Function Of MiR156 And Transcription Factor SPL In Secondary Metabolism Of Catharanthus Roseus

Catharanthus roseus is one of the most popular plants that have been studied in recent years.It is mainly planted in the provinces and regions such as southwest,middle and South China and East China.It has extremely rich medicinal value.The main secondary metabolites of Catharanthus roseus are alkaloids.Researchers have isolated more than 130 alkaloids from it,of which 6 have antitumor effects.However,in natural Catharanthus roseus,the content of these alkaloids is very small,and the growth environment and the growth cycle of the plant itself,the extraction of these alkaloids from the natural Catharanthus roseus is more limited,and the market is in short supply.Moreover,the synthetic routes of these secondary metabolites are extremely complex,and the chemical synthesis procedures are complex and costly.In recent years,researchers have explored how to use genetic engineering to regulate the synthesis of Catharanthus roseus alkaloids by analyzing the key enzymes in the secondary metabolic pathways their mechanisms of action.Studies have shown that MicroRNA(miRNA)is one of the most important regulatory elements of gene expression in many plants.It participates in the regulation of almost all plant physiological metabolic processes,such as miR156.Its target gene is called SPL(SQUAMOSA promoter binding protein-like)family.The SPL protein family is a large family of plant specific transcription factors,with a highly conserved-SBP domain in the DNA binding region,and most of them contain the identification sites of miR156.In Arabidopsis,miR156 controls the evolution of MYB-BHLH-WD40 transcriptional complex through the negative regulatory transcription factor SPL9,and further controls the expression of the anthocyanin synthesis gene DRF(Dihydro Flavonol Refuctase)and negatively regulates the synthesis of anthocyanins.This article expects to clone the gene of Catharanthus roseus transcription factor SPL9,predict and analyze its amino acid sequence structure,explore the mechanism between miR156 and the transcription factor SPL9 in Catharanthus roseus,and further study their regulation of the key enzymes in the secondary metabolic pathway of Catharanthus roseus,and finally to promote the production ofalkaloids.The research provides the basis and new ideas on the accumulation of secondary metabolites in Catharanthus roseus.In this study,wefirst extracted the total RNA of Catharanthus roseus,and reverse transcribed the cDNA.The designed specific primers to clone the SPL9 gene of Catharanthus roseus with cDNA as template,and named CrSPL9.Then weconstructed the recombinant plasmid pGMT-CrSPL9 and pBI121GD-CrSPL9,and compared the sequence of CrSPL9 gene with the original sequence,and predicted and analyzed its amino acid sequence.We transiently converted the plasmid pBI121GD-CrSPL9 and plasmid pCAMBIA2301-MIR156 fto Catharanthus roseusseedlings through agrobacterium mediated transformation.Then use QuantitativeReal-time PCR(qRT-PCR)technique to analyze the interaction between miR156 and transcription factor SPL9,and futher explore the regulation of miR156 and transcription factor SPL9 on the key enzymes in the biosynthetic pathwayof secologanin from geraniol inCatharanthus roseus.The results showed that the CrSPL9 gene of 1600 bp was successfully cloned from Catharanthus roseu leaves.After gene sequencing and homologous sequence alignment,it was found that the SPL9 gene of Catharanthus roseur was the most homologous to the gene of Durio zibethinustranscription factor SPL6(81%),and has a high similarity to 80% of the Olea europaea var.transcription factor SPL6.After the analysis of the amino acid sequence of the gene sequence,we found that CrSPL9 has a highly conserved-SBP domain and a highly conserved bi-directional nuclear location signal KRXXXRRRK.TheCrSPL9 gene sequence contains the miR156 recognition site,which indicates that CrSPL9 is also the target gene of miR156 in Catharanthus roseu.The results of qRT-PCR showed that the expression of miR156 and transcription factor SPL9 in the leaves of wild type Catharanthus roseus had temporal and spatial specificity: the expression of miR156 was the highest in the new leaf,and the expression amount decreased with the increase of leaf age,while the expression of CrSPL9 was in the opposite expression pattern with miR156..The expression levels of CrSPL9 and miR156 in Catharanthus roseu leaves transiently transformed pBI121GD-CrSPL9 and pCAMBIA2301-MIR156 f were higher than those in the control group.In the plants that overexpress CrSPL9 and miR156,one of the key enzymes in the iridoids pathway,is regulated accordingly: the expression of CrLAMT is inhibited in the leaves of theplants that overexpresses CrSPL9;In the leaves of the plants that overexpress miR156,the amount expressionofCrLAMT was obviously up-regulated.In conclusion,this study explored the amino acid structure of the transcription factor by cloning and analysis of the transcription factor gene CrSPL9,and confirmed the relationship between CrSPL9 and miR156 at the molecular level.And by overexpressing CrSPL9 and miR156,we found they have a regulatory effect on the key enzymes in the secondary metabolic pathway of Catharanthus roseus,loganic acid methyltransferase(LAMT).This study will provide some new ideas and lay a foundation for the regulation of miRNA and transcription factors in the secondary metabolites of Catharanthus roseus or other medicinal plants.