Persimmon ACC Oxidase Gene Cloning And Its Plant Expression Vector Construction

Persimmon fruit, a climacteric fruit, is very difficult to be storaged and transported because it is easy to be soften after harvested. A large body of evidence has demonstrated that ethylene is the hormone of catalyzing climacteric fruit ripening. After the starting of ethylene biosynthesis in fruits, ethylene production increased fast. Make use of trnasgenic technique to inhibit or decrease the ethylene biosynthesis of climacteric fruit, can decrease the content of ethylene in fruit. ACC synthase and ACC oxidase are the key enzymes during the biosynthesis of ethylene. We can improve the storage capability of climacteric fruit by inhibiting the expression of the two enzymes with transgenic method. In the present study, we have cloned the ACO gene fragment using total RNA extracted from the persimmon fruit, identified by sequencing and further constructed in the plant expression vector. The results were as follows:1. The quality RNA was extracted by Rneasy^? Plant Mini Kit. The value of OD₂₆₀/OD₂₈₀ was 2.0979.2. Reverse translation of mRNA was conducted with Omniscript^TM RT Kit. High quality RNA was obtainded by the kit, which was base for successful PCR.3. According to the amino acid sequences of Hiratanenashi' persimmon, a pair of specific primers were designed. One piece of ACO DNA sequence from persimmon fruit was cloned, which was amplified by Reverse Transcription Polymerse Chain Reaction (RT-PCR) from ripening persimmon fruit. Sequencing analysis showed that the PCR products were 592bp, which nucleotide acids and amino acids displayed 98% of identity with the sequences DK-ACO1 registerd in GenBank. The result proved that the cloned gene segment should be ACO gene segment of the persimmon.4. According to the ACO gene sequences analysis, the specific primers were designed respectively in both sides of introns, which had the restriction enzyme EcoRV. The two PCR products were digested by EcoRV and ligased as one gene segment by T4 DNA ligase. The gene segment was transformated in Escherichia coli DH5a and identified. The sequencing result showed that the introns were removed from the gene segment. 5. The PCR products and pBI221 plasmids were cut by restriction enzyme Xbaâ… and BamHâ… . The digested PCR products was inversely inserted into pBI221. The recombined expression vector was named as pBI-anti-ACO.6. The pBI-anti-ACO and pSMAK311 plasmids were digested by Smaâ… and Hindâ…¢. The CaMV 35S promoter and antisense PCR products from pBI-anti-ACO were inserted into pSMAK311 of 35S promoter excision. The antisense expression vector was constructed which was named as pSMAK-anti-ACO.7. The pBI-anti-ACO plasmids and PCR products were digested by BamHâ… and Smaâ… . The digested PCR products were forward inserted into pBI-anti-ACO. The recombined vector was named as pBI-ACO-RNAi.8. The pBI-ACO-RNAi and pSMAK311 were digested by Smaâ… and Hindâ…¢. The CaMV 35S promoter, sense PCR and antisense PCR products from pBI-ACO-RNAi were ligased into pSMAK311 of 35S promoter excision. The RNAi expression vector was constructed which was named as pSMAK-ACO-RNAi.9. Two plant expression vectors pSMAK-anti-ACO and pSMAK-ACO-RNAi were transformed into Agrobacterium EHA101 by freezing and melting method. Using the specific primers and transformed Agrobacterium EHA101 as template to PCR respectively, two PCR products same as pSMAK-anti-ACO and pSMAK-ACO-RNAi were appeared again, which proved recombined plasmids having been transformed to EHA101.