Selection Of Reference Genes In Carya Illinoinensis(Wangenh.) K. Koch

Carya illinoinensis is an important woody oil plant.Its fruit is rich in oil and unsaturated fatty acids,and the wood texture is beautiful.It is a high-quality fruit and timber dual-purpose woody plant.However,with the deterioration of environmental conditions,drought,soil salinization,cold damage and other stresses affect the growth and development of pecans and fruit yield.Therefore,it is of great significance to study the function of stress resistance genes in pecan for resistance breeding and genetic improvement.Real-time fluorescence quantitative PCR is an important means to study the molecular mechanism.In the study of gene expression analysis,appropriate reference genes are needed to calibrate the expression of target genes to obtain accurate results.In this experiment,the samples of eight-leaf stage seedlings of Carya illinoensis under different stress conditions(40℃ high temperature,4℃ low temperature,low phosphorus,10%PEG6000 drought,3%Nacl solution)were used as experimental materials.The reference genes that have been verified in other species were found from the literature.On the basis of the whole genome,and with the transcriptome of Carya illinoensis as a reference,10 reference genes ACT,PP1,PP2A,PR26S,TUB,UBC EF,RPL13,EIF4A,F-box were finally selected,and primers were designed across introns.Real-time fluorescence quantitative PCR technology was used for amplification.GeNorm,BestKeeper,NormFinder,ΔCt algorithm and comprehensive evaluation software RefFinder were used to obtain the comprehensive ranking of candidate gene stability was obtained.Finally,the most suitable reference genes and their numbers under different treatment conditions were selected,and the selected reference genes were verified to improve the accuracy of the test results.The specific research results are as follows:The expression of candidate reference genes in pecan under different stress conditions is as follows:PP2A,TUB,26S and PP1 can be selected as the corrected reference genes in leaves under high temperature stress,and PP2A,PR26S and EF are recommended as the combination of reference genes in roots under high temperature stress.The most stable reference gene combinations in leaves and roots under salt stress were PP2A and PR26S.It is recommended to use EF,PP2A,EIF4A,PR26S,and ACT as calibration reference genes in drought-stressed leaves,and the most stable combination of reference genes in drought-stressed roots is PP2A and EIF4A.RPL13 and EIF4A were recommended as the optimal reference genes in leaves under low temperature stress,and EIF4A and ACT were recommended as the optimal reference genes in roots under low temperature stress.The combination of EIF4A,UBC,PP2A and PR26S was recommended in low phosphorus stress leaves.The recommended reference genes in roots under low phosphorus stress were EIF4A and PP2A.Combined with all stress materials of pecan,the recommended internal reference gene combinations were PR26S,PP2A and TUB to calibrate the expression level of target genes and improve the accuracy of real-time fluorescence quantitative PCR results.