Selection Of Reference Genes And Clone Analysis Of Acetyl-CoA Transporter Gene(Rs-acatn-1) In Radopholus Similis

Radopholus similis(Cobb,1893)Thorne,1949,the harmful endoparasite nematode,is considered the most important plant pathogenic nematode.R.similis are seriously harmful to many important economic crops and ornamental plants.Chemical control is now the most common methods of prevention and control of R.similis,but overusing nematicides will seriously pollute the environment and endanger human health,so the new methods of sustainable control of R.similis are focusing on strategies of biological technology and on targets of netatode parasitic pathogenicity genes,which have become one of rescarch hotspots.In the functional analysis of target genes,it is necessary to select the ideal reference gene according to the experimental factors for correction and standardization,to ensure the accuracy of quantitative analysis.But so far there have been no reports on R.similis of reference genes.The acetyl-CoA transporter,ACATN is a multiple transmembrane protein in the endoplasmic reticulum,which is involved in the process of O-acetylation of sialic acid residues on gangliosides.Therefore,the study of ACATN of R.similis is helpful to obtain more potential target genes.But so far there have been no reports about acatn of plant parasitic nematodes.In this study,we used qPCR to select the ideal reference gene of R.similis,and to clone and analyze the acetyl-CoA transporter gene of R.similis,then the selected reference gene was used to quantitatively analyze the acetyl-CoA transporter gene of R.similis.The main conclusions of this study are as follows:1.The sequences of three traditional reference genes(Act1,eIF5A2,TUB,UBC)and three novel reference genes(CBR,?-PP1)were extracted from the database of R.similis transcripts by bioinformatics,and a traditional reference gene 18 S rRNA was selected on NCBI.The expression of these candidate genes was detected by real-time quantitative PCR in six populations of R.similis and in different development states(females,males,larvae,eggs)of GJ population.The expression stability of these genes was evaluated by BestKeeper,geNorm and NormFinder.It is suggested that eIF5A2 is the most suitable reference gene for the study of gene function of different populations of R.similis.CBR andeIF5A2 are the most suitable reference gene for the study of gene function of different development states in a group of R.similis,so eIF5A2 is the most ideal reference gene for the study of R.similis.2.In this study,we selected a transcript CL1740.Contig4 of the putative gland protein of Heterodera glycines from the database of R.similis transcriptome.The cDNA full length of R.similis acetyl-CoA transporter gene(Rs-acatn-1)was 1071 bp,the open reading frames of Rs-acatn-1 was 738 bp,which encoded 245 amino acid.The DNA coding region sequence was 1063 bp,including 5 exons and 4 introns.The prediction protein molecular weight was 27806.83.The protein also had a signal peptide,transmembrane region and other structures.3.There was no significant difference in the expression of Rs-acatn-1 at different development stages of R.similis.The expression of the gene in males and eggs were higher than other development stages,followed by females and larvae.In this study,we selected the ideal reference gene eIF5A2 of R.similis,which provided a guarantee for the accuracy of quantitative analysis of related genes of R.similis.In this study,we obtained the full-length sequence information of Rs-acatn-1,which is the first report in plant parasitic nematodes.This study is helpful to figure out a new way to control the plant parasitic nematodes.