Selection And Validation Of Reference Genes For Quantitative Analysis Of Gene Expression In Dove Tree (Davidia Involucrata Baill.)

Real-time quantitative PCR(qPCR)is widely used for gene expression research,with its advantages such as high sensitivity,strong specificity,good reproducibility and high-throughput capacity.Selection of suitable reference genes is the first step in analyzing the expression variation of target genes accurately by qPCR.Dove tree(Davidia involucrata Baill.)is a national(level I)protection plant,has a very high ornamental value and scientific research value.At present,the research on the gene expression in dove tree is increasing,but the study on reference genes is vacant.We choosed dove tree as the research material for the first time,and selected eight conventional housekeeping genes 18S rRNA(Ribosomal RNA 18S),ACT7(Actin 7),DNAJ(DnaJ-like protein),EF1a(Elongation factor 1-alpha),eIF(Eukaryotic initiation factor),GAPDH(Glyceraldehyde-3-Phosphat dehydrogenase),?-TUB(Beta tubulin),UBQ(Ubiquitin)and seven novel reference genes BADH(Betaine aldehyde dehydrogenase),CAC(Clathrin adaptor complexes),CSD(Coatomer subunit delta),hnRNP(Heterogeneous nuclear ribonucleoprotein),PP2A(Serine/threonine-protein phosphatase 2A),SAND(Sand family protein),TIP41(TIP41-like family protein)as candidate reference genes based on the transcriptome database of dove tree.In the present study,we measured the expression quantity of 15 candidate reference genes in seven different types of samples(vegetative organs,reproductive organs,different colored leaves,different bracts development,different seeds development,normal seeds and abortive seeds,total samples)of dove tree by qPCR.By using three kinds of software(geNorm,NormFinder,BestKeeper)and the geometric average comprehensive analysis method to evaluate the expression stability of candidate reference genes,we selected the reference genes which are suitable for different research in dove tree.To further demonstrate the accuracy of the results,the expression stability of the partial reference genes were verified by the target genes(DiLTP,DiMYB1,DiLAC)in different types of samples.In the end,two novel reference genes(BADH and CAC)which were identified as stable genes have been cloned from dove tree.Through the above research,we can draw the following conclusion:1.None of the reference genes was uniformly expressed across all the samples of dove tree,and the most suitable reference genes are tissue-specific-,and developmental-dependent.BADH,EF1? and hnRNP are the top three choices for vegetative organs.BADH,eIF and CAC are the top three choices for vegetative organs.SAND,18S rRNA and eIF are the top three choices for different colored leaves.TIP41,CAC and CSD are the top three choices for different bracts development.C4C,CSD and BADH are the top three choices for different seeds development.SAND,CAC and eIF are the top three choices for normal seeds and abortive seeds.BADH,CAC and DNAJ are the top three choices for all samples.In short,most of the novel reference genes performed better than the conventional housekeeping genes,in particular,the novel reference genes BADH and CAC were superior to traditional ones in terms of their expression stability.2.In the analysis of gene expression,the expression stability,expression quantity and the number of reference genes can affect the accuracy of quantitative results.In gene expression studies of dove tree,in order to improve the accuracy of quantitative results,we should choose the appropriate reference genes or reference genes combination which the expression quantity is stable and close to the target gene to adjust,according to the specific types of sample and target gene.3.The novel reference gene BADH named as DiBADH(GenBank accession number KX655718),encoding the betaine aldehyde dehydrogenase(BADH),has been cloned from dove tree.The fragment of DiBADH includes a ORF(open reading frame)of 966 bp,which encodes a protein with 322 amino acids.Homologous alignment analysis showed that it has the highest amino acid homology with tea tree(Camellia sinensis)BADH gene.The novel reference gene CAC named as DiCAC(GenBank accession number KX268525),encoding the clathrin adaptor complexes(CAC),has been cloned from dove tree.The fragment of DiCAC includes a ORF(open reading frame)of 1317 bp,which encodes a protein with 438 amino acids.Homologous alignment analysis showed that it has the highest amino acid homology with grape(Vitis vinifera)CAC gene.This study provides a theoretical basis for the reasonable selection of reference genes and accurate analysis of gene expression patterns in dove tree.Meanwhile,our findings provide referential basis for the development of novel reference genes resources.