【作者】 刘大丽；

【导师】 毛子军； 安志刚；

【作者基本信息】 东北林业大学 ， 植物学， 2013， 博士

【摘要】 随着社会经济和工业的发展,重金属污染成为全球范围内关注的环境问题。这种污染不仅会影响动植物生长发育、导致农产品质量下降,并且通过食物链直接威胁人类身体健康。因而,解决重金属污染问题迫在眉睫。生物修复（Bioremediation）技术由于其高效、经济、绿色、清洁、环保等特点,作为污染生态学的一个重要课题,在重金属污染治理中逐渐引起人们的关注。天然存在的具备重金属生物修复作用的微生物和超累积植物等往往由于它们在重金属逆境中的生长缓慢、生物量小等弊端,常常会影响生物修复的效率。本研究采用基因工程遗传转化、培养基模拟、转基因抗性微生物及植物筛选等技术手段,针对利用谷胱甘肽合成相关酶来提高大肠杆菌和能源甜菜生物修复重金属污染效果展开实验,系统地摸索了三种谷胱甘肽合成关键酶的重金属逆境中的响应机制,以及它们在转基因大肠杆菌、拟南芥和能源甜菜体内的表达特性；比较研究了三种基因型的重组大肠杆菌和转基因植物在重金属逆境下的表现型、生理生化差异及对重金属Cd、Zn、Cu的吸收规律；比较验证了这三种谷胱甘肽合成相关酶对提高大肠杆菌微生物、拟南芥、能源甜菜的重金属耐受性和重金属离子生物富集量的作用；初步探索了重组大肠杆菌和重金属耐性转基因植物在重金属生物修复方面的作用,从而为生物修复品种的开发提供了新思路。本研究的主要内容包括以下几方面：1.利用RT-PCR技术,本研究分别克隆了拟南芥（Arabidopsis thaliana L.）中编码谷胱甘肽合成酶的γ-GCS和GS的AtGCS （Genbank accession no. NM₁18439）、AtGS （Genbank accession no. NM₁22620）基因以及嗜热链球菌（Streptococcus thermophilus）中编码谷胱甘肽合成酶StECS-GS的StECS-GS （Genbank accession no. GQ848551）基因。这三个基因CDS全长分别为1569bp、1620bp、2265bp；所编码蛋白的分子量分别为58.56kDa、60.27kDa、85.12kDa;等电点分别为6.52、6.45和4.9；根据PSORT软件预测,三个蛋白在植物体内分别有56%、94%和45%的可能性位于过氧化物酶体、叶绿体膜以及细胞质中；同时在AtGCS和AtGS的5’非翻译区域存在着与干旱等环境逆境相关的顺式作用元件,说明它们可能与环境逆境之间存在着一定的应答关系。2.系统地比较研究了拟南芥AtGCS和AtGS基因在Cd、Zn、Cu重金属逆境下的转录表达特性。研究结果表明这两个基因均在转录水平上受到200μM Cd2+、Zn2+、Cu2+重金属逆境的诱导,并且与对照相比,它们在拟南芥地上部和地下部中表达量均有所增加。但在不同处理时间下受重金属逆境胁迫的影响,AtGCS和AtGS基因的表达量也出现了一定的变化和差异。在Cd逆境中,拟南芥地上部AtGS的诱导表达比较突出,而在地下部,AtGCS的诱导表达更明显一些；在Zn和Cu逆境下,AtGCS对前者的响应更加明显一些,AtGS则对后者更为敏感；在重金属方面,Cd对两个基因的诱导表达量的增幅最大,并且在Cd逆境下,两个基因在地上部的表达量要高于地下部,Zn和Cu逆境在这方面的差异不大。这说明拟南芥AtGCS和AtGS基因与重金属环境逆境之间存在着一定的应答关系,并且它们可能分别不同程度地参与到了拟南芥的抗重金属生理活动中。3.利用大肠杆菌外源蛋白重组技术,系统地比较了分别大量表达重组蛋白TrxA-AtGCS、TrxA-AtGS、TrxA-StECS-GS的重组大肠杆菌,在1mM CdCl2、ZnCl2、CuCl2重金属胁迫下的重金属耐受性和重金属离子富集能力。研究结果表明,过量表达TrxA的对照菌株在1mM重金属逆境下生长受到了严重的抑制,而过量表达TrxA-StECS-GS的E. coli对重金属逆境的耐受性最强,并且在提高细胞的存活率方面表现出StECS-GS>AtGCS> AtGS的趋势；同时,在重金属逆境下,重组菌的重金属富集能力也与对照相比具有明显的优势：过量表达TrxA-StECS-GS的E. coli对Cd、Zn、Cu的富集量分别是对照的15倍、8倍、9倍左右；TrxA-AtGCS过量表达细胞提高了10倍、5倍、7倍的大肠杆菌对重金属离子富集量；而过量表达TrxA-AtGS的大肠杆菌稍差一些,分别是对照的5、4、5倍左右。重组菌体内的GSH含量趋势（TrxA-StECS-GS>TrxA-AtGCS>TrxA-AtGS> TrxA）可以解释这种重金属高富集量的现象和差异。同时,研究将另外两种在重金属抗性机制中发挥重要作用的功能蛋白OsHSP90和OsCATb作为对比研究,结果发现过量表达GST-OsHSP90和GST-OsCATb的重组大肠杆菌尽管在生长势方面比过量表达GST的对照菌株高,并介于TrxA-AtGCS和TrxA-AtGS之间,但是它们的重金属富集能力和体内GSH含量却没有明显提升。这说明谷胱甘肽合成相关酶、分子伴侣和抗氧化酶在重金属抗性机制方面有所不同,并且在行使重金属污染生物修复功能方面,谷胱甘肽合成相关酶具有更加明显的优势。其中,StECS-GS的作用最为突出。4.利用转基因技术,将AtGCS、AtGS以及StECS-GS基因遗传转化到模式植物拟南芥中,以比较研究它们在植物体内的重金属耐受能力和在重金属生物修复中的潜力和分子机制。研究结果表明,在正常生长条件下,分别过量表达AtGCS、AtGS以及StECS-GS目的基因的转基因拟南芥株系（gcs2、gs2和ecs-gs4）和野生型（wt）在生长状态上没有明显差异；而随着CdCl2、ZnCl2、CuCl2的重金属浓度的增加,wt的生长受到了严重的抑制,并在根长及鲜重方面仅为ecs-gs4的1/4-1/3左右；过量表达AtGCS、AtGS以及StECS-GS的转基因拟南芥植株在Cd和Cu中均不同程度的提高了重金属耐受性,其中StECS-GS的作用最大；而在Zn逆境下,只有StECS-GS转基因植株表现出重金属耐受性的提高。在重金属离子富集量方面,总体表现出转基因植株地上部的重金属累积量高于地下部,转基因植株之间表现出StECS-GS基因型AtGCS、AtGS基因型>野生型累积趋势,并且ecs-gs4对Cd的富集量最高,是对照的4.5倍以上,Zn和Cu稍低一些。而在GSH和PC的含量上,总体趋势也同样呈现出类似的现象,并且随着逆境的施加,GSH在总体水平上下降,而PC含量上升。以上结果表明了三个谷胱甘肽合成关键酶在重金属逆境下是通过自身的过量表达解除相应的反馈抑制从而大量催化合成了GSH和PC,而GSH和PC可以大量螯合重金属离子,并具备抗氧化功能,因而使得转基因拟南芥可以表现出对重金属离子的高度富集和耐受性。