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Comparative transcriptome analysis using high papaverine mutant of Papaver somniferum reveals pathway and uncharacterized steps of papaverine biosynthesis.

ABSTRACT: The benzylisoquinoline alkaloid papaverine, synthesized in low amount in most of the opium poppy varieties of Papaver somniferum, is used as a vasodilator muscle relaxant and antispasmodic. Papaverine biosynthesis remains controversial as two different routes utilizing either (S)-coclaurine or (S)-reticuline have been proposed with uncharacterized intermediate steps. In an attempt to elucidate papaverine biosynthesis and identify putative genes involved in uncharacterized steps, we carried out comparative transcriptome analysis of high papaverine mutant (pap1) and normal cultivar (BR086) of P. somniferum. This natural mutant synthesizes more than 12-fold papaverine in comparison to BR086. We established more than 238 Mb transcriptome data separately for pap1 and BR086. Assembly of reads generated 127,342 and 106,128 unigenes in pap1 and BR086, respectively. Digital gene expression analysis of transcriptomes revealed 3,336 differentially expressing unigenes. Enhanced expression of (S)-norcoclaurine-6-O-methyltransferase (6OMT), (S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT), norreticuline 7-O-methyltransferase (N7OMT) and down-regulation of reticuline 7-O-methyltransferase (7OMT) in pap1 in comparison to BR086 suggest (S)-coclaurine as the route for papaverine biosynthesis. We also identified several methyltransferases and dehydrogenases with enhanced expression in pap1 in comparison to BR086. Our analysis using natural mutant, pap1, concludes that (S)-coclaurine is the branch-point intermediate and preferred route for papaverine biosynthesis. Differentially expressing methyltransferases and dehydrogenases identified in this study will help in elucidating complete biosynthetic pathway of papaverine. The information generated will be helpful in developing strategies for enhanced biosynthesis of papaverine through biotechnological approaches.

SUBMITTER: Pathak S

PROVIDER: S-EPMC3667846 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Comparative transcriptome analysis using high papaverine mutant of Papaver somniferum reveals pathway and uncharacterized steps of papaverine biosynthesis.

Pathak Sumya S Lakhwani Deepika D Gupta Parul P Mishra Brij Kishore BK Shukla Sudhir S Asif Mehar Hasan MH Trivedi Prabodh Kumar PK

PloS one 20130530 5

The benzylisoquinoline alkaloid papaverine, synthesized in low amount in most of the opium poppy varieties of Papaver somniferum, is used as a vasodilator muscle relaxant and antispasmodic. Papaverine biosynthesis remains controversial as two different routes utilizing either (S)-coclaurine or (S)-reticuline have been proposed with uncharacterized intermediate steps. In an attempt to elucidate papaverine biosynthesis and identify putative genes involved in uncharacterized steps, we carried out c ...[more]

PMID: 23738019

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Project description:INTRODUCTION:Papaver somniferum is the commercial source of morphine and codeine. The isolation of effective genes involved in the morphine biosynthesis of P. somniferum is very important in the production of specific metabolites achieved using metabolic engi-neering techniques. In this pathway, the key enzyme COR is involved in the conversion of codeinone to codeine and morphinone to morphine. METHODS:the gene encoding of this enzyme was isolated using primers designed on the base of gene sequence available on (NCBI) for P. somniferum. This gene correct size around (960 bp) was first subcloned into pTZ57RIT vector then cloned into expression vectors (pBI121) between BamHI and SacI sites to allow the expression of cor gene driven by the cauliflower mosaic virus 35S pro-moter. The result was confirmed through different molecular methods e.g. PCR and en-zyme digestion by BamHI and SacI. The recombinant plasmid was transformed into the E. coli strain DH5? using a freeze-thaw method. Having selected positive colones on selection medium, plasmid was extracted by miniprep method and recombinant plasmids were selected based on PCR and digestion. The construct was then mobilized in Agrobacterium tumefaciens C58/pGV3850 (KmR RifR). After gene transformation to P. somniferum plants, the agroinfiltration method was also used for transient expression of COR enzyme. RESULTS:evaluation results showed that morphine and codeine were detectable in the leaves of transgenic plants containing cor transgene and there was significant difference in the final production. After completing this experiment for three times, results showed that in 11 sets from 15 sets of leaves experiment tested, main alkaloids (codeine, morphine, papaverin, noscapine and thebaine) were detectable. CONCLUSION:Whereas no signal was detected in non-infiltrated control leaves or in leaves infiltrated with non-recombinant bacteria for morphine and codeine, others such as thebaine and papaverine were detectable.

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Comparative transcriptome analysis using high papaverine mutant of Papaver somniferum reveals pathway and uncharacterized steps of papaverine biosynthesis.

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Comparative transcriptome analysis using high papaverine mutant of Papaver somniferum reveals pathway and uncharacterized steps of papaverine biosynthesis.

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