Prokaryotic Expression And Tobacco Transformation Of ACC Synthase Gene In Cut Flower Of 'Luo Yang Hong' Tree Peony

In the pathway of Ethylene biosynthesis,ACC is the direct pre-cursor of Ethylene.The conversion of SAM to ACC was recognized as the rate-limiting step which was catalysed by ACS.Former study indicated that ethylene had a vital role in opening and senescence process of tree peony(Paeonia suffruticosa), besides that,ACS was also a main key which influenced dramatically during the process.According to above results,we can use transgenic techonology to control the process of opening and senescence of tree peony and prolong its vase life by reducing the production of ethylene.So,In present work,'Luo Yang Hong' tree peonyr,which produced ethylene in a climacteric-like pattern,was taken as material,During the study,a recombinant plasmid containing PsACS1cDNA for Prokaryotic Expression and other two plant vectors for transforming tobacco were constructed just to improve the life vase of tree peony in future.The main experimental results are indicated as follows:1.Obtained the cDNA fragment of ACC synthase gene in tree peony:Total RNA was isolated from the cut flower of tree peony'Luo Yang Hong'.A cDNA fragment of ACC synthase with a length of nearly 1.5kb was amplified by RT-PCR and cloned into a pGEM- T Easy vector.The recombination was marked as pACS1.Sequence analysis showed that the cloned 1476bp cDNA shares a homology 99%with PsACS in genebank.2.Constructed the Prokaryotic vector encoded ACSlgene for protein expression.Through recloning,the enzyme sites of BamHâ… and Hindâ…¢were transferred into the ends of ACC synthase gene fragment,such fragment and plasmid pET28a were both cut by these two enzymes.Through being purified and linked,it built the Prokaryotic vector encoded ACS1 gene for protein expression.The recombination can express the protein which weighed nearly 55KD by inducing with IPTG.3.Constructed the paint vectors encoded ACS1gene in sense and antisense transformed them into tobaccos mediated by Agrobacterium and obtained the transgenetic tobaccos initially:Two pairs of primers containing sites were designed and used to amplify sequenced plasmid,The PCR product was digested by BamHI andSalâ… restriction enzymes,and successfully constructed the plant expression pBInACS1s and pBInACS1s through linking the pBI121n which are digestesd by same enzymes.Using leaf method,the plant vectors had been transformed into tobacco mediated by Agrobacterium tumefaciens 3101.The transgenetic tobaccos had been identified by PCR amplication.