而StECS-GS因其具备的双重谷胱甘肽合成酶功能,在抗重金属逆境方面的独特优势使其成为利用基因工程培育人工超累积植物的优势基因,在重金属植物修复中具有潜在的优势。5.以上述研究为基础,以可以生产生物燃料乙醇的能源甜菜为生物修复基础材料,通过模拟实验研究了StECS-GS基因在提高转基因甜菜对单一或复合重金属Cd、Zn、Cu污染的耐受性和生物富集量方面的作用,初步探讨了该基因在能源甜菜体内的重金属响应机制和规律。研究结果表明过量表达StECS-GS基因的转基因甜菜（s2、s4、s5）,在不同浓度（50、100、200μM Cd、Zn、Cu）重金属逆境胁迫下,它们与对照植株wt相比均表现出明显的生长优势,并且在鲜重和根长方面得以具体体现；这种生长优势基本上与StECS-GS基因的表达量成正比。同时,在植株叶片叶绿素含量方面,重金属逆境下转基因植株体内的含量也比野生型植株有所提升,但转基因植株之间差异不大。在100μM重金属离子强度下,转基因植株s2、s4、s5地上部（Shoot）中的Cd、Zn、Cu的重金属累积量分别是对照甜菜的5倍至4倍左右,并且表现出s2>s4>s5>wt的趋势；在根中,转基因植株s2重金属的累积量要小于s4、s5,但却是对照植株2-3倍左右。总体来说,转基因植株地上部重金属离子的累积量是地下部的三倍以上,甚至更多。这说明在转基因甜菜体内对于重金属离子的累积主要集中在地上部。在正常生长条件下,转基因植株s2、s4和s5体内GSH的含量分别是wt对照甜菜的5倍、4倍和3.8倍左右,这说明StECS-GS的过量表达催化合成了大量的GSH；而在Cd、Zn、Cu胁迫下,s2、s4、s5和wt体内的GSH含量有所下降,PC含量上升,转基因植株体内的GSH和PC含量始终高于对照。因而,转基因植株所表现的高重金属耐受性和高重金属离子富集能力的现象基本可以解释为GSH一部分用于合成PC并与其一起螯合重金属离子,另一部分可能在抗重金属氧化胁迫中发挥重要作用。在双重Cd-Zn、Cd-Cu、Zn-Cu和多重Cd-Zn-Cu复合重金属胁迫条件下,转StECS-GS基因植株无论在表现型、生物量还是重金属富集能力方面,都比对照甜菜具有明显的优势,并且金属离子以加成的方式被富集在转基因甜菜体内。这说明过量表达StECS-GS基因的甜菜植株在重金属耐受性、生物量、重金属离子非特异性和富集量方面都具备了重金属污染植物修复的特质和潜力,具有极大的开发价值。更多还原

【Abstract】 With development of social, economy and industry, heavy metal pollution becomes a worldwide environment problem. It not only affects the growth and development of plants and animals, the decline in the quality of agricultural products, but also threats to human health through the food chain. Thus, it is time to solve such problem. Because of being high efficiency, economy, green, clean and environment-friendly, bioremediation technology raises more concern in heavy metal pollution treatment, as an important topic in pollution ecology. Microorganisms and hyper-accumulated plants naturally occurring with heavy metal bioremediation roles grew slowly and were with low biomass in heavy metal stress conditions. These defects often affected the efficiency of bioremediation. In this study, by using methods with genetic engineering, genetic transformation, medium analog, screening of transgenic resistant microorganisms and plant, the experiments were carried out by using glutathione synthetase to enhance the heavy metal remediation effects of E. coli and energy sugar beet, and explored the heavy metal stress responsive mechanism in these glutathione synthetase broadly, and their expression characteristics in transgenic E. coli, Arabidopsis and energy sugar beet. The research also focused on the comparison of phenotype, physiological and biochemical differences and absorption of Cd, Zn and Cu in these different genotypes of recombinant E. coli and transgenic plants under heavy metal stresses, and the functions of these glutathione synthetase in enhancing the tolerance and accumulation of heavy metal ions of E. coli, Arabidopsis and energy sugar beet, and the roles of recombinant E. coli and transgenic plants with resistance of heavy metal in bioremediation. The results will provide