Construction Of Overexpression And RNAi Vectors Of ACO And ACS Genes And Transformation Of Potato

ACC synthase (1-aminocyclopropane-1-carboxlic acid synthase, ACS) catalyzesthe synthesis of ethylene SAMS (S-adomet synthetase) synthetic ACC. ACS is arate-limiting enzyme in ethylene synthesis. ACC oxidase (1-aminocyclopropane-1-carboxlic acid oxidase, ACO) catalyzed ACC to ethylene. So ACO is also known EFE(ethylene-forming enzyme). Two enzymes in the synthesis of ethylene have played akey role. According to the gene sequences information which have been reported, thisstudy use potato cultivar "Gannongshu2" test-tube plantlet as material, RNA wasextracted from leaves of the potato-“Gannongshu2” to design gene special primers,the target genes ACS and ACO were cloned by RT-PCR and the target genes wereanalyzed by bioinformatics. This study built over expression vector pBI121-ACO andpBI121-ACS, and interference expression vector pHellsgate8-ACO andpHellsgate8-ACS. The vectors were introduced into Agrobacterium tumefaciens toprepare engineering bacteria, and obtained transgenic potato plants byagrobacterium-mediated method. This study lays the foundation for the study of ACOand ACS genes in potato growth and stress response process in the role, but also laidthe foundation for the use of genetic engineering ideal new potato varieties. Theresults are as following:1. With RT-PCR, design specific primers according to the gene sequence of ACCsynthase (ACS) and ACC oxidase (ACO)(http://solanaceae.plantbiology.msu.edu/,serial numbers are PGSC0003DMT400005971and PGSC0003DMT400014932) andits conservative fragments, obtain gene specific fragments after PCR amplificationand cloning. The full-length sequences of ACO and ACS will be attached to pMD18-Tcarrier. The interference fragments will be attached to carrier pDONRTM201by BPreaction. After transformation of Escherichia coli DH5α competent cells weresequenced. The results show that the full-length of ACS mRNA is1832bp in whichthe CDS district is1461bp, the homology is as high as97%compared with tomato.The full-length of ACO mRNA is1614bp in which the CDS district is906bp, thehomology is as high as96%compared with tomato.2. ACO was analyzed by bioinformation method, which indicated ACO encodeda protein of301amino acid, molecular weight almost34kDa, PI5.96containing9alpha helix,11beta pleated sheet,13beta turn and15nonregular coil.Bioinfornmation analysis showed ACS encoded a protein of486amino acid, molecular weight almost55kDa, PI6.76containing12alpha helix,22beta pleatedsheet,32beta turn and23nonregular coil. ACO contained26hydrophobic regionsand26hydrophilic regions. ACS contained39hydrophobic regions and41hydrophilic regions. Both ACS and ACO occupied the same evolutionary positionwith tomato.3. The plastid pBI121-ACO and pBI121-ACS was built by ACO and ACS genefragment, respectively, with Infusion method. The plastid pHellsgate8-ACO andpHellsgate8-ACS was built by ACO and ACS interference fragment, respectively, withLR reaction.4. The pBI121-ACO, pBI121-ACS, pHellsgate8-ACO and pHellsgate8-ACS wastransform to A.tumefaciens (LBA4404), respectively, then consequence infected thepotato “Gannongshu2”. Using PCR analysis,16transformed plants was detectedcontaining4pBI121-ACO infected plants,3pBI121-ACSinfected plants,4pHellsgate8-ACO infected plants and5pHellsgate8-ACS infected plants.