| Mori Folium are dried leaves of Morus alba L.,which is a traditional Chinese medicine and also one of the dual-use resources for food and medicine prescribed by the National Health Commission.Mulberry leaves have been used in the treatment of diabetes since ancient times,and it is recorded in the Dietetic Materia Medica of Tang dynasty that"mulberry leaves decoction can quench thirst,which is in the same way as drinking tea".Compendium of Materia Medica of Ming Dynasty records that"mulberry leaves are the medicines for Yangming disease,and drinking the decoction tea of mulberry leaves can quench thirst"（quench thirst is modern medical diabetes）.Modern studies show that the main active ingredient of mulberry leaves in treating diabetes is 1-deoxynojirimycin（DNJ）polyhydroxy alkaloids.DNJ alkaloids,as powerfulα-glycosidase inhibitors,can effectively inhibit the rise of blood glucose,reduce lipids to alleviate atherosclerosis,and anti-HIV infection,etc.Based on the unique chemical structure and remarkable pharmacological activity,DNJ shows great potential for the development of new drugs and functional foods for the treatment of diabetes.However,the content of DNJ alkaloids in mulberry leaves is very low,and it is difficult for the extraction and isolation,while chemical synthesis and microbial transformation methods have the problem of complicated routes,difficult separation of isomers and low yields.Therefore,it is urgent to find an effective new way to obtain DNJ alkaloids.Recently,the use of synthetic biology strategies by analyzing their biosynthetic pathways is expected to be a new way to obtain DNJ alkaloids.Our group has previously studied the upstream biosynthetic pathway of DNJ alkaloids in mulberry leaves,revealing the biosynthetic pathway from lysine to piperidine,but the methylation and hydroxylation processes from piperidine to DNJ remain unclear.In this study,the candidate genes for the methyltransferase and CYP450hydroxylase involved in DNJ alkaloids biosynthesis were screened through comparative transcriptome data of mulberry leaves.And then the candidate methyltransferase genes were cloned,expressed and their functions were studied in vitro and in vivo;finally,the methylation step of DNJ alkaloids biosynthesis was preliminarily clarified.In addition,some candidate genes of CYP450 hydroxylases were cloned and expressed.This study will lay a foundation for the complete analysis of the biosynthetic pathway of DNJ alkaloids,and also provide a basis for the eventual production of DNJ alkaloids by synthetic biology and alleviate their resource shortage.The main research contents and results are as follows:1.The mining of methyltransferase and CYP450 hydroxylase genes related to the biosynthesis of DNJ alkaloids in mulberry leavesNine methyltransferase differential genes related to the methylation step and 17 CYP450hydroxylase differential genes related to the hydroxylation step of DNJ alkaloids biosynthesis were preliminarily screened form transcriptomics data of mulberry leaves（SRA accession no.SR127713）and KEGG metabolic pathway.Analysis of the correlation between the differential genes expression level and DNJ content in mulberry leaves during different growth dates（from July 5 to October 25）showed that three methyltransferase genes（c34262＿g1,c41333＿g1 and c28557＿g1）and eight CYP450 hydroxylase genes（c11320＿g1,c29832＿g1,c36193＿g1,c29603＿g1,c39525＿g1,c42177＿g1,c82195＿g1,c46601＿g1）were significantly correlated with the change of DNJ content（P<0.05）,suggesting that they were candidate genes of methyltransferase and CYP450 hydroxylase involved in the biosynthesis of DNJ alkaloids.2.Cloning and prokaryotic expression of methyltransferase candidate genes involved in the biosynthesis of DNJ alkaloids in mulberry leavesCandidate methyltransferase genes MaMT1（Gen Bank accession no.OM140666）,MaMT2（Gen Bank accession no.ON152713）and MaMT3（Gen Bank accession no.OM140667）involved in the DNJ alkaloids biosynthesis were cloned from mulberry leaves for the first time.Bioinformatics analysis showed that MaMT1,MaMT2 and MaMT3 were annotated as serine hydroxymethyltransferase（SHMT）,sterol-C-24-methyltransferase（SMT）and ketopantoicacid hydroxymethyltransferase（KPHMT）families,respectively,and their amino acid sequences contained conserved structural motifs of the corresponding families.Phylogenetic tree results showed that MaMT1 has closest relationship with SHMT of Ostrinia furnacalis,while MaMT2and MaMT3 were closest related to SMT and KPHMT of Morus notabilis,respectively.Prokaryotic expression vectors of MaMT1/p ET30a,MaMT2/p ET30a and MaMT3/p ET30a were constructed by double enzyme digestion and T4 ligating method,and then were successfully expressed in Escherichia coli DE3 under the conditions of 0.2 mmol/L IPTG and cultured at 15℃for 16 h.The three recombinant proteins were all present as inclusion bodies.MaMT1,MaMT2and MaMT3 recombinant proteins with molecular weights of 38.0 k Da,39.6 k Da and 35.5 k Da were obtained by His-tag purification after denaturation and dialysis renaturation,respectively.3.In vitro functional studies of candidate