new ideas to the variety of bioremediation. The main contents included the following aspects:1. The three genes of AtGCS （Genbank accession no. NM₁18439）, AtGS （Genbank accession no. NM₁22620） and StECS-GS （Genbank accession no. GQ848551） were cloned by using RT-PCR, and they encoded three glutathione synthetase of Arabidopsis γ-GCS, GS and Streptococcus thermophilus StECS-GS separately. The CDS length of such three genes was1569bp,1620bp and2265bp, respectively; the molecular weight of protein was58.56kDa,60.27kDa and85.12kDa, respectively; the isoelectric point was6.52,6.45and4.9, respectively; according to PSORT prediction, such three enzymes were located in peroxisome, chloroplast membrane and cytoplasme with indentity of56%,94%and45%, respectively; meanwhile, there were cis-acting elements related to environmental stress such as drought in5’ untranslated region of AtGCS and AtGS, there may be certain responsive relationship between these two genes and certain environmental stresses.2. This study compared the transcriptional expression characteristics of AtGCS and AtGS under Cd, Zn and Cu heavy metal stresses comprehensively. The results showed that the two genes were induced by200μM Cd2+, Zn2+and Cu2+stress in their transcriptional level. Compared with the control plants without any heavy metal treatment, the expression level of AtGCS and AtGS were increased in shoots and roots, and there were some differences and changes in genes’expression under different processing time of heavy metal stresses. In Cd treatment, the expression of AtGS was higher than AtGCS in Arabidopsis shoots, and they were opposite in roots; in Zn and Cu stresses, the response of AtGCS was more obvious in Zn stress, and AtGS was more sensitive to Cu; in such three heavy metal stresses, Cd could enhance the inductive expression of the two genes more evidently, and there was no difference in Zn and Cu. These results exhibited that AtGCS and AtGS were responsive to heavy metal stresses, and they might participate in physiological activity of Arabidopsis resistance to heavy metal stress in different degrees.3. By using exogenous protein recombinant technology in E. coli, this study compared the heavy metal tolerance and accumulation of E. coli over-expressing TrxA-AtGCS, TrxA-AtGS and TrxA-StECS-GS under1mM CdCl2, ZnCl2and CuCl2stress, respectively. The results showed that the growth of control cells over-expressing TrxA was damaged severely, while E. coli over-expressing TrxA-StECS-GS exhibited strongest heavy metal tolerance, and the survival rate of recombinant strains performed a trend of StECS-GS>AtGCS>AtGS. Meanwhile, in heavy metal stresses, there were obvious advantages in heavy metal accumulation ability of recombinant bacteria than control cells:the Cd, Zn and Cu accumulation of E. coli over-expressing TrxA-StECS-GS were about15,8and9-fold of control