RNA-Seq For Transcript Profiling And Study On Signal Response Mechanism By Slicylic Acid In Stevia Rebaudiana Bertoni

Stevia belongs to the Asteraceae?Compositae?Stapleton Via genus of herbs,which is paid much attention as a kind of both sugar and medicinal plant.Its main component is stevioside?SGs?which is a mixture including rebaudioside A?Rebaudioside A,RA?,stevioside?Stevoiside,ST?and rebaudioside C?Rebaudioside C,RC?.RA is ideal for sweetening ingredients because its high sweetness and taste quality is close to sucrose.ST and RC have a bitter taste which largely affects the taste of stevia glycosides quality,so its application was limited.Therefore,it is the trends of stevia breeding and biotechnology improving the content and proportion of a certain glycosides such as RA glycoside in stevia.Although in recent years,glycoside synthesis-related genes have been reported,the molecular mechanism of glycoside synthesis remains unclear.Lacking of stevia genome sequence information greatly limits the biotechnology and molecular marker-assisted breeding.In this study,we studied the affect of salicylic acid?SA?on the genes expression related SGs biosynthesis,the content of SGs and the key enzyme in the SGs biosynthesis pathway.The main results are as follows:We did transcriptome sequencing for three different stevia strains:SR₁,SR₂ and SR₃ leaf.The SGs content among strains were significantly different,especially ST and RA.The ST content of SR₁,SR0₂ and SR₃ were 2.19%,12.87%and 1.23%,respectively;RA contents were 6.91%,0.02%and 9.35%,respectively.With Illumina HiSeqTM2000 sequencing,we respectively got 58,857,260 60,113,164;66,869,210 clean reads for SR₁,SR 2 and SR₃.After assembling and blasting,we obtained 80,160 unigenes.By pairwise comparison,we found that 636 differentially expressed genes between SR₁ and SR 2 which were enriched in 95 pathways.The metabolic pathway had the largest number of differentially expressed genes;2,464 differentially expressed genes between SR₁ and SR₃ which were mainly enriched in 179 pathways,and the secondary metabolism pathway had the largest number of differentially expressed genes;2,041 differentially expressed genes between SR₂ and SR₃ which mainly enriched in 189 pathways,and the metabolic pathway had the largest number of differentially expressed genes;.Combing ingredients analysis,we obtained 201 unigenes which might participate ST and RA biosynthesis pathway.In this transcriptome data,we found that there were 161 unigenes which were annotated to be UDP glycosyltransferase?UGTs?,including the reported glycosyltransferase genes UGT85C2,UGT74G1 and UGT76G1.A total of 10 070 SSRs were identified in 80 160 unigenes,with one SSR per 5.41 kb,accounting for 12.56%.Among all 60 SSR motifs,?A/T?n was the most frequent repeat motif?54.61%?,followed by?ATC/ATG?n?9.71%?and?AT/AT?n?8.03%?.The results of SSR-PCR amplification for randomly selected 20 primer pairs among 12 strains of stevia,showed that all pairs were polymorphic,accounting for 100%of the total design primers.There was correlation between the origins and cluster results of the tested S.rebaudiana lines based on UPGMA method to some degree.After 2mmol/L SA treatment,we found that the RA,ST,Rebaudioside C?RC?content of stevia were increased in varying degrees.The total glycosides?RA + ST + RC?content reached the maximum value of 17.2%after 24h treatment,comparing with 5.48%of CK,the difference was obviously significant.The growth of total glucosinolate was slower by the treatment time.RT-qPCR results showed that the expression of 15 genes involved in the synthesis of stevia glycosides was affected after SA treatment.The expression of UGT85C2,UGT74G1 and UGT76G1 participated in SGs synthesis reached the maximum level at 9h after SA treatment.At later stage,the expression of UGT85C2 and UGT76G1 was similar to CK,the expression of UGT74G1 was significantly higher than CK.Those results indicated that SA affected the synthesis of stevia glycosides,probably through regulating the gene expression related stevia glycosides biosynthesis.We also found that CAT activity showed "S" variation;POD,SOD activity and H2O2 content showed peak shape variation with 2mmol/L SA treatment.In the early induction?0-3h after treatment?,the activity of CAT and SOD was respectively decreased and increased;H2O2 content reached the maximum at 9h after treatment.Among 9-24h after SA treatment,The POD enzyme activity was rapidly increased in order to maintain H2O2 content balance.The endogenous SA of steiva was rapidly increased at 3h after SA treatment,but decrease with prolonged treatment time.The endogenous SA was not significantly different between treatment and CK.The expression of ICS-7and PAL-7 which participated in SA synthesis reached the maximum at 3h?9h after SA treatment,respectively,comparing with the control group increased 1.45 and 1.82 times respectively.The expression of NPR1-4 which was a key regulator of SA mediated resistance response was increased at 1.5h after SA treatment and reached the maximum at 24h after SA treatment,comparing with the control group increased 2.65 and 4.72 times,respectively.This experiment determined the expression level of CaMl and CaM2.RT-qPCR results showed that the expression of calmodulin gene was growing rapidly,reached the maximum at 9h after SA treatment,comparing with the control group increased 2.29 and 2.17 times respectively.The expression level was not significantly different between treatment and CK.With trancriptome sequencing,we investigated the genes with different expression between SA and water?control?treatment using the stiva leaves at 9h after treatment.The results showed that CK_leaf and SA_leaf respectively had 15,004,886 and 13,698,145 clean reads;We used the sequences obtained by trinity assembling as a reference gene sequence,then aligned each sample reads with reference gene sequence.After that,there were 11,967,707 of CK_leaf and 12,112,709 of SA_leaf clean reads which could be aligned to the reference gene sequences.Comparing the expressed genes of SA_leaf with CK leaf,we found 170 differentially expressed genes between them,including 52 upregulated genes,118 downregulated genes.The further analysis results showed that the expression of homologs of two abscisic acid?ABA?synthesis related genes?carotene lyase 4 and ?-glycoside hydrolases?was obviously upregulated in the SA-treated leaves.The investigation about the ABA biosynthetic homologs ZEP,NCED and SDR in Stevia showed:There were 518 and 24 genes which function were annotated as ZEP,NCED and SDR respectively.At the 9h point after SA treatment,the expression of 3 ZEP homologs,24 SDR homologs and 18 NCED homologs genes were regulated.The dynamic changes of ZEP,NCED,SDR in stevia leaves were verified by RT-qPCR.The expression of 3 genes were slightly decreased compared with the control at 1.5h after SA treatment and reached the maximum at 9h after SA treatment.Compared with the control group increased 3.73 and 2.18 times respectively.Therefore,SA signal pathway transduction related SGs biosythnsis was probably related ABA-mediated signal transduction pathways in stevia.However,the questions whether ABA signal pathway was directly related with the biosynthesis SGs or whether ABA affected the expression of SGs synthesis needed to further study.Our results provided a good theoretical basis for improving SGs content using biotechnology and molecular markers-assistant breeding,and laid a good foundation for understanding the relationship between SGs contenet and gene expression.