Cloning And Functional Analysis Of Key Morphine Synthesis Genes CODM And STORR In Papaver Somniferum L.

Objective:Based on the whole genome resequencing data,CODM gene and STORR gene in the synthesis pathway of alkaloid were cloned.Through the analysis of the gene sequence,all the sequences of CODM gene and STORR gene of Papaver somniferum L.were preliminary obtained.At the same time,the STORR gene and its mutant gene were expressed in eukaryotes,and the enzyme activity was measured in vitro.The molecular mechanism of alkaloid accumulation difference in different poppy resources was preliminarily discussed,which provided a research basis for the exploration of key enzyme genes in the synthesis pathway of Papaver somniferum L.Methods:On the basis of the cluster analysis of alkaloid content in the Papaver somniferum strains,nine representative strains were tested for Single-nucleotide polymorphism by whole genome resequencing,the key enzyme genes with large gene variation were selected as candidate genes.2.The CODM gene sequences of Papaver somniferum and Papaver rhoeas were obtained by cloning and sequencing.The differences of CODM gene expression between the Papaver somniferum and Papaver rhoeas were revealed by using nucleotide and amino acid sequence alignment,phylogenetic tree construction and other bioinformatics methods.3.Through cloning and sequencing,the sequence of the Storr gene of Papaver somniferum was obtained,and the mutant of STORR gene was selected by sequence comparison.The expression of the STORR gene and its mutants was carried out in Pichia pastoris and Saccharomyces cerevisiae,and the activity of the STORR gene and its mutants was detected.Results:1.Based on the analysis of SNP data of whole genome re sequencing,it was found that there were significant differences in SNP results of several genes in different chemical types of poppy and its related species.Combined with the literature,we selected the STORR gene with the most gene variation and the CODM gene without amino acid variation for representative study.2.The results showed that there were three genotypes in CODM of Papaver Somniferum L.and five genotypes in CODM of Papaver rhoeas L.,including three genotypes of Papaver somniferum L.,and the consistency of five sequences was 99.89%.Bioinformatics analysis showed that all CODM proteins had no signal peptide sequence,and these proteins were predicted to be non-secretory proteins,belonging to the Pcbc superfamily supergene family.The amino acid sequence of CODM in poppy is the same,but the expression of CODM in different poppy resources is significantly different.It is speculated that the variation of transcription level of CODM may be related to its non-coding region sequence.3.The results of sequence alignment showed that there were two kinds of STORR genes,homozygous and heterozygous,and there were four different genotypes,the sequence consistency was about 97.89%.The expression vector of Saccharomyces cerevisiae was successfully transferred into WAT11,but the activity of the enzyme was not detected;Pichia pastoris expression vector was successfully transferred into GS115 strain,and after induced expression,it was found that the mutant STORR gene can catalyze the transformation of(S)-Reticuline into a new compound,but the reaction conditions and product need to be further optimized and identified.Conclusion:In this study,based on the whole genome resequencing,the CODM gene and STORR gene sequence were selected as the specific research object,and the nucleotide sequences of two genes were cloned.Different strains of Papaver somniferum contain the same CODM genotypes,but their expression levels are different.It is speculated that the variation of codm gene transcription level may be related to its noncoding region sequence.The STORR gene was expressed by eukaryotes and the STORR mutant was found to have a certain activity,which laid a foundation for further research on the synthesis and regulatory mechanism of alkaloids from Papaver somniferum.