Molecular Cloning And Characterization Of Genes Which Catalyze The Scopolamine Biosynthetic Pathway In Anisodus Acutangulus

A few genera of the plant family Solanaceae including Anisodus, Atropa, Datura, Duboisia, Hyoscyamus and Scopolia are able to produce biologically active tropane alkaloids simultaneously. Tropane alkaloids such as hyoscyamine and scopolamine, which are widely used as anticholinergic agents, can act on the parasympathetic nervous system. Because of their low contents in plants, the supply of hyoscyamine and scopolamine is unable to meet the need of the pharmaceutical market. The development of modern biotechnology provides a fantastic way to genectically improve tropane alkaloids production by transferring genes involved in the tropane alkaloids biosynthetic pathway into plants and culturing transgenic hairy roots in a large scale.In this research, two new genes codoned putrescine N- methyltransferase and hyoscyamine 6β-hydroxylase in A acutangulus were cloned, which are both key enzymes involved in the tropane alkaloids biosynthetic pathway. In order to further investigate the biosynthetic pathway and the moculeular basis for higher alkaloids content in A acutangulus, characteristics of genes and proteins, the expression regulation and effects of the inductor were carried out and reported as following:1.A new full-length cDNA encoding putrescine N- methyltransferase which catalyzes the first committed step in the scopolamine biosynthetic pathway was isolated from A. acutangulus by rapid amplification of cDNA ends (RACE) for the first time, namely AaPMT. The full-length cDNA of AaPMT was 1237bp and contained a 960bp open reading frame (ORF) encoding a deduced protein of 319 amino acid residues. The deduced protein had an isoelectric point (pI) of 5.51 and a calculated molecular mass of about 35.1 kDa. Sequence analyses showed that AaPMT had high homology with other PMTs isolated from some scopolamine-producing plants. Bioinformatics analyses results indicated that AaPMT belonged to the S-adenosylmethionine-dependent N-methyltransferase superfamily. Phylogenetic tree analysis showed that AaPMT had the closest relationship with PMT from A belladonna and A tanguticus. Southern hybridization analysis of the genomic DNA revealed two PMTs belonged to a multi-copy gene family .2. A new full-length cDNA encoding hyoscyamine 6β-hydroxylase (designated as AaH6H, GenBank Accession No. EF187826), which catalyzes the last committed step in the scopolamine biosynthetic pathway was isolated Anisodus acutangulus by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of AaH6H was 1394bp and contained a 1032bp open reading frame (ORF) encoding a deduced protein of 344 amino acid residues. The deduced protein had an isoelectric point (pI) of 5.09 and a calculated molecular mass of about 38.7kDa. Sequence analyses showed that AaH6H had high homology with other H6Hs isolated from some scopolamine-producing plants such as Hyoscyamus niger, Datura metel and Atropa belladonna etc. Bioinformatics analyses results indicated that AaH6H was in the 2-oxoglutarate-dependent dioxygenase superfamily. Phylogenetic tree analysis showed that AaH6H had the closest relationship with H6H from A tanguticus. Southern hybridization analysis of the genomic DNA revealed that AaH6H belonged to a multi-copy gene family. Tissue expression pattern analysis found that AaH6H expresses in all the tested tissues including roots, stems and leaves and indicated that AaH6H is a constitutive-expression gene, which was the first reported tissue-independent H6H gene compared to other known H6H genes. Methly Jasmonate inducement tests showed that expression of H6H in A acutangulus was regulated differently in various parts.3. A pCABIA 1304~+ plant expression vector contained AaPMT gene and AaH6H gene, which was driven by the CaMV 35S promoter respectively, was constructed and transferred into Agribacterium tumefaciens C58C1.