Molecular Modification Of The AaH6H And Function Investigation In Anisodus Acutangulus Hairy Roots

Naturally occurring tropic alkanes alkaloids including cocaine,anisodine,hyoscyamine,6-beta hydroxyhyoscyamine,scopolamine,including scopolamine and anisodine unique to our country.This kind of alkaloids is widely used to fight choline medicine,can combine with acetylcholine competitive toadstool receptors,can block certain nerve impulses,achieve stabilization effect,this kind of medicine acts on the parasympathetic nerve for treatment.Anisodus acutangulus is a medicinal plant that is endemic to the solanaceae in yunnan province,and is valued for its high content of topoalkane alkaloids.Plant metabolic process,by scopolamine 6 beta hydroxylase(H6H),hydroxyl reaction after step and step epoxidation reaction,scopolamine can convert form scopolamine,but in the vast majority of the amount of the latter in the solanaceae plant is much lower than the former.Scopolamine is characterized by high pharmacological activity and small side effects,so the demand for scopolamine is growing rapidly on the basis of the original large market.In recent years,by means of genetic engineering,established the rapid accumulation of secondary metabolites in the hairy root culture system,and obtained some valuable natural secondary metabolites,and have achieved encouraging results,but the use of genetic mutations transformation combined with gm technology to improve the research of secondary metabolites is less.This topic in screening of early cooperation with east China university of science and technology to improving the AaH6 H hydroxyl in vitro dynamic Ser14 Pro and Lys97Ala(mutations)molecular genetic mAaH6 H,on the basis of further its genetic into three points from three hair root,inspects its synthesis and metabolism of scopolamine biological transformation.Through molecular modified mAaH6 H gene as a template,cloned in the molecular modified genes,build mAaH6 H containing molecules modified genes of plant expression vector,and taking the three points of three key enzyme genes not AaH6 H construction of plant expression vector for reference;By hair root,agrobacterium mediated genetic transformation of three points and three explant for molecular modified mAaH6 H gene and modified AaH6 H gene expression of three points to three transgenic hairy root strain;Preliminary purpose gene has been detected by using PCR is inserted into the transgenic hairy root genome,analysis by using the method of quantitative rt-pcr gene expression in transgenic hairy root,and using high performance liquid chromatography(HPLC)determination oftropic in transgenic hairy root alkanes alkaloids,scopolamine,anisodamine and scopolamine)content;The effects of the three-trifoliate root on the biosynthesis and transformation of anisodamine were analyzed.The results are as follows.1.On the NCBI BLAST tool,analyzing AaH6 H find 11 different species H6 H homologous genes,the phylogenetic tree,can be found AaH6 H and Atropa belladonna(AbH6H)has the highest similarity,reached 92%,the second is to Brugmansia arborea(BaH6H)similarity of 91%.Multiple sequence alignment results showed that H6 H of Anisodus acutangulus and the species of alkaloids in toluitane was highly homologous.2.The obtained pMON530-AaH6H-GFP and pMON530-GFP vector are expressed in tobacco.The scanning laser confocal microscope was used to observe,and the air carrier control was established.The results indicate that AaH6 H hydroxylase is expressed in cell membrane.3.The amino acid sequence of AaH6 H was input into PDB protein structure database for pBLAST comparison,comparing the results found that protein is the highest homology from arabidopsis anthocyanin in synthase,with the protein crystal structure as a template to use Swiss-model homology modeling,analysis of the amino acid mutations,and found two hot Ser14 amino acids and Lys97 are located at the surface of the protein structure.4.The organization expression spectrum analysis results show that AaH6 H as a type of expressed genes,different expression in different organizations,AaH6 H expressed in root quantity highest,followed by leaves,these results suggest that AaH6 H main function in the root and exist most alkaloids.5.HPLC analysis results show that:mAaH6H overexpression of total alkaloid content in the hairy root was significantly improved compared with AaH6 H overexpression of the hairy root.The content of scopolamine and hyoscyamine were increased in different degrees compared with AaH6 H,the content of anisodamine was slightly lower than that in AaH6 H.In summary,the molecular modification of mAaH6 H express total tropic alkaloid content in the hairy root than AaH6 H overexpression of hairy root increases markedly,and the possible molecular mAaH6 H to participate in the regulation of alkaloids of molecular key enzyme activity was improved after reforming to promote the synthesis of alkaloids.The changes of scopolamine and scopolamine were significantly improved and the contents of scopolamine were significantly increased.