Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent.Here,we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme pu-trescine N-methyltransferase(PMT)and the downstream enzyme hyoscyamine6β-hydroxylase(H6H)of scopolamine biosynthesis in transgenic henbane(Hyoscyamus niger)hairy root cultures.Transgenic hairy root lines expressing both pmt and h6h produced significantly higher(P<0.05)levels of scopolamine compared with the wild-type and transgenic lines har-boring a single gene(pmt or h6h).The best line(T 3 )produced411mg/liter scopolamine,which was over nine times more than that in the wild type(43mg/liter)and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184mg/liter).To our knowledge,this is the highest scopolamine content achieved through genetic engi-neering of a plant.We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield,which was more efficient than plants harboring only one of the two genes.It seems that the pulling force of the downstream enzyme(the faucet enzyme)H6H plays a more important role in stimulating scopolamine accumulation in H.niger whereas the functioning of the upstream enzyme PMT is increased propor-tionally.This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.

Molecular Cloning and Characterization of Enolase from Oilseed Rape （Brassica napus）

An enolase-encoding cDNA clone in oilseed rape (Brassica napus L.) was isolated. This gene (accession number: AY307449) had a total length of 1 624 bp with an open reading frame of 1 335 bp, and encoded a predicted polypeptide of 444 amino acids with a molecular weight of 47.38 kD. The deduced amino acid sequence shared identity with a number of enolases ranging from Bacillus subtilis to human beings and had much higher identity with other plant enolases than with enolases from Bacillus, yeast and human beings. Comparison of its primary structure with those of other enolases revealed the presence of an insertion of five amino acids in enolase of B. napus. Southern blotting analysis of genomic DNA indicated that enolase was likely to be a low-copy gene in the oilseed rape genome. Expression of the cloned enolase gene increased under salt stress, but decreased in response to low temperature. Our studies suggested that the cloned gene was a new member of plant enolase gene family, which contributed to the energy supply in stress-treated tissues.

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.