Molecular Cloning And Functional Characterizing Of N-Methylputriscine Oxidase Genes In Atropa Belladonna

Tropane alkaloids(TAs)are among the oldest traditional medicines,having been used for nearly 3000 years.The main TAs are hyoscyamine and scopolamine.These two alkaloids are widely used as anticholinergic drugs for pain relief,anesthesia,drug detoxification,alleviating of motion scicknes,and treatment of Parkinson's disease.The production of medicinal TAs requires extraction from specific medicinal plants of the Solanaceae family,including Atropa belladonna(belladonna),Datura stramonium,and Duboisia hybrid.These medicinal plants have very low TA contents,which cannot meet the market demand.For example,in belladonna,hyoscyamine accounts for 0.02–0.17% of the dry weight of leaves,and scopolamine for only 0.01–0.08%.Improving the TA content of medicinal plants by biotechnology is the most effective way to resolve the disparity between supply and demand of TAs;thus,this goal is being pursued by the relevant industries.Molecular biology research to elucidate the biosynthesis pathway of TAs is a prerequisite of developing biotechnology for this pathway.Atropa belladonna is a medicinal plant containing TAs that is used in the Chinese pharmacopoeia.In recent years,many genes involved in the biosynthetic pathway of TAs in belladonna and other medicinal plants have been isolated and identified,making it possible to improve the TA content of some plants by genetic engineering technology.However,in some key aspects of TA biosynthesis,research is still relatively lacking.The oxidative deamination of N-methylputrescine oxidase,a key step in TA biosynthesis,may be catalyzed by N-methylputrescine oxidase(MPO),a member of the copper amine oxidase superfamily.However,the gene encoding MPO has not been cloned and identified in TA medicinal plants,and it remains unknown whether it is involved in TA biosynthesis.In this study,two MPO genes(AbMPO1 and AbMPO2)were cloned from belladonna,and their bioinformatic data and tissue expression patterns were analyzed.Transgenic roots of belladonna were obtained using RNA interference(RNAi)technology to investigate their effects on TA biosynthesis.The results of this study are as follows.1.Cloning and sequence analysis of AbMPO1 and AbMPO2Two full-length cDNAs of MPO genes in A.belladonna were cloned by rapid amplification of cDNA end(RACE),and named AbMPO1 and AbMPO2,respectively.The full-length sequences of AbMPO1 and AbMPO2 were 2747 bp and 2706 bp,respectively.The amino acid sequences encoded 767 amino acids and 782 amino acid residues,respectively.The amino acid sequences of AbMPO1 and AbMPO2 had 75.3% identity and 83.5% similarity;compared with 84.9% and 90.1%,respectively,for AbMPO1 and tobacco MPO1(NtMPO1),and 75.0% and 83.6%,respectively,for AbMPO2 and tobacco MPO1(NtMPO1).BLASTP analysis showed that both MPOs belonged to the copper amine oxidase family and had copper ion binding sites.2.Analysis of tissue expression profiles of AbMPO1 and AbMPO2The expression levels of AbMPO1 and AbMPO2 in the secondary root,primary root,mature stem,young stem,mature leaf,young leaf,flower,and flower bud of belladonna were analyzed using real-time fluorescence quantitative PCR.The results showed that AbMPO1 was only expressed in roots,and that its expression in fibrous roots was significantly higher than that in main roots.Digital expression profiling showed that the tissue expression pattern of AbMPO1 was consistent with it being a component of the TA biosynthesis pathway,i.e.,high/specific expression in primary roots.AbMPO2 was expressed in all tested tissues,and the expression level in roots was lower than that in above-ground parts.Digital expression profiling showed that the expression pattern of AbMPO2 in tissues was inconsistent with it being part of the TA biosynthesis pathway.The tissue expression profiles of AbMPO1 and AbMPO2 indicated that AbMPO1 might be involved in TA biosynthesis.3.Inhibition of AbMPO1 resulted in a decrease in hyoscyamine and scopolamine contents in belladonna hairy rootsIn order to further study whether AbMPO1 is involved in TAs biosynthesis,belladonna hairy roots that were obtained using transgenic and RNAi techniques.The PCR results showed that the 626 bp rolC gene fragment and 423 bp rolB gene fragment were present in all hairy roots;a 521 bp 35S:: iAbMPO1 fusion fragment and 597 bp NPTII gene fragment could also be detected in iAbMPO1 hairy roots,but not in control hairy roots.The expression of AbMPO1 was detected by real-time fluorescence quantitative PCR.The results showed that the expression of AbMPO1 was significantly decreased in iAbMPO1 hairy roots compared with control roots,whereas the expression of AbMPO2 did not change.Molecular detection results showed that hairy roots of transgenic belladonna,in which AbMPO1 was specifically inhibited,were obtained.High-performance liquid chromatography(HPLC)results showed that inhibition of AbMPO1 significantly reduced the contents of hyoscyamine and scopolamine.The content of hyoscyomine and scopolamine in the control hairy roots was 2.85 mg/g dry weight and 1.53 mg/dry weight,respectively.In the hairy roots with hairy roots were 0–0.50 mg/g dry weight.These results indicate that inhibition of AbMPO1 can significantly reduce TA contents,suggesting that AbMPO1 is involved in TAs biosynthesis.4.Inhibition of AbMPO2 had no effect on the synthesis of hyoscyamine or scopolamine in belladonna hairy roots.In order to investigate whether AbMPO2 is involved in TAs biosynthesis,hairy roots of belladonna with inhibition of AbMPO2 were obtained by transgenic and RNAi techniques.PCR results showed that 626 bp rolC gene fragments and 423 bp rolB gene fragments were present in all hairy roots;a 514 bp 35S:: iAbMPO2 fusion fragment and 597 bp NPTII gene fragment were also detected in iAbMPO2 hairy roots,but not in control hairy roots.Real-time fluorescence quantitative PCR was used to detect the expression of AbMPO1.The results showed that the expression of AbMPO2 was decreased significantly in iAbMPO2 hairy roots compared with control roots,but the expression of AbMPO1 did not change.Molecular detection results showed that transgenic belladonna hairy roots with specific inhibition of AbMPO2 were obtained.TA content analysis by HPLC showed no significant difference between levels of scopolamine in control and iAbMPO2 hair roots.Relevant studies on inhibition of AbMPO2 showed that AbMPO2 is not involved in TA biosynthesis.In conclusion,two genes belonging to the copper amine oxidase family,AbMPO1 and AbMPO2,were cloned and identified from belladonna.Although the amino acid sequences of AbMPO1 and AbMPO2 were highly similar,their expression and role in TA biosynthesis were completely different.AbMPO1 showed root-specific expression in belladonna with the highest expression level in fibrous roots,which is consistent with the tissue expression pattern of a TA biosynthetic gene.Inhibition of AbMPO1 resulted in the blockage of TA biosynthesis.AbMPO2 had a different expression pattern from that of TA biosynthetic genes,and inhibition of AbMPO2 had no effect on TA biosynthesis.This study not only confirmed that AbMPO1 is involved in TAs biosynthesis,but also provides a valuable gene for TA metabolic engineering in the future.