Strawberry Fruit-specific Expression Of The Gene (annfaf) Cloning, Expression In Vitro And Genetic Transformation

A complete cDNA of annfaf expressed specifically in strawberry fruit was cloned via RT-PCR and was subcloned to pMD18-T vector to construct the recombined pMD18-T(annfaf) vector. The inserted fragment was sequenced and the character of deduced amino acids was analyzed To obtain the antiserum. the fusion expression vector and the non-fusion expression vector were constructed by sub-cloning die annfaf cDNA to pET-30(a) plasmid. annfaf was expressed high-effectually in E.coli. BL21(DE3) in the form of inclusion body. The target proteins with molecular weights of 35kD and 44kD were detected by SDS-PAGE. The 35kD target protein recovered from SDS-PAGE was used to immunize rabbit and the annsemm with high titer was obtained. The ELISA and Western Blot analysis indicated that the reaction between annserum and antigen was high specifically. To block the annfaf expression in strawbern.' fruit, the antisense gene of annfaf. was constructed by sub-cloning the annfaf cDNA fragment to pROKII plasmid. The annfaf cDNA with CaMV35S promoter and Nos terminal formed the antisense fusion gene. The engineered stain EHA105 (pROKII -annfaf) was constructed by conducting the pROK II -annfaf to A. tumefaciem EHA1 05 and was used to transform strawbern'. The position clones were obtained via using selection marker and PCR anah sis .