Construction And Genetic Transformation Of Double-antisence Expression Vector Of ACC Oxidase And ACC Synthase Gene In Phalaenopsis

In this experiment, ACC oxidase(1-aminocyclopropane-1-carboxylate oxidase,ACO) gene and ACC synthase(ACS) gene were cloned from Phalaenopsis, then the fusion antisense plant expression vector was constructed. The vector was introduced into Phalaenopsis protocorm by Agrogacterium-mediated and Gun- mediated transformation. The presence and expression of the transgenes in Phalaenopsis orchid was assessed by histochemical GUS assay. The molecular assays, PCR were carried out to verify Phalaenopsis transformants. The main results are as follows:(1)In order to obtain Phalaenopsis hybrid new strain which possesses the feature of longer florescence, two pair of specific primers were designed and synthesized according to the reported ACC oxidase (1-aminocyclopropane-1-carboxylate oxidase,ACO) gene and ACC synthase(ACS) gene cDNA sequences ,which were obtained from total RNA of Phalaenopsis by RT-PCR. The RT-PCR products were cloned to the pMD19-T vector and identified by enzyme cutting analysis. Then we obtained the positive clones,pMD-RACS and pMD-RACO.(2)Clone the fragments 461bp ACS gene and 333bp ACO gene to the middle vector pBI221 in antisense orientation between 35S promoter and nos terminator. The 461bp ACS gene fragment was obtained from pMD-RACS digested by BamHI and SacI,and 333bp ACO gene fragment was obtained from pMD-RACO digested by XbaI and BamHI.(3)Digest the middle vector pBI221 from 35S promoter to nos terminator by PstI and EcoRI, then clone the fragment into plant expression vector pCAMBIA3301,the fusion antisense plant expression vector pCAMBIA3301-RACSACO was constructed,and the recombinant plasmid was identified with PCR and enzyme digestion by PstI and EcoRI.The plant expression vector was introduced into Phalaenopsis protocorm by Agrogacterium-mediated and Gun-mediated transformation.(4)The plant regeneration system of Phalaenopsis was established and optimized. The protocorm-like bodies (PLBs) and regenerating planets were obtained from embryos. The results show that the suitable medium for germination of embryos was 1/3MS+ KT 0.5 mg/L + sucrose 20.0g/L + CW15% + AC 2.0 g/L + agar 8.0g/L.(5)The best transient expression of GUS was obtained under the conditions as follows: pre-culture for 3d,OD600≈0.5, infection for 5-15min, and co-culturing for 3d and appended 100 pmol/L AS.(6)After extracting the genome DNA of transgenic plant, The presence and expression of the transgenes in Phalaenopsis orchid was assessed by histochemical GUS assay. The molecular assays, PCR were carried out to verify Phalaenopsis transformants.