Cloning And Characterization Of Tropinone Reductase I From Atropa Belladonna And Its Overexpression For Enhancing The Tropane Alkaloids Production In Atropa Belladonna

Tropane alkaloids (TAs) are a class of low molecular weight nitrogen-containing substances mainly extracted from Solanaceae, which offer clinically commonly used anticholine drugs hyoscyamine and scopolamine widely for the pain relief, anesthesia, refraining drug addiction, control of motion sickness and cure of Parkinson’s disease with an enormous maket damands. Due to the expensive cost price in industral synthesis, hyoscyamine and scopolamine for now are acquired mainly by extracting from three Solanaceae plants Atropa belladonna, Hyoscyamus niger and Datura stramonium, and the production is limited, among which A. belladonna is the only commercial plant species (authorized by pharmacopoeia of The People’s Republic of China2011) that can be used to extract TAs, so it is of great significance to carry out metabolic engineering to enhance TAs contents in A. belladonna. In this study, we cloned the full-length cDNA of tropinone reductase I (TRI) from medicinal plant A. belladonna using the RACE method and made a detailed bioinformatic analysis, simultaneously expression profiles of AbTRI in different tissues and induced expression profiles were constructed; after activity validation through prokaryotic expression, the plant expression vector was constructed to genetically transform belladonna to obtain transgenic hairy root, and then liquid cultured to the end of the exponential growth to detect the total TAs content and the corresponding AbTRI gene expression. The final results showed that AbTRI functioning as a check point enzyme in A.belladonna, its overexpression can greatly promote the flow of metabolic flux towards hyoscyamine and scopolamine, thus significantly enhancing the production of the total TAs. TRI is the check-point enzyme which locates at the middle branch of TAs biosynthetic pathway, catalyzing the reduction of carbonyl in tropinone molecules to generate α-hydroxytropane (tropine). HnTRI gene in H. niger plant is strongly expressed only in the root inner cortex and outer cortex with specificity, and the TRIs in D. stramonium and Anisodus acutangulus are also cloned from the cultural root tissue. In this study, A. belladonna root was adopted for material and we successed in obtaining the full-length cDNA of AbTRI gene of1079bp based on homology cloning method, using RACE technology. The cDNA consists of a coding sequence of819bp,5’UTR of14bp,3’UTR of234bp and polyA of13bp with termination codon of TAA. The coding region encodes a272-amino-acid-residues protein (AbTRI) which was predicted to have a molecular weight of29.3KDa and an isoelectric point of6.05.Bioinformatics analysis of the deduced amino acid sequence of AbTRI showed that the AbTRI shared an amino acid sequence similarity of more than80percents with other Solanaceae species reported, and on the whole is very conservative, indicating that their TRIs start to differentiate from a common ancestor protein occurred in relatively recent time; in phylogenetic tree, TRIs from the Solanaceae, Brassicaceae, Monocots and Bacteria are clearly clustered into four branches, among which AbTRI have a closest genetic relationship with TRI from A. acutangulus which belongs to different genus from A. belladonna’s and followed by H. niger TRI. Through conserved domain prediction, we found in AbTRI amino acid sequence a typical characteristic domain of short-chain dehydrogenase (short-chain dehydrogenase/reductase, SDR_c), which is consistent with the fact that TRIs from D. stramonium and H. niger have been identified as members of the SDR family. Further tertiary structure prediction found that there is binding region of NADPH in the N-terminal SDR of AbTRI, and the conserved amino acid residues Lys31and Arg53can prioritily combined to NADPH. At the same time, it found out that there are conservative amino acid residues His112and Va1168at the C-terminal end of tropine ketone binding region, which can make sure that tropinone combined to TRI protein correctly. Tissue Expression and induction expression analysis showed that this gene specifically expressed in A. belladonna old leaves, little expressed in flowers and roots, and slightly expressed in other tissues. At the same time, its expression level was strongly affected by the ABA, ASA, MeJA, UV-B and ethanol induction and SA inhibition.Take PET28/E. coli as genealogical tree to prokaryotically identify the function of AbTRI gene. Functional verification induced by1mM IPTG induction for4h,, AbTRI recombinant protein content of more than50%of the total cellular protein, mainly in the form of inclusion bodies, the molecular weight of about30Kda consistent with the forecast. Recombinant protein inclusion bodies were purified on AbTRI and dialysis refolding, atropine ketone as a substrate in the presence of NADPH, the detection of the catalytic activity of the RcTYDC recombinant protein by HPLC detected the formation of reaction products tropine, show that the The cloned Belladonna’s the function AbTRI gene.To build carrying the A6TRI gene of a plant effective expression vector p1304+-AbTRI Agrobacterium tumefaciens C58C1was transformed into the "Resurrection" Engineering bacteria C58Cl-p1304+-AbTRI genetic transformation of belladonna leaves by detection on rol B of rolC Hygr and AbTRI, gene after successful screening seven transgenic roots, respectively, the liquid medium to expand cultivation and then detect the content of tropine the alkane alkaloids (TAs) and gene expression.5TAs content of the hair root has been greatly improved in the detection of seven genetically modified hair roots, followed by the T9> T16> of T23> T11, T9TAs content than the empty bacteria hair root control and no-load The hair root control were increased by3.2times and8.3times, the improved rate of scopolamine on average higher than that of scopolamine; three roots appear with varying degrees of gene silencing of TAs were significantly lower than the control. The AbTRI seven rounds of the roots of the real-time fluorescent quantitative PCR gene expression results with the alkaloid content of the test results are basically the same:four alkaloid content to improve the hair root (T9, T16, T23, T11) of The gene expression levels were significantly higher than control and other transgenic hair root, the expression level; three low levels of hair roots (T10, T5, T15) gene expression levels were significantly lower than control, indicating that indeed the gene co-suppression.This study the experimental results show that overexpression AbTRI can contribute to the metabolic flux to the target product and significantly increase the content of total TAs in belladonna, provides a new candidate genes and theoretical support for further belladonna TAs metabolic engineering, combined with the laboratory before the research results in the overexpression of TAs way downstream speed limit further overexpression of TRI gene the pmt and h6h of belladonna alkaloid synthesis capabilities can maximize the potential development TAs way, and encouraged scopolamine ultra-yielding the belladonna lines, to provide quality drug source material for the corresponding industries.