Mining And Analysis Of The Key Genes In DNJ Polyhydroxy Alkaloid Biosynthesis Of Mulberry Leaves Based On Transcriptomics Technology

Abstract:Mori Folium are dry leaves of Morus alba L.,which is commonly used as traditional Chinese medicine for expelling pathogenic wind-heat,clearing away lung heat and moisturizing,and clearing up heat from the liver to improve acuity vision.Mulberry leaves are also one of the medicine and food resources prescribed by the Ministry of Health.Mulberry leaves have been used for the treatment of diabetes since ancient times.It is recorded in"Compendium of Materia Medica"that"Mulberry leaves juice decocted in tea,can quench thirst."it is also recorded in the Japanese ancient book"The Tea Health Notes"that Mulberry leaves can help improve the"drinking water disease."Modern studies have shown that the main active ingredient for treatment of diabetes in mulberry leaves was a kind of polyhydroxy alkaloids based on1-deoxynojirimycin?DNJ?.However,the content of polyhydroxy alkaloids of DNJ in mulberry leaves is very low,making it difficult to obtain a large number of DNJ by traditional separation methods.Therefore,the need to find new methods for obtaining a large number of DNJ polyhydroxy alkaloids is urgent.Recently,the use of transcriptomics technology has become an important approach to excavate the key enzyme genes for the biosynthesis of numerous natural active ingredients based on synthetic biology.In this dissertation,the key enzyme genes involve in DNJ polyhydroxy alkaloids biosynthesis were excavated by transcriptomics method,the genes were cloned,expressed and verified by biological technology.This study will lay the foundation for further elucidation of the biosynthetic pathway of DNJ polyhydroxy alkaloids and devising synthetic biology methods for the production of DNJ in large quantities.In addition,the effects of hypoglycemic and effect of clearing away lung heat and moisturizing,mulberry leaves before and after frost were studied to provide basis for functional food development and as guidance for clinical application.The main research contents and innovative results are as follows:1.Dynamic changes of polyhydroxy alkaloids in mulberry leaves at different growth stages The dynamic changes of DNJ alkaloid content in mulberry leaves at different growth stages were analyzed by the pre-column derivatization high performance liquid chromatography.The results showed that the content of DNJ in mulberry leaves was highest in July and August and lowest in November.The mulberry leaves studies in 2016 and 2017 showed that the dynamic variation of DNJ content was consistent.In 2016,the highest DNJ content in mulberry leaves was 3.43?0.15 mg/g on July 31 and the lowest DNJ content was 0.19?0.05 mg/g on November15.Similarly,the highest DNJ content in mulberry leaves obtained in 2017 was 3.22?0.06 mg/g on July 25 and the lowest content was 0.17?0.01 mg/g on November 15.This provided a basis for the samples selection of mulberry transcriptome sequencing.2.Transcriptome sequencing and comparative transcriptomic analysis of mulberry leaves Illumina HiSeq 2500 was used to sequence the mulberry leaves transcriptomes.A total of 16 Gb raw data were obtained.105,189,326 and 65,166,690 original sequences were obtained,respectively.The average sequence length was 150 bp.High-quality reads were De Novo assembled to 112,481 transcripts,with an average length of 766 bp.The assembled transcripts were BLASTed against nine public databases?CDD,KOG,NR,NT,PFAM,Swiss-Prot,TrEMBL,GO,and KEGG?and annotated to 89,606 sequences.In the COG/KOG database,20,725 sequences were annotated,30,716 sequences were annotated in the GO database,13,268sequences were annotated in the KEGG database,in which 323 metabolic pathways were involved,131 pathways were associated with secondary metabolism,and 157 genes were involved in the biosynthesis pathways of alkaloids.A total of 11,318 differentially expressed genes were obtained through analysis,of which 6,606 were up-regulated and 4,712 were down-regulated.KEGG metabolic pathway annotation analysis revealed that differentially expressed genes were involved in 294 metabolic pathways,mainly enriched in carbon metabolism and amino acid biosynthesis and oxidative phosphorylation pathways,and 15differentially expressed genes involved biosynthesis pathways of alkaloids.3.The