RNA-seq Of Hyoscyamus Niger And Functional Characterization Of HnODC

Hyoscyamine and scopolamine,belonging to tropane alkaloids（TAs）,are clinically used as anticholinergic drug.Hyoscyamine,as well as scopolamine,has pharmaceutical effects on parasympathetic nervous system and shows the functions associated with anesthesia and pain relief.These TAs are specifically produced in secondary roots of the medicinal plants of Solanaceae,such as Hyoscyamus niger,Atropa belladonna,Datura species,Scopolia lurida and so on.The TA biosynthesis enzymes/genes,including putrescine N-methyltransferase（PMT）,tropinone reductase I（TRI）and hyoscyamine 6β-hydroxylase（H6H）,have been functionally characterized.However,the TA biosynthetic pathway has not been completely understood due to more unknown enzymes.Ornithine decarboxylase（ODC）catalyzes the decarboxylation of ornithine to produce putrescine that is the general precursor for TA biosynthesis.To understand the ODC roles in the TA biosynthesis,we sequenced the transcriptomes of Hyoscyamus niger,and isolated the gene encoding ODC from H.niger（HnODC）,analyzed its expression pattern and investigated its enzymatic activity.The transcriptomes of secondary roots and leaves of H.niger were respectively sequenced.The clean reads for secondary root and leaf transcriptomes were 99.75%and 97.94%,respectively.Differential gene expression analysis indicated that 8107 genes were differentially expressed between secondary roots and leaves,and 4186 genes among the 8107ones had higher expression levels in secondary roots.The characterized TA biosynthesis genes,including PMT,TRI and H6H are highly/specifically expressed in secondary roots,and further their expression was validated using qPCR.High/specific expression in secondary roots of these TA biosynthesis genes was consistent with the fact that TAs were synthesized in secondary roots.Based on the transcriptomes of Hyoscyamus niger,7 transcripts were found to be the members of pyridoxal-dependent decarboxylase group 4.Among them,the transcript with the record number of comp39535_C0_Seq1 was predicted to be ornithine decarboxylase of H.niger（HnODC）.This transcript has similar digital expression pattern to the identified TA biosynthesis genes,and all of them were highly expressed in secondary roots.Then the full-length cDNA of HnODC was cloned using RACE technology.Bioinformatic analysis showed that HnODC was highly similar to other plant ornithine decarboxylases,especially the ODC enzyme of Datura stramonium.High expression of HnODC in secondary roots was confirmed using qPCR.Like HnPMT,HnODC was also induced by methyl jasmonate（MeJA）.To confirm that HnODC catalyzes ornithine to yield putrescine,HnODC was expressed in E.coli to generate the recombinant protein.His-tagged HnODC protein was purified from E.coli using Ni²⁺-chelating column and the molecular weight of HnODC was approximately 50kDa,consistent with its calculated molecular weight and similar to those of ODC from tobacco（NgODC）and cocao（EcODC）.When ornithine was fed to HnODC,the product of putrescine was detected,suggesting that HnODC did have the function of ornithine decarboxylase.Finally,the kinetics of HnODC was determined.The values of Km,Vmax and Kcat of HnODC were 3.40 mM,2.095 nmol.min^-1.μg^-1 and1.809 s^-1,respectively.The results of enzymatic assays indicated that HnODC had lower affinity to ornithine than NgODC and EcODC,but it had higher catalytic efficiency than NgODC and EcODC.To be summarized,HnODC was isolated based on Hyoscyamus niger transcriptomes and it was highly expressed in secondary roots and could be induced by MeJA.HnODC could efficiently catalyze ornithine to produce putrescine.Molecular cloning and enzymatic identification of HnODC facilitated understanding of its roles in the TA biosynthesis.