Genetic Transformation Of Antisense4CL1Gene And Cloning Of Cu/ZnSOD Gene From Eucalyptus Grandis X E.urophylla

Eucalyptus is widely cultivated in tropical and subtropical regions. In this paper,the Eucalyptus tissue culture system was established by the optimization oforthogonal test, half-lignification Eucalyptus stems with bud as initial explants. Thebest adventitious bud induction medium was1/2MS+6-BA0.75mg/L+NAA0.05mg/L+Cys10mg/L. The best rooting medium was1/2MS+ABT12.5mg/L+AC0.15mg/L+sucrose20g/L and the rooting rate reached100%. After acclimatizationand transplanting to the greenhouse in15d, it grow good. The leaf explants ofAgrobacterium tumefaciens mediated transformation system was optimized. Theresult shows the theoretical optimal differentiation medium was WPM+6-BA0.3mg/L+NAA0.15mg/L and the bud differentiation rate reach26.7%. Kanamycin with20mg/L in rooting culture medium is used to select transgenic plant.4-coumarate: CoA ligase （4CL） plays a key role in the lignin phenylpropanoidmetabolism by the means of genetic engineering. The Plant expression binary vectorcontained the glycine rich protein （GRP1.8） fused with antisense4CL1gene fromPopulus tomentosa.was transformed to Eucalyptus by agrobacterium-mediated.Regenerated eucalyptus plants were verified by PCR procedure, Three seedlingsshowed positive, proved antisense4CL1gene has been successfully transferred toEucalyptus. The Eucalyptus was directly cultivated to reduce lignin content forpapermaking.Superoxide dismutase （SOD） is capable of scavenging superoxide anion freeradical in organism. The full length mRNA of cytosolic copper/zinc superoxidedismutase （Cu/Zn SOD1） was cloned from Eucalyptus grandis×E. ophylla throughRT-PCR and PCR amplification method （GenBank Accession Number:JX138573）.The sequence of cDNA contains459bp nucleotide encoded polypeptide152aminoacids with the predicted molecular mass15.2kD and theoretical pI6.42. Molecularformula is C₆₅₂H₁₀₄₈N₂₀₀O₂₁₁S₄including a total of2115atomic. There are18kinds ofamino acids in Cu/Zn SOD1protein, which glycine （Gly） accounts for the highestproportion（19.7%）, cysteine （Cys） and methionine （Met） for lowest percentage（1.3%）.Instability index23.51and fat index76.32, the protein belongs to a stable kind. Bybioinformatics analysis, The nucleotide sequence was similar with upland cotton upto99%,and the amino acid sequence up to98%.The full-length gene is amplified to construct expression vetor pET-Cu/Zn SOD1.The Escherichia coli is induced by1mM IPTG in37℃for3hours and theexpression protein molecular weight is about33kD by SDS-PAGE. Enzyme activity assay result shows that enzyme activity of the translated bacteria has increased19.9%than the control in28℃.