Expression Of Sugarcane ACC Oxidase CDNA In Escherichia Coli And Construction Of Plant Expression Vector Of Sugarcane ACC Oxidase CDNA

Primers were designed according to the ACO cDNA sequence of sugarcane in GenBank (AY521566). We got the sugarcane ACO cDNA ORF of fμLl length 969bp via RT-PCR by taking the total RNA from ROC20 leaves which was induced by 75mmol/L LiC1+0.5mmol/L IAA+0.5mmol/L BA as template. The nucleotide sequence and amino acids sequence of pMD18-AC0 and AY52566 alignment resuLts showed that their nucleotide sequence shared 97.5% identity and their amino acids sequence shared 97.5% identity. Comparison of amino acids sequence of Isopenicillin N synthase (IPNS P05326)and ACO from pMD18-ACO Oryza sative (OS-ACO CAA59749)and tomato (LE-ACO1 P05116) showed that they shared three conserved His residues H43 H183 and H240 in Box 1 Box 2 and Box 3.And the sugarcane A CO DNA of fuLl length 1075bp was amplified via PCR by taking the genome DNA from ROC20 leaves as template. Squencing analysis of ACO cDNA and DNA indicated that the ACO DNA consisted of two exons of fuL1 length 972bp and interrupted one 103bp intron.The two exons encode 324 amino acids. The intron is a repetitive sequence rich in AT (61%). To get further knowledge of the mechanism of ethyle biosynthsis in ACO regμLation, the sense plant expression vector pCBUSACO and the antisense plant expression vector pCBUantisaco were constructed by the binary vector pCAMBIA3301 via the middle vector p JUS ACO and pJUantisaco for genetic transformation of sugarcane.The recombinant expression vector pET30a-Sgaco was constructed via sugarcane ACO cDNA. PET30a-Sgaco was transformed in E.coli BL21 (DE3) plysS, and the sugarcane ACO cDNA was high expressed. The recombinant protein had a molecμLar mass of 40KD and it was insoluble inclusion bodies. The recombinant protein was purified with Ni²⁺ -NTA column affinity chromatography. The resuLts showed that the approach for one-step...