strains; cells over-expressing TrxA-AtGCS could accumulate Cd, Zn and Cu with10,5and7-fold of control cells; while Cd, Zn and Cu accumulation of stains over-expressing TrxA-AtGS was relatively low, and it was5,4and5-fold of control cells, respectively. The trend in GSH content of （TrxA-StECS-GS>TrxA-AtGCS>TrxA-AtGS>TrxA） could explain the phenomenon of high heavy metal ions accumulation in recombinant bacteria. Meanwhile, the functional proteins involving in heavy metal resistance mechanisms of OsHSP90and OsCATb were introduced as comparative experiments. It was showed that, although the growth of E. coli over-expressing GST-OsHSP90and GST-OsCATb was better than control cells over-expressing GST, and between cells over-expressing TrxA-AtGCS and TrxA-AtGS, their ability of heavy metal accumulation and GSH content were not improved significantly. These results indicated that there were differences among glutathione synthetase, molecular chaperone and antioxidant enzyme in heavy metal resistance mechanisms, and glutathione synthetase had more obvious advantages in exercising the functions of heavy metal bioremediation. The role of StECS-GS was more prominent.4. AtGCS, AtGS and StECS-GS genes were separatively transformed into model plants-- Arabidopsis thaliana by transgenic technology, and were analyzed their tolerance capacity of heavy metal stresses and the potential and the molecular mechanisms of heavy metal bioremediation comparatively. The results showed that, under normal growth conditions, there were almost no differences among transgenic Arabidopsis （gcs2, gs2and ecs-gs4） over-expressing AtGCS, AtGS and StECS-GS, and wild-type （wt）. As increase of concentrations of CdCl2, ZnCl2and CuCl2stresses, the growth of wt received serious inhibition, and its root length and fresh weight were only1/4-1/3of ecs-gs4; the tolerance ability of transgenic Arabidopsis over-expressing AtGCS, AtGS and StECS-GS genes was enhanced in different degree under Cd and Cu stresses, and StECS-GS lines were most obviously. While in Zn stress, only StECS-GS transgenic plants exhibited improved heavy metal tolerance. In the aspect of heavy metal accumulation in transgenic plants, it was much higher in shoots than in roots, and it was showed the trend of StECS-GS genotype>AtGCS、AtGS genotypes>wt. The accumulation of Cd in ecs-gs4was highest, and the level was more than4.5-fold of control plants. ecs-gs4accumulated less Zn and Cu than Cd. GSH and PC contents exhibited the similar tendency, and with treatment of heavy metal stresses, the level of GSH was declined, while the PC content was increased. These results declared that the three glutathione synthetases released the feedback inhibition by their over-expression, and thus could produce more GSH and PC. GSH and PC could chelate heavy metal ions and functioned as antioxidant. These features made the