Identification Of Curcin By Western-blot In Calli Generated From Explants Of Jatropha Curcas L. And Study On Its Tobacco Transgene

Jatropha curcas L., a member of the family Euphorbiaceae, is widespread in tropical and subtropical areas. The toxicity of the whole seed of the species has been known for a long time, rendering them potentially toxic to sheep, goat, rat, mice and human being, especially to children. A toxic protein, designated as curcin, was isolated and purified from J. curcas seeds. It's a ribosome-inactivating protein. This curcin has a similar mechanism to trichosanthin in damaging ribosomes with rRNA N-glycosidase activity. It has a wide application future in anti-fungus, anti- virus and anti-cancer.The curcin was isolated and purified from J. curcas seeds by precipitation with 60 % saturation of (NH4)2SO4 followed by chromatography on Sephedex G-75(Lin juan,2002). And the purified protein was used to immunize rabbits to prepare antiserum. The antibody was detected by techniques of ELIST and Western-blot. The titer of antiserum was 1:12800. Western blot analysis confirmed antiserum only recognized the curcin. The proteins from endosperm, root, stem, leaf, leafstalk of Jatropha curcas L. and their calli were hybridized the seed toxin protein (curcin) of Jatropha curcas L. by Western blot. The result showed that curcin signaling wasspecifically appeard in the endosperms and their calli, while it was not detected in roots, stems, leaves, and leafstalks of Jatropha curcas L. and their calli . Therefore, the calli induced from endosperms can be another source for producing curcin, which is an effective way to solve the problem of poor natural resources, and have the advantages of simplicity, short production period and not limited by geographic area, season or climate conditions.Primers were designed in accordance with the cDNA sequence of curcin. By using total DNA of young leaf as template, the ORF of curcin gene was amplified, which is almost identical with its cDNA sequence. This ORF was inserted into vector pBI121 . Plant expression vector pBI121-curcin was constructed in which the expression of curcin was under the control of Camv35S promoter and T-nos terminator. Curcin was successfully introduced into tobacco plant by Agrobacterium-mediated (Agrobacterium Tumefaciens EHA105) transformation. The leafdiscs were infected by Agrobacterium Tumefaciens, then polyshoots were induced on plates containing MSS2 medium supplemented with kanamycin. The roots were induced on plates containing MSS3 medium from polyshoots. The rate of roots-polyshoots were 81%. The regenerated plants were transferred to soil, the . survival rate is 100%. And the transgenic plants were obtained and confirmed by PCR and RT-PCR analysis. The transformation rate is about 47%.