Functional Analysis Of SOD Gene From Puccinellia Tenuifolra And Its Genetic Transformation In Populus × Euramericana 'Guariento'

Plants frequently encounter stresses such as drought,high temperature,low temperature, they adversely affect plant growth,development and produetivity.SOD is the first enzyme of resist oxidation in the scavenging course.It can catalyze O²..into H₂O₂ and O₂.Later,H₂O₂ can be decomposed into H₂O and O₂.SOD plays a important role on scavenging ROS,protecting the function and the strcture of the cells,escaping damage of oxidation.With the development of the industrial and the city at present,ecological environment has been seriously damaged,the air pollution become worsing(mainly car exhaust,dust-type), severe soil compaction,radiation,noise,these adversities are seriously impact the growth and development of the ornamental plants.Therefore,it is important to cultivate a large number of garden plants with stress tolerance.In this paper,we cloned the SOD gene from PuccineUia tenuiflora(PutSOD).We focused in the biochemistry characteristic,the responsive relationship between SOD and environmental stress,the relationship between transgenic yeast and environmental stress,we select the Populus×euramericana 'Guariento'as the research material,the PutSOD was transformed into it.The aim is to explore the suitable conditions and create a more practical platform of ornamental plants by genetic transformation.The results are as followed:1.We cloned a gene from a Puccinellia tenuiflora root cDNA library whose expression improved the carbonate tolerance in E.coli;The gene open read frame is 615bp.The deduced amino acid sequence of the gene has high homology with the Cu/ZnSOD of the rice,zea, Arabidopsis thaliana.2.The SOD gene and GFP were together cloned in plant expressional vector pBI121,then the plasimed pBI121-SOD was transformed in Arabisopsis.Observing the cell by Confocal Laster Scanning Biolgical Microscopr(OLYMPUS).The result indicated that the SOD gene encoding protein lied in Arabidopsis chloroplast,this result is identical with predictive result initially.3.The SOD enzyme active of Puccinellia tenuiflora was detected under stress.The results indicated that SOD enzyme active of Puccinellia tenuiflora in the leaves and roots was higher than the control.This suggests that SOD gene expression has connected with the stress environment.4.The SOD gene was cloned into the prokaryotic expression vector pQE-30,the plamid pQE-30-SOD was transformed into E.coli M15.After the inducement with IPTG and the His-SOD fusion protein was finally purdied by Ni⁺-sepharose affinity column. 5.The PutSOD was constructed yeast express vector,and induced for expressing protein, Analysis of the tolerance,transformant yeast expression PutSOD protein increased compared with the control.The results indicated that the yeast highly expressiong PutSOD enhanced the resistance to stresses.6.The PutSOD gene was cloned into pBI121,and introdued into A.thaliana by Agrobacterium tumefaciens-mediated transformation using the vacuum infiltration method.The stable expression lines of over-expressing different PutSOD was confirmed by PCR analysis.Salt toleranee of the independent transgenic lines in germination was examined,and the wide type plants were restrained more heavily than the transgenic plants under NaCl stress.This results suggest the transgenic plants harboring PutSOD have a stronger resistanee than the wide-type Arabidopsis thaliana.7.we select the Populus×euramericana 'Guariento' as the research material.Several factors,such as pre-eultivated time,Agrobaeteria concentration,infection time,eo-eultivated time were investigated in the researeh.The explant was leaves of Populus×euramericana 'Guariento',The highest differentiation medium was MS+0.5mg/L 6-BA+0.1 mg/L.The rate of inducement was 90%.The regenerated plants were obtained in the rooting medium(WPM + 0.4mg/L IBA).2%sucrose concentration was the suitable culture medium to induct leaves differentiation.500mg/L-700mg/L of Cefazolin Sodium concentration was selected on bud inducing and rooting for Populus×euramericana 'Guariento' and 700mg/L concentration of Cefazolin Sodium was selected on rooting for Populus×euramericana 'Guariento' during the experiment.The Kanamycin concentration of 30 mg/L was determined when leaves differentiation.20 mg/L was determined when rooting culture medium.The pre-culture time of 3-4days,the Agrobaeterium concentration of OD₆₀₀=0.3-0.4,infection time of 15min,co-cultre time of 3-4days were the suitable for transformation.Some transformants were identified by using PCR molecule detections and seventeen positive transformants were achieved.It is about 3.7%in the number of the plant to be filtrated by antibiotics.The SOD activation of the some transgenic plants were mensurated at the same time,the result was that the SOD activation of transgenic plants were higher than the normal ones.