Isolation of Promoters Strongly Expressed in Various Tissues of Switchgrass

In order to introduce new genes into switchgrass, constitutive and tissue specific promoters are needed to direct the expression of target genes. In switchgrass, we identified six constitutive promoters and one bundle-sheath specific promoter through comparison with previous studies and our micro-array database. Eight primer pairs were designed to amplify about 2 kb of genomic DNA. The PCR products were cloned into pGEM-easy vector and sequencing was performed. Based on these sequencing results, seven sequences corresponding to promoters for PvAct1, PvUbi1, PvCons1, PvCons2, PvCcO, PvAPX and PvRbcS were identified 2 kb upstream from the ATG site. These seven promoters and a positive control promoter, ZmUbi1, were cloned into the binary vector pCXGUS-P. Because stable transformation can take up to six months to complete, we first bombarded each construct into switchgrass callus to confirm transient expression of these chimeric genes in plant cells. Stable transformations for each construct were performed using our established protocol. Embryogenic calli were co-cultivated with Agrobacteria carrying each gene construct. Resistant calli were obtained after hygromycin selection. Green shoots were obtained after transferring the resistant calli onto regeneration medium. PCR screening of the regenerants showed expected bands. Detailed analyses of expression patterns in different tissues and at different stages will be carried out in switchgrass.