Screening Of Stable Reference Genes Under Different Stress Conditions In Parsley

Parsley(Petroselinum crispum L.),is a kind of biennial herbaceous plant in Apiaceae family,also known as coriander or french parsley.As a kind of spicy vegetable,its tender leaves can be eaten,and fresh root and stalk juice can be used for medicinal.Though there are many methods for detection of the gene expression level,such as qRT-PCR,all need to use some reference genes for normalization to get relatively more reliable results.In this study,the qRT-PCR assays for quantification of each reference gene had been performed.The further researches on the gene expression stability under different stress treatments were evaluated.In this study,to further verify the expression stability of the reference genes,we also investigated the expression profiles of the pcDREBl gene in parsley under high temperature stress treatment.The specific findings are as follows:(1)Part of fragments of 8 reference genes(GAPDH,ACTIN,eIF-4?,SAND,TIP41,UBC,EF-1? and TUB)in parsley were selected.The BLAST results indicated that all the genes showed highly similarity with homologous genes in other species,and the base homology was more than 80%.These results showed that the candidate reference gene are highly conserved in different species,and they had a good application universality for quantitative analysis.(2)qRT-PCR assays of each gene were established.Standard curves showed highly sensitivity and correlation when we used 10-fold dilution of cDNA as templates to calculate the amount of gene in samples.This will provided more accurate data for subsequent experiment.(3)The cDNAs under different stress treatments were served as temples for analyzing the expression stability of 8 reference genes using qRT-PCR.The analyses were performed by three biology software(geNorm,BestKeeper,and NormFinder).The result showed that the TUB and ACTIN under high temperature treatment displayed relatively stable level of expression.Under low temperature stress,eIF-4? and GAPDH could be used for stability analysis.Under drought treatment,the genes of EF-1? and ACTIN were the best reference gene.EF-1? and GAPDH were the best choice under drought stress treatment.EF-1?performed best under GA and ABA treatment.Under SA and MeJA treatments,ACTIN and EF-1? worked best treating with SA and MeJA.