Transformmation And Expressing Of Bt Gene Cry1Ia8 In Cabbage

Head cabbage (Brassica oleracea L. var. capitata L.) is severely damaged by insect-pests. Among them cabbage worm (Pieris rapae L.) and diamondback moth (Plutella xylostella L.) are the primary pests. It is the best way to obtain the cultivars with insect-resistance in pest control because the resistance to insecticide is easily induced.cry1Ia8 gene has been cloned from Bacillus thuringiensis strains Btc008 and has no homology with cry1-type which is commonly used in pest control. After codon optimization, new cry1Ia8 sequence was artificially synthesized and transferred into E.coli. The result of prokaryotic expression in vitro bioassays showed that Cry1Ia8 protein have relatively broad insecticidal controlling spectrum, strong insecticidal activity to corn borer and diamondback moth, and the pests didn't have cross-resistance on both of them.The regeneration system of Cabbage had been optimized with 21-3 and 24-5: The regeneration frequency was the highest with explants from seedings after 5 days from sowing. Both hypocotyls of 21-3 and cotyledons of 24-5 were perfect explants for regeneration. We could obtain high regeneration frequency by supplementing 6-BA 2 mg/L and NAA 0.1 mg/L on the MS medium. The higher regeneration rate of adventitious buds could be obtained by adding Silver nitrate and silver thiosulfate. Moreover, silver thiosulfate was better than silver nitrate.The transformation system of cabbage had been improved through the cry1Ah gene transferred into plants with plant constitutive expression vector pCS1Ah. The results showed: non-transgenic green shoots regenerated from explants of cabbage could be completely inhibited by medium with 10 mg/L kanamycin. There are no obvious effects to add acetosyringone on transformation frequency of cabbage. The experiment detected that the optimal infection time was 8 min. The research indicated that the highest transformation frequency reached to 16.13%.The plant constitutive expression vector pCSIaN including cry1Ia8 gene was transferred into hypocotyls and the cotyledons of cabbage by Agrobacterium tumefaciens-mediated method. 38 transformants were obtained and PCR analysis demostrated that cry1Ia8 gene was integrated into the genome of cabbage. Moreover, Southern blot, RT-PCR, and Western blot analysis showed that cry1Ia8 gene was expressed in DNA, RNA and protein levels. The results of bioassay against diamondback moth (DBM, Plutella xylostella) displayed that 9 transgenic plants were mostly resistant to the larvae of susceptible diamondback moth and also to the insect strain which is resistant to Cry1Ac toxin.