mining of key enzyme genes for the biosynthesis of DNJ polyhydroxy alkaloids in mulberry leaves Based on the transcriptome results combined with the KEGG pathway,a lysine synthesis pathway?ko00300?and a piperidine ring synthesis pathway?ko00960?involving the biosynthesis of DNJ polyhydroxy alkaloids were found.A total 38 related enzyme genes were involved,and coding 9 enzymes,9 differentially expressed genes,one gene up-regulated and 8genes down-regulated.Three candidate key enzyme genes for the biosynthesis pathway of DNJ polyhydroxy alkaloids were found,and the two enzymes encoded are lysine decarboxylase and primary amine oxidase.Analysis of transcriptome data yielded 103 CYP450 genes.There were20 differentially expressed genes,of which 11 genes were up-regulated and 9 genes were down-regulated.Correlation analysis showed that 5 genes expression level were significantly related to DNJ content.A total of 110 methyltransferase genes were analyzed and 17 genes were differentially expressed,of which 8 were up-regulated and 9 were down-regulated.4.Cloning,expression and functional verification of lysine decarboxylase and copper oxidase genes from mulberry leaves The ORF full-length sequences of lysine decarboxylase?MaLDC?gene?GeneBank accession number:MG727866?and copper amine oxidase?MaCAO?gene?GeneBank accession number:MH205733?were cloned from the mulberry leaves for the first time.The bioinformatics results showed that MaLDC contains a conserved domain of lysine family PGGXGTXXE,MaCAO contains a copper amine oxidase domain and belongs to the Cu_amine_oxid subfamily;Protein tertiary structure prediction results showed that the similarity of MaLDC protein and lysine decarboxylase?PDB ID:2Q4D?was 69.12%,and the similarity of MaCAO protein to copper amine oxidase?PDB ID:1W2Z?was 45.52%.Recombinant pET28a?+?/MaLDC and pET-22b?+?/MaCAO recombinant expression vectors were constructed by double digestion and transformed into E.coli BL21?DE3?for expression of recombinant proteins.The in vitro enzymatic reaction was used to verify the function of the recombinant protein.The results showed that Ma LDC recombinant protein can effectively catalyze the decarboxylation of lysine to produce cadaverine.The K_m and V_max values of the substrate lysine were 19.18 mmol/L and 3.31 mmol/min,respectively.The functional verification of Ma CAO recombinant protein showed that this protein can catalyze the formation of5-aminopentylaldehyde from 1,5 pentamethylenediamine.MaLDC gene expression level and enzyme activity as well as MaCAO gene expression level were significant positively correlated with DNJ content in mulberry leaves,indicating that MaLDC and MaCAO may be the key enzyme genes for the synthesis of DNJ polyhydroxy alkaloids.5.Study on hypoglycemic effect and clearing away lung heat and moisturizing of mulberry leaves before and after frost The clearing away lung heat and moisturizing and hypoglycemic effect of green mulberry leaves?August?and frost mulberry leaves?November?were studied.The hypoglycemic effect of diabetes mice showed that green mulberry leaves can significantly reduce the fasting blood glucose levels of diabetic mice,increase the synthesis of hepatic glycogen,and significantly reduce serum alanine aminotransferase?ALT?and malondialdehyde?MDA?levels in diabetic mice.The results of pathological tissue section analysis showed that the green mulberry leaves had protective and repairing effects on mouse liver and pancreas,indicating that the hypoglycemic effect of the green mulberry leaves was better than that of the frost mulberry leaves.In contrast,mouse ammonia stimulating cough,guinea pigs electrical stimulation cough,rat tracheal capillary drainage,phenol red excretion of mice airway and diabetic mice pharmacological models and experimental methods were used.The results showed that frost mulberry leaves had a significant inhibitory effect on cough induced by concentrated ammonia in mice and electrical stimulation of guinea pig cough.Frost mulberry leaves can significantly promote the amount of sputum evacuation and airway phenol red secretion in mice within 4 hours.These results indicated that the anti-tussive and expectorant effects of frost mulberry leaves were better than green mulberry leaves.This result provided guidance for development of functional foods and the rational use of the mulberry leaves.