Cloning And Functional Characterization Of The Key Reductase Genes Involved In The DNJ Biosynthetic Pathway In Medicinal And Edible Mulberry Leaves

Mulberry leaves(mori folium)are dry leaves of Morus alba L.(family moraceae),which is a kind of medicinal and edible plant resource.Mulberry leaves have been recorded in ancient materia literatures for"mulberry leaves can quench thirst",that is,the modern prevention and treatment of diabetes.The alkaloid 1-deoxynojirimycin(DNJ)is one of the main active components in mulberry leaves with hypoglycemic effect.DNJ is a strong?-glucosidase inhibitor,and has the effects of anti-hyperglycemia,anti-tumor,and anti-virus.The development of health food and drugs with DNJ as the active ingredient has broad application prospects.DNJ from natural sources is the most abundant in mulberry leaves,however,the content of DNJ in mulberry leaves is low,so it is difficult to obtain a large amount of natural DNJ by traditional separation and preparation methods.With the development of synthetic biology technology,it is expected to provide a new method to obtain a large amount of natural DNJ.DNJ is a piperidine alkaloid derived from lysine.The entire DNJ biosynthetic pathway has not been well-understood.In our previous study,we speculated the biosynthesis pathway of DNJ in mulberry leaves and clarified the synthetic pathway of DNJ from lysine to?~1-piperideine by cloning and functional characterization of lysine decarboxylase and copper amine oxidase in mulberry leaves,but the process from?~1-piperideine to piperidine and its methylation and hydroxylation are not elucidated.In this paper,we mainly studied the reduction process of?~1-piperideine to piperidine in the DNJ biosynthesis pathway.Three candidate reductase genes Ma SDR1,Ma SDR2 and Ma P5CR1 were screened out and used for cloning,expression,and in vitro functional characterization.The genetic transformation system by agrobacterium-mediated in mulberry was constructed,and the technologies of gene overexpression and virus-induced gene silencing were used to verify the in vivo function.The biological process of reduction from?~1-piperideine to piperidine of DNJ biosynthesis in mulberry leaves was successfully revealed,and the biosynthetic pathway from?~1-piperideine to piperidine in mulberry leaves was elucidated.This study laid a foundation for elucidating the entire DNJ biosynthesis pathway in mulberry leaves and realizing the production of DNJ polyhydroxy alkaloids by synthetic biology.The main research contents and results are as follows:1.Screening of key reductase genes involved in the DNJ biosynthetic pathway in mulberry leaves.The transcriptome data(SRA accession:SRP127713)of mulberry leaf samples with the highest DNJ content(M7)and mulberry leaf samples with the lowest DNJ content(M11)in different seasons was analyzed by comparative transcriptome method,according to the annotation information of the transcriptome and related metabolic pathways in KEGG,a total of one hundred and twenty four possible carbon-nitrogen double bond reductase genes involve in the DNJ biosynthetic pathway from?~1-piperideine to piperidine were screened,they encoded short-chain dehydrogenase/reductase(SDR)and delta1-pyrroline-5-carboxylic acid reductase(P5CR).We found ten related reductase differentially expressed genes(DEGs),including nine significantly down-regulated reductase DEGs and significantly one up-regulated reductase DEG.Correlation analysis between DNJ content and expression levels of reductase DEGs showed that Ma SDR1,Ma SDR2 and Ma P5CR1were significantly and positively correlated with DNJ content in mulberry leaves.It was speculated that these three genes might be the key reductase genes for the pathway from?~1-piperideine to piperidine in biosynthesis of DNJ in mulberry leaves.2.Cloning and expression of reductase genes Ma SDR1,Ma SDR2 and Ma P5CR1 involved in the DNJ biosynthetic pathway in mulberry leaves.Reductase genes Ma SDR1(Gen Bank accession:MT989445),Ma SDR2(Gen Bank accession:MT989446)and Ma P5CR1(Gen Bank accession:MT989447)were cloned from mulberry leaves.The results of bioinformatics analysis showed that the proteins encoded by Ma SDR1 and Ma SDR2 genes had similarity of 93%and 99%with short-chain dehydrogenase(PDB ID:6y4d.1.A),belonging to the family of short-chain dehydrogenase;the protein encoded by Ma P5CR1 gene had similarity of 100%with delta1-pyrroline-5-carboxylic acid reductase(PDB ID:5bse.1.A),belonging to the family of PLN02688.Enzymes in these two families may reduce the carbon-nitrogen double bond.The results of multiple sequence alignment with other species suggested that Ma SDR1 and Ma SDR2 both contained the cofactor binding motif TGXXXGX.Ma P5CR1 had a conserved domain of the delta1-pyrroline-5-carboxylic acid reductase family.The phylogenetic tree results showed that Ma