Studies On Cloning Of ACS And ACO Full Length CDNAs, Construction Of The ACS Antisence Expressive Vector And Genetic Transformtion

In this experiment, the leaves of Chinese narcissus （Narcissus tazetta var. chinensis） wereused as the materials for cloning the key genes for ethylene biosynthesis that named ACC synthasegene and ACC oxidase gene, and further prediction of the function and structure through thebioinformatics analysis. In the meantime, the construction of the antisense expression vector ofACS gene and the genetic transformation in tobacco were conducted. Besides, the slivers of buldof Chinese narcissus （Narcissus tazetta var. chinensis） were used to induce callus, and theoptimization of induction medium were also carried out, which was of great reference values inboth theoretical and practical applications for Chinese narcissus genetic transformation. The mainresults were as follows:1Cloning the gene of ACS from Chinese narcissusThe5’cDNA sequence of ACS was obtained by the RACE method from the leaves of Chinesenarcissus. The full length cDNA of Chinese narcissus ACS gene （GenBank GU182503） wasobtained through assembling by DNAMAN6.0. It was about1790bp, consisted of5’and3’utranslated regions （93bp and224bp）, including a poly （A） tail of17bp. There were1473bp of theORF through prediction of the DNAMAN6.0. It encoded490amino acids, and the initiationcodon was ATG, and TAA was the terminatior codon.The bioinformatics analysis about the protein structure and prediction of ACS gene werestudied, the results showed that: the molecular weight of the protein was54899.6, isoelectric pointwas8.19; the protein was a kind of alkalescence and hydrophilic protein, with transmembranestructure, without signal peptide; it was likely located in the cytoplasm or nucleus, the secondarystructure of the protein was constituted by32.22%alpha helices and18.71%extended strand and49.06%irregular curly, irregular curly and alpha helis were the main structures; the homologouspercentage of the ACS protein was96%～100%when compared with those of the other plants;the ACS protein contained a aminotransferase class-I pyridoxal-phosphate attachment site, itbelonged to ACC SYNTHASE family, containing aminotransferase, class I/class II structuredomains, pyridoxal phosphate-dependent transferase, major region, subdomain2structuredomains, and aminotransferases, class-I，pyridoxal-phosphate-binding site. Bacterium had themost members in this protein family. There were14phosphorylation sites.on Ser,7on Thr and1 on Tyr. Using the isogeny modeling method, the ACS protein was made up beta sheet andirregular curly, the identity was66.90%when compared with the reference protein.2Cloning the gene of ACO from Chinese narcissusThe conserved region,3’cDNA and5’cDNA sequence of ACS were obtained by the RACEmethod from the leaves of Chinese narcissus. The full length cDNA of Chinese narcissus ACOgene （GenBank JQ002569） was obtained through assembling by DNAMAN6.0. It was about1208bp, consisted of5’and3’ utranslated regions （74bp and192bp）, including a poly （A） tail of14bp. There were942bp of the ORF through prediction of the DNAMAN6.0. It encoded313amino acids, and the initiation codon was ATG, and TAA was the terminatior codon.The bioinformatics analysis about the protein structure and prediction of ACO gene wasstudied, the results showed that: the molecular weight of the proein was35521.5, isoelectric pointwas5.20; it was a kind of alkalescence and hydrophilic protein, wihout signal peptide and with notransmembrane structure, and it was likely located in the cytoplasm, or endoplasmic reticulum; thehomologous percentage of the ACO protein was96%～100%when compared with those of theother plants; it contained a Fe (²⁺)2-oxoglutarate dioxygenase domain profile and there was acoiled coil between100to150amino acids; the secondary structure of the protein was made up30.19%alpha helices and17.21%extended strand and52.60%irregular curly. Irregular curly andalpha helis were the main structures; the protein contained Oxoglutarate/iron-dependentoxygenase structure domains, not belonging to any isogeny family, with8phosphorylation siteson Ser,2on Thr and6on Tyr. Using the isogeny modeling method, the ACO protein of Chinesenarcissus was made up beta sheet and irregular curly, the identity was73.62%when it wascompared with the reference protein.3Construction of the ACS of Chinese narcissus Antisense Gene ExpressionVectorIn this experiment pCAMBIA1302was selected as the expression vector, and the conservedregion sequence of the ACS gene in Chinese narcissus was inserted into the vector by doubledigestion and ligation reaction. The recombinant plasmid was identified by detecting on the targetgene. The recombinant plasmid was introduced into the Agrobacterium tumefaciens strainLBA4404and then transformed into tobacoo. The PCR detection result showed that the targetgene was introduced successfully.4Induction of the callus from buld in Chinese narcissus In the experiment, the effects of different factors on the callus induction in the Chinesenarcissus were studied.The results showed that：the order of the impact on the callus induction was2,4-D>6-BA> KT> NAA. The best medium for the callus induction was MS medium supplemented with2.0mg· L^-12,4-D,1.0mg·L^-1KT and1.0mg·L^-1NAA.