Transcriptome Analysis Of ER Stress–related Genes And Validation Of Reference Genes In Gene Expression RT-qPCR For Cyclocarya Paliurus(Batal.) Iljinskaja

The normal growth and development of plants depend on the external environment.Under adverse conditions,the endoplasmic reticulum(ER)in plants is enriched with a large number of proteins that cannot be folded or misfolded,causing ER stress.It is an effective strategy of cells responding to ER stress by activating conserved unfolded protein response(UPR).Cyclocarya paliurus(Batal.)Iljinskaja is a species endemic to China and has many important pharmacological activities.However,abiotic stresses,such as drought,salt,cold or heat,can lead to the accumulation of misfolded or unfolded proteins in ER,resulting in reduced yield.UPR induced by ER stress plays an important role in plant development and adaptation to stress.At present,the genome of Cyclocarya paliurus(Batal.)Iljinskaja is unknown,and there are few related studies.It is one of the most important methods to study ER stress related genes in Cyclocarya paliurus(Batal.)Iljinskaja by transcriptome sequencing.In this study,1)A total of 452,300,260 clean reads and 196,207 unigenes were detected by RNA-seq in Cyclocarya paliurus.Compared with the control group(TM-0h),there were 1,867 down-regulation and 3,040 up-regulation in the experimental groups(TM-6h and TM-14h);2)A batch of commonly used candidate reference genes were screened from the RNA-seq database of Cyclocarya paliurus: UBQ4,TUB,TUA,GAPDH,18 SrRNA,ACT2 and ACTF;3)RT-qPCR(Real-time quantitative PCR)policy and common internal stability analysis software(geNorm,NormFinder)were used to analyze and evaluate the stability of internal reference genes treated by TM and under other abiotic stresses(drought,salt,low temperature and high temperature).It was found that ACT2 and TUB were the most suitable internal reference genes in TM treatment,18 srRNA and ACTIN2 were the most appropriate internal reference genes under drought stress,18 srRNA and GAPDH were the most suitable internal reference genes under salt stress.GAPDH and ACTIN2 had the most stable expressions under low/high temperature stress;4)Based on the results of DEG and RNA-seq sequencing,nine upregulated genes were randomly selected,and RT-qPCR was conducted to verify the validity of RNA-seq data using dual-reference genes(TUB and ACT2).It was found that the expression of nine genes increased with the length of treatment time,which proved the reliability of RNA-seq results.In summary,RNA-seq analysis was used to compare the expression of ER stress-related genes in Cyclocarya paliurus under TM treatment in this study.The number and types of internal reference genes that can be used for RT-qPCR standardization were found.The most suitable internal reference genes in TM treatment and under other abiotic stresses were identified as well.The results of this study may provide scientific basis for the ER stress studies.