methyltransferase genes involved in the biosynthesis of DNJ alkaloids in mulberry leavesThe functions of MaMT1,MaMT2 and MaMT3 proteins were verified by enzymatic reaction in vitro.The results showed that MaMT1 protein could catalyze piperidine to form 2-methylpiperidine with piperidine as substrate and S-adenosine-L-methionine（SAM）as methyl donor,while MaMT2 and MaMT3 could not catalyze the conversion of piperidine to 2-methylpiperidine.Molecular docking further verified that MaMT1 protein had a good interaction with piperidine and SAM,while MaMT2 and MaMT3 had no interaction with piperidine and SAM.Using piperidine as substrate and hydroxymethyl as donor,it was found that MaMT1 protein could not catalyze the formation of 2-methylpiperidine from piperidine in vitro.These results indicate that MaMT1 has the function of catalyzing piperidine to form 2-methylpiperidine in vitro,but could not catalyze the formation of 2-hydroxymethylpiperidine.The correlation between expression levels of MaMT1 gene and DNJ content in mulberry leaves in different growing seasons was analyzed,and the results showed that thay had a significant positive correlation（P<0.05）,indicating that MaMT1 gene may be a key enzyme gene involved in the methylation step of piperidine to 2-methylpiperidine in the biosynthetic pathway of DNJ alkaloids in mulberry leaves.The substrate specificity of MaMT1 protein was investigated,and the results showed that MaMT1 could not catalyze the methylation reaction of 4-hydroxypiperidine andΔ1-piperideine,while MaMT1 catalyzed the methylation of 3-hydroxypiperidine to produce a new substance,which was identified as 3-hydroxy-N-methylpiperidine by similarity comparison of mass spectrometry database,but the DNJ in N-position had no-CH3,indicating that DNJ could not be synthesized with 3-hydroxypiperidine as substrate.These results may provide reference for other synthetic pathways of DNJ alkaloids.4.In vivo functional studies of key MaMT1 gene involved in the biosynthesis of DNJ alkaloids in mulberry leavesThe regulation function of the key MaMT1 gene on the biosynthesis of DNJ alkaloids in mulberry leaves was verified by gene silencing experiment using the VIGS transformation system of mulberry.The recombinant viral vector V-MaMT1/p TRV2 was constructed by enzyme digestion and T4 ligating method and then transformed into Agrobacterium GV3101.The sterile mulberry seedlings were infected by vacuum osmosis method.After culture,positive VIGS mulberry leaves were identified by PCR.The expression levels of MaMT1 gene and DNJ content in mulberry leaves of VIGS groups and control groups were analyzed,and the results showed that the expression levels of MaMT1 gene in VIGS mulberry leaves was significantly decreased compared with control groups（P<0.001）,and the average inhibitory efficiency of MaMT1 gene was 80.7%;the average content of DNJ in mulberry leaves of control group and VIGS group was0.27 mg/g and 0.16 mg/g,respectively,with a reduction rate of 42.1%（P<0.01）,indicating that the accumulation of DNJ alkaloids in mulberry leaves was significantly reduced when MaMT1gene was silenced.And it is suggested that MaMT1 gene was involved in the biosynthesis of DNJ alkaloids.5.Cloning and eukaryotic expression of candidate CYP450 hydroxylase genes involved in the biosynthesis of DNJ alkaloids in mulberry leavesFive CYP450 hydroxylase genes,Ma P450-A1（Gen Bank accession no.ON152708）,Ma P450-A2（Gen Bank accession no.ON152709）,Ma P450-A3（Gen Bank accession no.ON152709）,MAP450-A4（Gen Bank accession no.ON152711）and MAP450-A5（Gen Bank accession no.ON152711）,were firstly cloned from mulberry leaves,which belongs to P450 71A1,P450 724B1,P450 77A1,P450 89A2 and P450 734A1 subfamilies,respectively.Multiple sequence alignment showed that Ma P450s protein contains FGx Gxxx Cx G,Exx R and（A/G）Gx（D/E）T of CYP450conserved domains.Phylogenetic tree results showed that the five candidate genes belonged to different subfamilies of CYP450,which were clustered into one class,respectively,and all the five enzyme genes were closely related to the corresponding CYP450 subfamily enzyme genes in Morus notabilis.The Ma P450s/p ESC-TRP eukaryotic expression vector was constructed by double enzyme digestion and T4 linkage method,respectively,and then were transformed into Saccharomyces WAT11 strain.The protein expression was induced by galactose,and the Ma P450-A1/A2/A3/A4/A5 microsomal protein was extracted from WAT11 strain.These results will lay the foundation for the follow-up study of its in vitro function.In conclusion,this study screened,cloned and heterologous expressed methyltransferase candidate genes based on mulberry leaves transcriptome data,and initially clarified the key methylation step of piperidine to 2-methylpiperidine that involved in DNJ alkaloid biosynthetic pathway in mulberry leaves,and the CYP450 candidate genes related to hydroxylation step were screened,cloned and eukaryotic expressed,which will lay the foundation for the complete elucidation of DNJ alkaloids biosynthetic pathway in mulberry leaves. |
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