Cloning Of Genes Related To Alkaloid Synthesis And The Expression Analysis In Mulberry

1-deoxynojirimycin DNJ is the most marked piperidine alkaloid in mulberry.DNJ has good pharmacological effects such as lowering blood glucose,lowering blood lipid,anti-virus,improving immunity and anti-aging.Because of the huge medicinal value of DNJ,the exploration of DNJ synthesis pathway has become a research hotspot in the field of alkaloids.This study with lu in the mulberry mulberry 9703 as material,cloned 4 genes associated with DNJ synthesis,cytochrome P450 reductase,CPR-5,3 hydroxy methyl 3 glutaric acyl coenzyme A reductase,HMGR,aspartic acid kinase 2,LysC,4-hydroxy-4 hydrogen pyridine synthase,DapA,and successfully build A expression vector,and successfully expressed in E.coli.q RT-PCR was used to analyze the expression of these four genes in different organs(tissues),leaves at different leaf positions and young leaves of different germplasm of mulberry,and DNJ extraction was carried out on different organs(tissues),leaves at different leaf positions and young leaves of different germplasm of mulberry,so as to analyze the correlation between gene expression and alkaloid content.The results can provide some reference for the determination and exploration of DNJ biosynthesis pathway in mulberry.According to the sequence information of existing plants in Gen Bank,design CPR-5,HMGR,LysC,DapA primers,PCR method for mulberry 9703 homologous gene cloning,in CPR-5 protease gene coding sequence length of 1812 bp,encoding 603 amino acids,predict protein molecular weight of 66.57 k D,isoelectric point is 8.54,the sequence homology comparison and evolutionary tree analysis found that the gene with sichuan and the highest similarity,with ulmaceae and bombacaceae plant relatively close relatives.The sequence length of the coding region of HMGR gene was 1647 bp,encoding 548 amino acids,and the predicted protein molecular mass was 59.0 k D,and the isoelectric point was 6.68.Homologous comparison and evolutionary tree analysis of the sequence showed that this gene had the highest similarity with cymbalaceae,and was relatively close to the family ulaceae.The LysC coding region was 1683 bp in length,encodes 560 amino acids,predicts a protein molecular weight of 61.65 k D,and has an isoelectric point of 6.26.Homologous comparison and evolutionary tree analysis of the sequence revealed that the gene had the highest similarity to cymbalaceae,and was relatively close to elmaceae and kapokaceae.The sequence length of DapA gene coding region was 1098 bp,encoding 365 amino acids,the predicted protein molecular mass was 40.18 k D,and the isoelectric point was 8.2.Homologous comparison and evolutionary tree analysis of the sequence showed that the gene had the highest similarity to the mulberry and was relatively close to the family ulmaceae.When ethanol-hydrochloric acid solution is used to extract DNJ in mulberry tissue,and the content of DNJ is detected by spectrophotometry,found in mulberry 9703(organs),different organizations of DNJ content in different leaf blade gap is obvious,at the root of DNJ content in different organs(tissues)is the highest,followed by bud and bast,DNJ content in the stem is the lowest,different leaf alkaloid content shows that it can be seen that is also with the increase of leaf number,the content of DNJ present a downward trend,24 different germplasm of DNJ content in mulberry leaves were 40?60?g/g,The content of yu71-1 DNJ was the highest.The difference of alkaloid extraction amount between mulberry varieties was not obvious.The expression levels of CPR-5,HMGR,LysC and DapA were significantly different in different mulberry germplasm,different organs(tissues),and different leaf positions.According to the correlation coefficient,the alkaloid contents of CPR-5 and HMGR genes in different mulberry tissues were closely correlated with the gene expression levels.The alkaloid content of CPR-5and DapA gene in different leaf positions of mulberry was closely correlated with gene expression level,and LysC content in different tissues and germplasm of mulberry was negatively correlated with gene expression level.There was a significant correlation between the expression amount and alkaloid content of these four genes in different organs(tissues),leaves of different leaf positions and the expression amount and alkaloid content in leaves of 24 mulberry germplasm.