Cloning Of CDNA Encoding Annexin In Strawberry (Fragaria×ananassa Duch.) Fruit And Genetic Transformation Of The Antisense Gene

During fruit ripening, the physiological changes, especially biological synthesis of ethylene and reaction to exoethylene, are distinct different between climateric and non-climateric fruits. The research on the molecular biology of the ripening of strawberry fruit, a typical non-climateric one, would be helpful to reveal the ripening mechanism of the non-climateric fruit. The mRNA expressing specifically and encoding annexin in ripening strawberry fruit has been identified, and the protein (AnnFaF) encoded by it might play an important role in strawberry fruit ripening as ion channel protein or signal peptide. The complete reading frame of the AnnFaF cDNA has not been obtained and the protein function on strawberry fruit ripening has not yet been characterized. Therefore, we cloned the known and 5'-end unknown sequence of the AnnFaF cDNA. The AnnFaF antisense fusion gene was constructed and was transformed into tobacco and strawberry to obtain the AnnFaF deficiency plant. This would be helpful for studying the function of the gene in strawberry fruit ripening and senescence, and for studying mechanism of non-climateric fruit ripening. The isolation of RNA from strawberry fruit is made difficult by the presence of contaminating carbohydrate polymers and polyphenols. To isolate translatable RNA from strawberry fruit, the procedure of isolation of RNA was studied, and a new method of total RNA isolation by a single extraction with acid phenol was obtained. The method was suitable to the plant tissue rich in contaminating carbohydrate polymers and polyphenols. With this method, translatable RNA can be obtained in 3~4 hours. The 836bp fragment was obtained by RT-PCR using primers corresponding with RJ4 cDNA sequence published in GenBank from total RNA of strawberry fruit, and was cloned to pGEM-3Z(f+) vector to construct the recombined pGRJ4 vector. Then, the inserted fragment was sequenced by using SP6 and T7 promoter primers with Sanger's method The homology of DNA sequence and that of amino acid sequence were 99% and 99.63% compared with the published ones. Furthermore, the 5'-end unknown region of AnnFaF cDNA was cloned by 5'-end rapid amplification of cDNA ends (RACE). It consisted of 180bp containing initiation sequence and encoded 43 amino acids. The complete reading frame of AnnFaF cDNA consisted of 945bp nucleotide and encoded 314 amino acids. To get the antisense gene of AnnFaF, the plant expression pRRJ4 vector was constructed by cloning the RJ4 fragment to pROKII plasmid. The RJ4 fragment with CaMV35S promoter and Nos terminal formed the antisense fusion gene. Then, the engineered strain EHA105 (pRRJ4) was constructed by conducting the pRRJ4 to A.tumefaciens EHA105 and was used to transform tobacco and strawberry M14. Sixty-one clones rooting in medium containing Km were obtained from tobacco and four ones were obtained from strawberry M14, the transformation frequency reached 31.9% and 1.9% respectively. The Southern blotting and PCR-Southern analysis indicated that the antisense fusion gene of AnnFaF was integrated into the genomes of tobacco and strawberry. The antisense gene expression of AnnFaF in strawberry fruit and the regulation function of it during strawberry fruit ripening are being studied.