【作者】 吴建慧；

【导师】 柳参奎；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2008， 博士

【摘要】 植物在遭受干旱、高温、冷（冻）害等逆境侵袭时,细胞内活性氧产生与清除的动态平衡被破坏,产生氧化胁迫,影响植物的生长、发育,甚至导致细胞或植株死亡。超氧化物歧化酶（superoxide dismutase,SOD）是活性氧清除反应过程中第一个发挥作用的抗氧化酶,能将超氧物阴离子自由基(O^2.-)快速歧化为过氧化氢（H₂O₂）和分子氧。SOD对于清除氧自由基,防止氧自由基破坏细胞的组成、结构和功能,保护细胞免受氧化损伤具有十分重要的作用。目前,随着工业的迅速发展,城镇化建设的步伐加快,城市生态环境遭到严重破坏,城市大气污染日趋严重（主要为汽车尾气,粉尘类）,土壤严重板结,辐射、噪音增加,所有这些逆境都严重地阻碍了园林植物的正常生长发育。因此,大量培育抗逆园林植物、创造园林植物新品种显得尤为重要。本研究克隆了碱茅超氧化物歧化酶SOD基因（PutSOD）,对SOD基因生理生化特性、基因与盐耐受性之间的关系、转基因酵母做了初步探讨,同时选用欧美杨108为转化受体材料,通过遗传转化手段将碱茅SOD基因转入杨树,旨在提高杨树抗逆境胁迫的能力,同时,也期望在实验中摸索树木转基因的各项合适条件,为我国其它园林树木的分子育种建立一种较为实用的操作平台,最终达到通过分子手段改造园林植物的目的。主要得到以下结果:1、从碱茅根cDNA文库中克隆出SOD基因（命名为PutSOD）,阅读框架（ORF）为615bp。通过BLAST对该基因编码蛋白的氨基酸序列进行同源性检索（DDBJ）:该基因编码蛋白的氨基酸序列与水稻Cu/ZnSOD、与玉米Cu/ZnSOD、与拟南芥Cu/ZnSOD都具有较高同源性,推测该基因就是碱茅Cu/ZnSOD基因之一。2、将PutSOD基因与绿色荧光蛋白GFP基因融合,构建到植物表达载体pBI121上。提取转基因拟南芥的原生质体,利用共聚焦显微镜观察,结果证明:该基因编码的蛋白存在于细胞的叶绿体中,这个结果与我们最初预测的PutSOD基因编码的蛋白在细胞内的存在位置结果一致。3、对碱茅不同器官SOD酶活性在60mMNaCl、20mMNaHCO₃、10%H₂O₂等逆境条件下的活性进行了测定。结果表明:在盐逆境（NaCl,NaHCO₃）,氧化逆境（H₂O₂、ABA）条件下PutSOD的酶活性与对照相比明显增高,表明碱茅PutSOD基因表达与逆境存在着一定的应答关系。4、利用大肠杆菌体外表达技术,将碱茅SOD基因构建到原核表达载体pQE-30中,并将pQE-30-SOD质粒导入E.coli M15菌株,通过对诱导温度及IPTG浓度等条件的调整,经Ni⁺凝胶亲和层析获得了纯化的His-SOD融合蛋白。5、把PutSOD基因构建在酵母表达载体上,诱导表达后,观察遗传转化酵母的耐逆境能力。结果表明,强化表达PutSOD基因的酵母与对照相比,对各种逆境的耐性有了一定的增强。6、将PutSOD基因功能区构建到双元表达载体pBI121中,经农杆菌介导的真空浸润法将基因转入拟南芥中。通过卡那霉素筛选,PCR鉴定,获得了能够稳定遗传的转基因株系。对转基因拟南芥,进行抗盐性试验,结果表明,转PutSOD基因拟南芥都比野生型有更好的生长势。表明PutSOD在提高植物耐受性上有更强的作用。7、本研究同时选用欧美杨108作为转基因的受体材料,对预培养时间、工程菌液浓度、侵染时间、共培养时间对其遗传转化效率的影响进行了实验。以欧美杨108叶片作外植体,在MS+0.5mg/L 6-BA+0.1 mg/L NAA的培养基上诱导叶片愈伤组织率和分化率较高,最高可达90%。不定芽在WPM+0.4mg/L IBA的生根培养基上很快生成不定根。2%蔗糖浓度较适合欧美杨叶片愈伤组织的诱导及分化。筛选培养中欧美杨叶片产生愈伤组织和分化的头孢唑林钠抑菌浓度选用500mg/L-700mg/L;诱导生根的头孢唑林钠抑菌浓度选用700mg/L。采用30mg/L卡那霉素作为叶片转化筛选的临界浓度。20mg/L的卡那霉素是生根筛选的最佳临界浓度。进行欧美杨遗传转化时,预培养时间以3-4d为最好。欧美杨转化的菌液液度以OD₆₀₀=0.3-0.4,侵染时间15min,共培养3-4d,侵染效率比较高。经PCR分子检测共获得17株阳性植株,占经抗生素筛选所得到的植株数的3.7%。同时对4株阳性植株进行了超氧化物酶活性检测,转基因植株超氧化物酶活性均高于对照植株。更多还原

【Abstract】 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.更多还原