Cloning And Transformation Of A CDNA Fragment Of Senescent-associated Gene Of Oryza Sativa

Rice "Liang You Pei Jiu" was used as experiment material to study themolecular mechanism of crop senscence, and to improve and perfect theories aboutsenescence in this paper. First, total RNA was isolated from the Oryza sativasenescence leaves. By using the specific primers designed on the conservativesequence of Arabidopsis thaliana SAG₁₂(senescent-associated gene) gene inGenBank, a fragment was produced by reverse transcription polymerase chainreaction (RT-PCR). The amplified fragment was cloned to pGEM-T easy vector, andit was confirmed by sequencing. Second, the amplified fragment of SAG₁₂ wasdigested by SalI and EcoRI, then it was inserted into expression vectorpCAMBIA1300 in reverse orientation, resulting in a plasmid named pCAMBIA1300-anti-SAG₁₂. Then by using the specific primers designed on the promotersequence of the Arabidopsis thaliana SAG₁₂ in GenBank, a fragment was obtainedby PCR with PS-T (the Promoter sequence of SAG₁₂ in pGEM-T Easy Vector) astemplate. The amplified fragment was digested by SalI and PstI, and inserted into thevector pCAMBIA1300-anti-SAG₁₂, structuring in an expressing plasmid namedP_SAG12-anti-SAG₁₂- Last, the vector was isolated and transformed into Agrobacteriumtumefaciems, and it was identified by PCR with S2F and S2R as the primers. TheP_SAG12-anti-SAG₁₂ was transformed into the mature embryo calli of "Liang You PeiJiu" rice by using an Agrobacterium tumefaciems-mediated transformation system,and 27 resistant plants were abtained after selected with the NB media addedhygromycin.