Cloning of ACC Synthase Gene from Paeonia suffruticosa and Construction of Antisense Plant Expression Vector

According to the DNA full-length sequence of ACC synthase gene fragment of Paeonia suffruticosa(FJ769773)in GenBank database,a pair of specific primers,P1 and P2,were designed and synthesized.Based on the optimized PCR amplification system and high fidelity DNA polymerase KOD-Plus,ACS gene fragment PsACS-4 was successfully amplified from the total DNA which was extracted from leaves of peony cultivar Luo Yang Hong.Sequencing result indicated that the length of PsACS-4 fragment was 970 bp,and the similarity of the sequence of PsACS-4 and the target sequence was 100%.Two sequences alignment analysis revealed that sequence of PsACS-4 didn′t contain any intron sequence.The recombinant plasmid and the plant expression vector pBI121 were digested with the Sac I and Sma I digest enzyme.After the fragment of ACC synthase gene and pBI121 were recovered and ligated,PsACS-4 gene fragment was inserted into 35S promoter downstream of plant expression vector pBI121 reverse in orientation,and an antisense plant expression vector pBI121-anti-PsACS-4 was constructed successfully.