Construction And Genetic Transformation Of Antisence Expression Vector Of ACC Synthase Gene In Phalaenopsis

In this experiment, ACC synthase(ACS) gene were cloned fromPhalaenopsis, then the antisense plant expression vector was constrcted. Theantisense ACS gene was introduced into Phalaenopsis protocorm byAgrobacterium-mediated.The presence and expression of the transgenes inPhalaenopsis orchid was assessed by histochemical GUS staining. The molecularassays, PCR were carried out to verify Phalaenopsis transformants. The main resultswere described as follows：（1）Acquiring and cloning ofACS in PhalaenopsisIn order to obtain Phalaenopsis hybrid new strain which possesses the featre oflonger florescence, two pair of specific primers were designed and synthesizedaccording to the reported ACC synthase(ACS) gene cDNA seqences in NCBI,whichwere obtained from total RNA of Phalaenopsis by RT-PCR. The ACS gene wasconnected to the cloning vector pMD19-T vector and transferred into E.coliDH5α.,Then the recombinant plasmid pMD19-ACS products was verified by PCR、double-enzyme cleavage and gene sequence analysis.(2) Constrction and identification of the middle vector pBI221-RACSUsing gene recombination technique, inserting the fragments461bp ACS gene tothe middle vector pBI221in antisense orientation between35S promoter and nosterminator. Then transfer pBI221-RACS into E.coli DH5α Identified the recombinantplasmid via PCR and double-enzyme cleavage.（3）Constrction and identification of the Antisense gene expression vectorpCAMBIA3301-RACSDigest the middle vector pBI221from35S promoter to nos terminator by PstIand EcoRI, then inserting the fragment into plant expression vector pCAMBIA3301,and transfer pCAMBIA3301-RACS into E.coli DH5α.,Identified the recombinantplasmid identified with PCR and enzyme digestion by PstI and EcoRI.（4）Transferring pCAMBIA3301-RACS to Agrobacterim tumefaciensThe recombination plasmid pCAMBIA3301-RACS was introduced intoAgrobacterim tumefaciens EHA105by freezing and thawing. The positive clone was screened with Rif and Kana.Through plasmid PCR and double-enzyme cleavageidentification, it is proved that pCAMBIA3301-RACS was transferred toAgrobacterim tumefaciens EHA105.（5）Constrction of Phalaenopsis regeneration systemThe protocorm-like bodies (PLBs) and regenerating planets were obtained fromseed.MS was the basic culture medium. The medium1/3MS+KT0.5mg/L+AC1.0g/L+peptone1g/L+coconut15.0%was selected for seeding;1/3MS+NAA0.5mg/l+6-BA5.0mg/l for differentiation; Hyponex12.5g/L+Hyponex20.5g/L+6-BA5.0g/L+coconut10%was the optimum for multiplication of PLBs; Hyponex13.0g/L+NAA0.3mg/L was suitable for rooting.（6）The screening of transformation body and plant regenerationAfter pre-cultured for2-3days, the explants were dipped in suspension ofAgrobacterim tmefaciens for90-120mintes, And then trained for2-3days. Thentransfer them to the medium that containing Mer and PPT for differentiation andselection.The transformants transfered into foreign genes would be germinated、multiplication and took root. Thus the transgenic plants were obtained.（7）PCR test of transgenic PhalaenopsisAfter extracting the genome DNA of transgenic plant, The presence andexpression of the transgenes in Phalaenopsis orchid was assessed by histochemicalGUS staining. The molecular assays, PCR were carried out to verify Phalaenopsistransformants.