transgenic Arabidopsis acquire a higher heavy metal accumulation and tolerance. The unique advantages of StECS-GS resistance to heavy metal stress made it a dominant gene in artificial hyper-accumulated plants by using genetic engineering, and it had a potential advantage in heavy metal phytoremediation.5. On the basis of the above study, and as the bioremediation basic materials of energy sugar beet which could produce bio-fuel ethanol, the experiment studied on functional role of StECS-GS in transgenic sugar beet tolerance and bio-accumulation under single or multiplex heavy metal stresses. The results showed that transgenic sugar beet over-expressing StECS-GS （s2, s4and s5） exhibited significant growth advantage on root length and fresh weight compared with control plants （wt） under different concentrations of50,100,200μM Cd, Zn and Cu. This advantage was proportional to the amount of StECS-GS expression. Meanwhile, the chlorophyll contents of transgenic sugar beet were higher than control plants, but the differences between the transgenic plants were slight. In100μM heavy metal stresses, the Cd, Zn and Cu accumulation concentrations in transgenic sugar beet s2, s4and s5shoots were5-4-fold of the control sugar beet with the trend of s2>s4>s5>wt. In roots, although heavy metal contents of s2were lower than s4and s5, they were about2-3-fold of control plants. In general, the heavy metal accumulations of shoots transgenic plants were more than3-fold of roots. These indicated that the accumulation of heavy metal in transgenic plant were mainly concentrated in shoots. Under normal growth conditions, GSH contents of s2, s4and s5were5,4and3.8-fold of wt, respectively, and it could be deduced that over-expression of StECS-GS catalyzed and formed large amount of GSH in transgenic sugar beet. Under Cd, Zn and Cu stresses, GSH concentrations decreased, and PC contents increased in s2, s4, s5and wt, but the GSH and PC level of transgenic plants were always higher than that of control. This phenomenon could be explained that some GSH was used for synthesis of PC to chelate heavy metal ions, and some GSH might play an important role in heavy metal anti-oxidative stress. Under double Cd-Zn, Cd-Cu and Zn-Cu and multiple Cd-Zn-Cu multiplex heavy metal stressful conditions, StECS-GS transgenic sugar beet exhibited obvious advantages on phenotype, biomass and heavy metal accumulation, compared with the control sugar beet, and these heavy metals were accumulated in transgenic plants as the manner of addition. It could be concluded that sugar beet over-expressing StECS-GS had characteristics of heavy metal tolerance, high biomass, non-specific accumulation of heavy metal ions, and potential of heavy metal pollution phytoremediation. There will be a great development value by using transgenic energy sugar beet in heavy metal pollution bioremediation.更多还原