Genetic Transformation Of Sugarcane ACC Oxidase Gene Mediated By Agrobacterium Tumefaciens

Primers were designed according to the reported sequence of ACO gene in sugarcane.A 969bp fragment of ACC oxidase was amplified from cDNA of sugarcane through RT-PCR and then cloned.The cloned ACC oxidase gene was then inserted into a binary vector pBI121 with the forward or reverse orientation between the CaMV35S promoter and NOS terminator sequence respectively,to construct the sense plant expression vector pBIaco and the antisense plant expression vector pBIantiaco,both contained a Km resistance gene(NPTâ…¡).The recombinant vectors of pBIaco and pBIantiaco were introduced into Agrobacterium tumefaciens strain EHA105 with freeze-thaw method, respectively.Then the leaf discs of tobacco plants were transformed with ACO gene and ACO antisense gene.34 sense transgenic plants and 28 antisense transgenic plants had been confirmed by PCR analysis,with the transformation efficiency of 24%and 31%,respectively.Parts of regenerated kanamycin-resistant plants were selected and proven to be positive by Dot-Southern bloting,indicating that the ACO gene and the antisense gene had been integrated into tobacco genome.The ethylene production from leaves of transgenic and nontransgenic tobacco were determined by gas chromatography(GC).The ethylene productions from ACO sense transgenic plants was increased by 77.22%,and that from ACO antisense transgenic plants was reduced by 68.33%,respectively,in compared with that from nontransgenic plantlet.The results confirmed the expression of sugarcane ACO gene in heterologous eukaryotic system.The tobacco transformed with sense ACO grew faster and flowered earlier than the antisense transformers.Subsequently,sugarcane ROC22 was transformed with ACO antisense gene mediated by Agrobacterium tumefaciens.The transformed shoots were screened under the selective pessure of G418 and 19 resistants were obtained, and two of which were further proven to be positive through detecting NPTâ…¡by PCR.The positive plants grew slowlier and were dwarf than the wild type and the untransformed sugarecane as well.