Preliminary Study On Protein S-nitrosylation-Mediated Triterpenoids Synthesis In Betula Platyphylla Treated With Nitric Oxide

Nitric oxide is a necessary signaling molecule for the synthesis of secondary metabolites in plant cells,and protein S-nitrosylation is one of the most important protein translation mediated by NO.It has not been reported whether NO-mediated protein S-nitrosylation is involved in the triterpenoid accumulation.Therefore,the effect of NO and H2S on the triterpenoid accumulation in birch suspension cells,protein S-nitrosylation mediated by NO on triterpenoid accumulation in GSNOR transgenic plants will be revealed.The main results are as follows:1.Endogenous NO/H2S mediated triterpenoid accumulation under H2S/NO treatmentThe H2S donor NaHS and its inhibitors hydroxylamine(NH2OH),NO donor sodium nitroprusside(SNP)and nitric oxide synthase inhibitors(L-NAME)were added to the 8d cultured birch suspension culture system treated by 2d.We analyzed the content of H2S and NO,key enzyme activity of H2S and NO synthesis,and the content of betulin in birch suspension cells.The results showed that exogenous H2S promoted the accumulation of endogenous NO and betulin,in which NO content was increased by 60.84%,and betulin content was increased by 71.03%;Exogenous NO had an induction effect on the accumulation of endogenous H2S and betulin,and the content of H2S was 28.23%higher than that of the control,and the content of betulin was 234.41%higher than that of the control group;The content of H2S and betulin with the treatment of SNP+ NH2OH was lower than the treatment of SNP,NaHS+L-NAME had no obvious effect on NO and betulin compared with that of NH2OH.The above results showed that the interaction between endogenous NO and H2S was involved in the triterpenoid synthesis in birch suspension cells.2.Differently expressed gene of GSNOR transgenic plants in Betula platyphyllaUsing Illumina high throughput sequencing technique,the GSNOR silencing transformation plants were used to sequence and assemble,and the Unigenes were analyzed by functional annotation,classification and metabolic pathway.The results showed that,there were 61,136 Unigenes with the N50 value was 1,753bp and the average length was 950bp.Compared with Nr,KEGG?Swiss-prot and COG/KOG database,31803 Unigenes were annotated.There were 7,038 differently expressed gene in GSNOR transgenic plants,and the expression quantity up-regulated genes were 3,464,and the expression quantity decreased genes were 3,574.The Unigenes were selected according to coefficient with gene expression value of GSNOR((?)0.99).The results showed that most of the Unigenes that were negatively correlated with gene expression value of GSNOR were derived from the transcription factor family,including NAC,BZIP,MYB,BHLH,ABC,while significantly correlated derived from nucleic acid metabolism and amino acid metabolism.According to the analysis of correlation between gene expression value of GSNOR and the triterpenoid synthesis Unigenes,the correlation value between the CAS,?-AS and the GSNOR was 0.8 and 0.5,respectively.3.Effect of NO-mediated protein S-nitrosylation on the triterpenoid accumulation510 unigenes were further analyzed with a difference of more than 10 times between wild and GSNOR silencing transformation plants.Among of them,there are 472 downward Unigenes,mainly distributed in disease-resistant proteins;there are 30 rise Unigenes,mainly distributed in the cytochrome P450 family and riterpenoid synthesis pathway.The key gene expression of FPPS,LUS,CAS2 and VIT in triterpenoid synthesis and the content of the triterpenoid were confirmed by real-time quantitative PCR and colorimetry,respectively,which were the same with the results of transcriptome sequencing.It is concluded that NO-mediated protein S-nitrosylation was involved in the triterpenoid accumulation.