SDR1,Ma SDR2 and Ma P5CR1 all had the closest relationship with Morus notabilis.Recombinant plasmids of Ma SDR1/p ET28a(+),Ma SDR2/p ET28a(+)and Ma P5CR1/p ET28a(+)were constructed by double-enzyme digestion method,and transformed into E.coli BL21(DE3)to induce expression and obtain recombinant proteins.The recombinant proteins were all soluble proteins.The molecular weight of the recombinant proteins were all about 34 k Da.The proteins were purified by method of His-tag protein purification kit.3.In vitro functional characterization of reductases Ma SDR1,Ma SDR2 and Ma P5CR1 involved in the DNJ biosynthetic pathway in mulberry leaves.In vitro enzymatic reactions were used to verify the functions of reductases Ma SDR1,Ma SDR2 and Ma P5CR1.The results showed that both Ma SDR1 and Ma SDR2 could catalyze the reaction of?~1-piperideine with the coenzyme NADPH to generate piperidine.Ma P5CR1 could catalyze the reaction of?~1-piperideine with the coenzyme NADH to generate piperidine.The Km and Vmax values of Ma SDR1 to the substrate?~1-piperidene were 82.52?mol/L and 11.55?mol/L/min,respectively;the Km and Vmax values of Ma SDR2 to the substrate?~1-piperideine were 60.61?mol/Land 24.65?mol/L/min,respectively;the Km and Vmax values of Ma P5CR1 to the substrate?~1-piperideine were 84.79?mol/L and 23.67?mol/L/min,respectively.The kinetic parameters of Ma SDR1 and Ma SDR2indicated that Ma SDR2 had a higher binding ability to?~1-piperideine than Ma SDR1.The expression levels of reductase genes Ma SDR1,Ma SDR2 and Ma P5CR1 in mulberry leaves in different seasons were significantly positively correlated with DNJ content,indicating that Ma SDR1,Ma SDR2 and Ma P5CR1 might be the key reductase genes for the synthesis of DNJ,and might have regulatory effect on DNJ biosynthesis.4.In vivo functional characterization of reductase genes Ma SDR1,Ma SDR2 and Ma P5CR1 involved in the DNJ biosynthetic pathway in mulberry leaves.The overexpression genetic transformation systems mediated by C58C1 Agrobacterium rhizogene and virus-induced gene silencing(VIGS)transformation systems mediated by GV3101 Agrobacterium in mulberry leaves were established to verify the in vivo functions of reductase genes Ma SDR1,Ma SDR2 and Ma P5CR1 involved in the DNJ biosynthetic pathway in mulberry leaves.The A.rhizogene O-Ma SDR1/p BI121-EGFP/C58C1,O-Ma SDR2/p BI121-EGFP/C58C1 and O-Ma P5CR1/p BI121-EGFP/C58C1 were obtained by double-enzyme digestion and ligation methods and infected the explant of aseptic mulberry seedlings.After co-culture,selective culture,and expansion culture,the positive transgenic hairy roots were identified by PCR verification.The gene expression levels of O-Ma SDR1,O-Ma SDR2 and O-Ma P5CR1 in their respective transgenic hairy roots were significantly increased than those in O-Control hairy roots(P<0.001),indicating that the overexpression systems of O-Ma SDR1,O-Ma SDR2 and O-Ma P5CR1 genes in mulberry roots were successfully constructed.When O-Ma SDR1,O-Ma SDR2,and O-Ma P5CR1 genes were overexpressed,the average content of DNJ in hairy roots corresponding to O-Control,O-Ma SDR1,O-Ma SDR2,and O-Ma P5CR1 were 0.06,0.27,0.36,and 0.19 mg/g,respectively.The average content of DNJ in transgenic hairy roots was significantly increased than that in non-transgenic hairs roots(P<0.001).The agrobacterium V-Ma SDR1/p TRV2/GV3101,V-Ma SDR2/p TRV2/GV3101 and V-Ma P5CR1/p TRV2/GV3101 were obtained by double-enzyme digestion and ligation methods and infected aseptic mulberry seedlings,and the positive VIGS mulberry leaves were identified by PCR.The gene expression levels of V-Ma SDR1,V-Ma SDR2,and V-Ma P5CR1 in the respective VIGS mulberry leaves were significantly lower than that of V-Control(P<0.001),indicating that the V-Ma SDR1,V-Ma SDR2 and V-Ma P5CR1 genes in the VIGS systems in mulberry leaves were successfully constructed.When V-Ma SDR1,V-Ma SDR2and V-Ma P5CR1 genes were silenced,the average DNJ content in VIGS mulberry leaves corresponding to V-Control,V-Ma SDR1,V-Ma SDR2 and V-Ma P5CR1 were 0.19,0.14,0.14,0.15mg/g,respectively,the average content of DNJ in VIGS mulberry leaves was significantly reduced than that in non-VIGS mulberry leaves(P<0.001).The above results further characterized the regulatory roles of reductase genes Ma SDR1,Ma SDR2 and Ma P5CR1 in the biosynthesis of DNJ in mulberry leaves.When these three reductase genes were overexpressed,the DNJ content increased.When these three genes were silenced,the gene expression was blocked and the DNJ content decreased,indicating that these three genes participated in DNJ biosynthesis,they were the key reductase genes involved in the DNJ biosynthetic pathway in mulberry leaves.This result laid the foundation for further research on the hydroxylation and methylation of the DNJ piperidine ring in mulberry leaves,and the formation of DNJ polyhydroxy alkaloids.