Genetic Transformation And Antimicrobial Activity Of Parthenocissus Tricuspidata

Parthenocissus tricuspidata, worldly distributed, belongs to Parthenocissus ofVitaceae. With the greening, environmental protection and medicinal value, its advancedutilization is prospective.This dissertation focus on P. tricuspidata genetic transformation mediated byAgrobacterium tumefacien and its antimicrobial activity.Binary plasmid p2326 was constructed based on pCAMBIA2301. It conferred theforeign genes such as dehydration-responsive element binding protein gene (DREB1B),β-glucuronidase gene (gus) and neomycin phosphotransferase gene (nptⅡ). DREB1Bcloned from Arabidopsis was under the control of the promotor of responsive dehydrationgene rd29B. Both gus and nptⅡgenes were controled by 35S promotor of Cauliflowermosaic virus. This vector was transferred into A. tumefaciens EHA105, followed bytransforming calli or stems of P. tricuspidata.After 3-4 rounds of seletion, the calli with resistance to kanamycin were obtained.PCR and GUS histochemical assay displayed that the foreign genes were integrated intohost genome and expressed.Under the normal condition without stress, the transgenic calli grew well as the wildtype control. For high salinity treatment assay, 150 and 250 mmoL/L NaC1 were added intothe media for calli culture. The results showed that the tolerance to salt tress of transgeniccalli had improved greatly. Although compared to non-transgenic calli, after 20d treatment,the growth of transgenic ones was restrained, and they still survived. However the wild type control was withered away and died. It implied that overexpression of the cDNAencoding DREB in transgenic calli activated the expression of many stress tolerance genes.Moreover, we found that the stems of P. tricuspidata collected from nature couldstrongly restrained the growth of A. tumefaciens with p2326, which carried rifampin andkanamycin resistant genes.The leaves and stems of P. tricuspidata were collected from garden wall aroundChengdu institute of biology in April, August and December. The test of antimicrobialability showed that leaves collected in Dec. had stronger antimicrobial ability than theothers, and followed being extracted by polar-different organic solvents such as petroleumether, ethyl acetate, n-butanol and water. The results showed that antimicrobial ability ofthe n-butanol extract from leaves was strongest in different fractions.And the n-butanol extract was examined for antimicrobial activity against fivemicrobes by using filter paper method. The results showed that the extract restrained thegrowth of Escherichia coli, A. tumefaciens, Bacillus subitilis and Bacillus pumilus, whileinhibited the growth of yeast at a certain extent. And the minimum inhibitoryconcentrations (MIC) were 0.25, 0.3, 0.25, 0.3, 1 g/mL respectively. It was proved thatthe higher the concentration of the extraction, the stronger the antimicrobial activity it has.Moreover the n-butanol extract also could restrained some bacterium strains, which hadantibiotic resistances. After heated at 95~C for 2-3h, the n-butanol extract from P.tricuspidata leaves still presented strong bacteriostasis to germs. This result indicated thatthe substance had heat-stability.