Analysis Of The Expression Of VQ Genes And Selection Of Novel Reference Genes In Poplar

Poplar,is an important fast-growing and bio-energy tree,whose growth is constrained by drought,salinity and other abiotic stress.Therefore,researching the regulation mechanism of poplar resistance plays an important role in defensing the abiotic stress.VQ protein,a highly conserved protein family in plants,involved in the regulation of plants under drought stress,salinity,etc,which has vital research value for improving poplar resistance.Choosing a suitable reference gene is an effective method to improve the accuracy and reduce the experimental error.However,we have not found a suitable reference gene for multiple experimental conditions so far.Nowadays,no information available describes the functions of the VQ genes in Populus trichocarpa.In our study,comprehensive analysis of poplar VQ genes were performed including genome-wide identification,characterization,and expression analysis under salt and drought treatment.Reference genes play a crucial role in quantitative RT-PCR(qRT-PCR)which is one of the effective ways to detect gene expression.Choosing a suitable reference gene is an effective method to improve the accuracy and reduce the experimental error.However,we have not found a suitable reference gene for any experimental conditions so far.With the development of gene chip and high-throughput sequencing technology,it has become an important technique to select novel reference genes using gene expression data.Nevertheless,this method has not been widely applied in woody plants.Therefore,the purpose of our study is to select the novel reference genes which are expressed stably under salt and drought stress in poplar using multiple microarray data.In the study of novel reference gene in poplar,first of all,first of all,the data of poplar genes under different growth periods and different stress treatments were obtained by gene chip technique.Then,the data was be 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 out(PtRG1,PtRG2,PtRG3,PtRG4,PtRG5,PtRG6).These six novel reference genes were employed to be assessed by qRT-PCR with six traditional reference genes(PtUKN1,PtUBQ,Actin,EF1α,18 S rRNA,TUA8).And then using the program of geNorm,NormFinder and BestKeeper to analyze the experimental data in order to compare the stable expression of the 12 genes and accordingly select the suitable novel reference genes.To further verify the stability of the novel reference gene obtained by selecting,the PtVQ6,PtVQ13 and PtVQ37 expressed highly in the poplar VQ gene family were chosen as the target gene.The following is the result of our study:(1)Fifty-one VQ genes were identified and classified into seven subfamilies(I–VII),distributed randomly on 17 of the 19 chromosomes in poplar.(2)The poplar VQ genes expanded primarily due to segmental duplication.(3)Gene structure and protein motif analysis indicated that these genes were relatively conserved within each subfamily;especially 39 of the 51 VQ genes had no introns.(4)The results of quantitative real-time RT-PCR(qRT-PCR)analysis indicated that the VQ genes were variously expressed under different stresses.(5)This study selected six stable expression of the novel reference genes PtRG1-6.(6)It was found that PtRG1,PtRG3 and PtRG5 are expressed more stably at different periods under salt and drought stress.