Formulation Development For Microbial Flocculant HBF-3 And Genetic Transformation Of Vip3Aa Into Paecilomyces Lilacinus

Two independent studies are described as follows:1 Formulation development for microbial flocculant HBF-3The novel bioflocculant HBF-3 is an acidic extracellular polysaccharide, which is produced by the deep-sea bacterium Halomonas sp. V3a'. For a further development of fiocculant HBF-3, the study on dosage forms of HBF-3 was performed. The supernatant of the fermentative culture was made into solid formulation by freeze-drying with 5% sucrose as cryoprotectant, yielding a lyophilized power, which can be easily rehydrated and therefore keep a flocculating activity as high as 82.4%. Simultaneously, liquid formulation was made through rotary evaporation process, with a little flocculating activity changed. The results also demonstrated that HBF-3 was thermostable. When added 1.0 g/L sorbic acid potassium to the concentrated solution and stored at 4℃,28℃, 37℃, and 50℃, respectively, the sample maintained high flocculating activities even one month later.2 Genetic transformation of vip3Aa into Paecilomyces lilacinusPaecilomyces lilacinus (Thom.) Samson 1974 is an effective parasitic fungus of phytopathogenic nematodes such as Meloidogyne spp. and Heteridera spp.. It is accepted that P. lilacinus is one of the most promising biocontrol fungus against phytopathogenic nematodes. On other hand, Vip3Aa is one kind of soluble protein expressed during the vegetative growth phase of Bacillus thuringiensis. It is confirmed that Vip3Aa has specifically high toxicity to Agrotis ipsilon and Spodoptera exigua.The plasmid pVIP was constructed based on expression vector pET28b(+) and transformed into Escherichia coli strain BL21(DE3) for expression of the Vip3Aa protein. A ployclonal antibody against Vip3Aa was obtained from New Zealand rabbits.The genetic transformation vector puPVNT of vip3Aa gene was constucted using pcDNA3.1(-) and pcDNA3.1(+) as intermedium plasmids, and transformed into Agrobacterium tumefaciens LB4404. Then, the vip3Aa gene was transformed into P. lilacinus 9410 strain via ATMT(Agrobacterium tumefaciens-mediated transformation) using the resistance of G418 as selective marker. PCR assays confirmed that vip3Aa gene had been integrated into the genome of P. lilacinus 9410 strain.