preliminary has the accuracy and the feasibility.3.Using reference gene standardization analysis the transcription and expression characteristics of NcSUT4 in different time,tissues,and the photosynthetic cycle at different time points,the research showed that it expressed in the phloem,xylem,young leaves,mature leaves,roots and other parts of Neolamarckia cadamba.With the change of time and environment,NcSUT4 was expressed in temporal and spatial specificity.