Mining And Functional Study Of Genes Involved In Crocins Biosynthesis Or Stigma Development In Crocus Sativus L.

Crocus sativus L.is a kind of herbaceous plant belonging to the genus Crocus(monocot)of the Iridaceae family.It is wildly used in China because of its functions of promoting blood circulation,cooling blood,detoxifying the body,and relieving depression.C.sativus is clinically used for the treatment of cardiovascular diseases and depressive disorder.Medicinal research suggested that apocarotenoids like crocins,crocetin,picrocrocin,safranal are important material basis of significant pharmaceutical effects of C.sativus.Stigmas are sites for crocins biosynthesis and accumulation,which has a small biomass,leading the low production.Integrate analysis of multiomics data to identify genes involved in stigma development or related to glycoside synthesis,then uncovering accurate crocins biosynthesis pathway and verifying genes function which involved in stigma development.On this basis,creating the cultivation of high-yield and high-quality strains which will effectively alleviate the conflict between supply and demand in the saffron market.To discover potential key catalytic steps and the basis of molecular regulation,we performed deep transcriptomic sequencing and dynamic metabolome analysis using different developmental stage stigmas.In particular,metabolic flux associated with crocins biosynthesis was monitored.269,943 unigenes of stigma were obtained by comprehensive de novo assembly.The transcripts were distinct at different stages,and all differentially expressed genes could be classified into 7 clusters according expression patterns.According to the expression patterns,all enzyme genes(181)and 63 transcription factors involved in biosynthesis of apocarotenoids were screened out.15 important metabolites were targeted and hierarchically clustered into 4 groups by analysing metabolomics data.To further explore the relationships between metabolites and genes,a “TF-gene-metabolite” coexpression network was constructed though integrate the results of transcriptome and metabolome.From the network,32 carotenogenic genes and 28 transcription factor were captured with the highest Kcore values.More importantly,the number of transcripts encoding β-GS /ADH / UGT was reduced to 7/4/4.Comprehensive analysis of "gene-compound" co-expression network and fluorescence quantitative PCR results indicated that the expression of AGL6 b gene was positively correlated with the content of crocetin developed with style.The AGL6 gene belongs to the family of MADS-box regulators,which have been shown to regulate floral development in a variety of plant species.The saffron AGL6 b gene is closely related to the accumulation of secondary metabolites in the synthesis pathway of crocin,possibly through regulating the development of the floral organs of Crocus sativus,causing the change of the style of the saffron to cause changes in the content of secondary metabolites.The composition and properties of amino acid sequences,signal peptide,leader peptide,transmembrane topological structure,subcellular localization,hydrophilicity or hydrophobicity,secondary structure,tertiary structure and functional domain of the 17 full-length MADS-box proteins were analyzed by systematic bioinformatics methods.The Homology analysis of the Cs MADS-box and other plants was also compared.Simultaneously,the phylogenetic tree of Cs MADS-box and Arabidopsis was constructed.These results will provide reliable information for further research on the biological functions of the Cs MADS-box.Integration of co-expression networks and C.sativus transcriptome,5 MADS-box genes with high specific expression in the C.sativus stigma were obtained,named SCR(Stigma and Crocins Regulator).To verify the role of SCR transcription factors in the regulation of C.sativus stigma development or glycoside synthesis,all sequences were cloned and analyzed.And also subcellular localization experiments and gene-overexpression in Arabidopsis were initially performed.The molecular weight of the amino acid encoded by the Cs SCR gene is about 25.0 k Da,and the isoelectric point is approximately 9.15,which showed that they were all basic proteins.At the same time,the domain analysis showed that all five proteins contained a MADS domain consisting of 60 amino acids,followed by a KBOX domain contain 93 amino acids.This demonstrated that all five proteins belong to the MADS-box transcriptional family.The results of subcellular localization experiments confirmed that five Cs SCR proteins are located in the nucleus act as transcriptional regulators.The experiment of gene-overexpression in Arabidopsis found that Cs SCR3 and Cs SCR5 can regulate the development of Arabidopsis flower,leading early flowering.In general,several candidate genes involved in the biosynthesis of crocins were reported by integrating multiomics data in this study,which laid the foundation for the accurate analysis of the crocins biosynthesis pathway in C.sativus.It also provides a new perspective of metabolic engineering research to improve the content of crocins from regulating the development of stigma.Based on this,it will cultivate a high-yield and highquality C.sativus line,which could provide a solid upstream support for strengthening the saffron industry.