Cloning, identification and expression analysis of stylopine synthase )STS) gene in Chelidonium majus L.

Document Type : Research Paper

Authors

1 Department of Genetics and Plant Breeding, Imam Khomeini International University, Qazvin, Iran

2 Associate Professor, Department of Genetics & Plant Breeding, Imam Khomeini International University, Qazvin, Iran.

10.22092/ijrfpbgr.2023.357538.1405

Abstract

Background and objectives
Sanguinarin is an important secondary metabolite of the benzyl isoquinoline alkaloids, which is widely found in Papaveraceae family plants such as Chelidonium majus. Sanguinarin has many properties such as antimicrobial, antifungal, and anti-inflammatory. The use of abiotic elicitors is one of the effective ways to induce and increase of secondary metabolites. The elicitors such as methyl-jasmonate induce the biosynthesis and accumulation of secondary metabolites by stimulating the plant's defense system. Since there is no report on the isolation of cDNA encoding the stylopine synthase in sanguinarine biosynthesis pathway in Chelidonium majus, the full length of this gene was isolated to provide an opportunity to identify and investigate the pathways of benzylisoquinoline that have not been known so far. Also, the expression pattern of this gene was surveyed under the treatment of methyl-jasmonate to improve its production.
Methodology
 In this research at the first stage, the cDNA encoding the stylopine synthase gene (STS) was identified and isolated from the root of Chelidonium majus. STS is one of the genes in sanguinarin biosynthesis pathway that converts (S)-Cheilanthifoline to (S)-Stylopine in the methyloxy bridge, which belongs to CYP719 subfamily. In the next stage, the expression pattern of STS gene was examined in a completely randomized design with four replicates by applying methyl-jasmonate (concentration of 100 μM) at four treatment levels (foliar, irrigation, foliar + irrigation and without methyl-jasmonate as control) in different organs (leaf, root and stem) and different sampling times (6, 24 and 48 hours after methyl-jasmonate treatment) In a pot experiment under greenhouse condition.
Results
 Isolation of STS gene cDNA was done in two steps. At first, the incomplete sequence (1000 bp) was isolated, and then, by designing new primers based on the information from the primary sequence, the full length (1500 bp) of the coding region of gene was isolated and successfully cloned into pTG19-T plasmid. After sequencing and necessary modifications, the obtained gene sequence was registered in the NCBI database with the accession number KY550671.2. By evaluating the sequence characteristics and studying the phylogenetic relationships, it was determined that CYP719A3, the product of the STS gene, belongs to the P450 protein family. The results of variance analysis showed the methyl-jasmonate treatment affect significantly (P≤0.01) STS gene expression at different times in different organs of Chelidonium majus. The highest level of gene expression was observed in methyl-jasmonate foliar spraying + irrigation 48 hours after treatment in the root. The mean comparison showed that the expression level of STS gene was different in the root, leaf and stem of Chelidonium majus, so it was 1.5 and 2.5 times higher in the root than leaf and stem, respectively. The high expression of the STS gene in the root of Chelidonium majus indicates the effectiveness of the plant organ in the level of gene activity, on the other hand, the use of methyl-jasmonate could play an effective role in increasing gene expression.
 
Conclusion
 Methyl-jasmonate increased the expression of STS gene in the sanguinarine biosynthesis pathway so that the highest expression was observed in the root. Therefore, the sequence of the gene encoding stylopine synthase in the sanguinarine biosynthesis pathway and its expression pattern in Chelidonium majus can be used in the pathway engineering of this valuable metabolite.

Keywords

Main Subjects

References

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