【作者】 陈珍；

【导师】 朱诚；

【作者基本信息】 浙江大学 ， 植物学， 2013， 博士

【摘要】 汞(mercury, Hg),是人类食物链中毒性最强的重金属之一。汞矿的开采、含汞废弃物的排放、化肥和农药的不合理施用等,已造成环境汞污染日益严重,对动植物的生长发育和人类的健康造成了严重威胁。Hg易被植物吸收,其与巯基(-SH)有很强的亲和力,能与亲水蛋白中的半胱氨酸(Cys)残基结合,扰乱任何未受保护蛋白质的功能；抑或是作为二价阳离子,取代金属酶的辅基如Mg2+、Mn2+、Zn2+等,导致酶活性下降或丧失；此外汞诱导的活性氧自由基会引起巯基氧化和二硫键交联等损伤,从而抑制植物的生长发育。因此,如何保护巯基与二硫键,防止蛋白的变性与聚集,保证正常的代谢,亟待研究。蛋白质二硫键异构酶(protein disulfide isomerase, PDI; EC5.3.4.1)及其类蛋白(PDIL),是硫氧还蛋白超家族的一员,具有催化蛋白质二硫键氧化、还原及异构的功能；且具有分子伴侣活性,可促进蛋白质的折叠与装配,并可防止变性蛋白的聚集。但迄今为止PDI在植物响应汞胁迫中的作用还未见报道。本文首次研究了汞胁迫下水稻PDILs(OsPDILs)的表达变化,并证实过表达来自嗜热自养甲烷杆菌的PDIL (MTH1745)可提高植物对汞等胁迫的耐性,实现了深海基因资源的利用。具体结果如下：1)利用生物信息学方法分析了水稻自身二硫键异构酶类蛋白(OsPDILs)的基本信息、结构域、分类、定位及表达,结果表明12个OsPDILs氨基酸长度从148aa到563aa不等,具有1至2个Trx (CXXC)结构域,分布在9条染色体上,基因表达分析(UniGene数据库和MPSS数据)表明这12个OsPDI基因表达具有组织差异性与生长发育阶段差异性。2)以水稻‘日本晴’(Oryza sativa L., cv.Nipponbare)为材料,采用荧光定量PCR分析了12个OsPDILs基因在汞胁迫下的表达变化,结果表明,75μM HgCl2胁迫可诱导水稻幼苗根中OsPDIL1-1、OsPDIL1-3、OsPDIL5-1、OsPDIL5-2及OsPDIL5-3表达显著上调,OsPDIL1-2表达下调,而OsPDILs2-1、OsPDILs2-3釉OsPDILs5-4等无显著变化。3)将来源于嗜热自养甲烷杆菌杆菌(Methanothermobacter thermoautotrophicum)的PDIL蛋白编码基因(MTH1745),通过农杆菌介导的花序转化法导入拟南芥,获得含目的基因的纯合株系,逆境处理实验结果表明转基因拟南芥能提高对汞胁迫的耐性,且对高温和盐胁迫也具有较强的耐性。4)将MTH1745通过农杆菌介导导入水稻‘日本晴’中,获得了MTH1745组成型高表达(CaMV35S启动子)的纯合株系。结果表明,在不同浓度HgCl2处理下转基因水稻生长优于野生型,具有更强的抗性。转基因植株的光合效率(净光合速率Pn、气孔导度Gs、蒸腾速率Tr)、抗氧化酶(SOD、POD等)活性、非蛋白硫醇(NPT)和还原态谷胱甘肽(GSH)含量均明显高于野生型,这意味着转基因植株有可能通过维持蛋白质的合成,提高含巯基(-SH)类物质和二硫键化合物活性,保护光合作用过程中关键酶等,防止膜脂过氧化损伤,从而起到缓解汞引起的毒害作用。更多还原

【Abstract】 Mercury (Hg) is one of the most toxic heavy metals. It has become a major contaminant through mining and smelting, untreated industrial wastes, mercury by-products and other livelihood activities, and with significant impacts on growth and development of organisms even human health. Hg is easily taken up by plants and is biochemically toxic because it binds to sulfhydryl groups (-SH) of proteins, resulting in displacement of essential elements or disruption of structure. Further, Mg2+, Mn2+, or Zn2+can be replaced by Hg2+, so the activities of the enzymes with these metals may be disturbed. Anymore, Hg can trigger the generation of reactive oxygen species (ROS) and induce oxidation of-SH or cross-linking of disulfide bonds (-S-S). So it is important and urgent to find the ways for protection of-SH and-S-S, preventing the aggregation of denatured proteins and maintaining the metabolism in order.Protein disulfide isomerase (PDI, EC5.3.4.1) and disulfide isomerase-like protein (PDIL), one member of the thioredoxin super-family, can catalyze thiol-disulfide interchange, resulting in the formation, reduction, or isomerization of protein disulfide bonds in protein substrates. In addition, PDI or disulfide isomerase-like protein (PDIL) display chaperone activity that promote protein folding or prevent protein aggregation. But the relationship of PDIL and plant response under Hg toxicity has not been reported until now. The expression profile of OsPDILs was first investigated in this study, and the enhanced tolerance to mercury was observed in transgenic plants by over-expression of PDIL. The results in details are as follows:1) In silico identification and analysis, including basic information, structural domain, classify, chromosomal location, and expression, of the protein disulphide isomerases in rice were performed, and the results showed the amino acid length of the12OsPDILs was about148aa to563aa. They have one or two domain of the Trx (CXXC) and located in9chromosomals. And the expression analysis from UniGene database or the massively parrellel signature sequencing proved the differences in genes, tissues or stages of growth and development.2) The expression of OsPDILs under Hg treatment was determined by real time quantity-PCR with Oryza saliva L., cv. Nipponbare. The results showed that OsPDIL1-1, OsPDIL1-3, OsPD1L5-1, OsPDIL5-2and OsPDIL5-3were up-regulated in roots of rice seedlings under75μM HgCl2treatments, compared to normal condition, while OsPDIL1-2was down-regulated. And OsPDILs2-1, OsPDILs2-3and OsPDILs5-4showed no significant difference whenever the Hg2+treatment or not.3) MTH1745, a PDIL from Methanothermobacter thermoautotrophicum, was reconstructed and transformed into Arabidopsis thaliana by inflorescence. The homozygous lines were chosen and presented the resistance to Hg stress. They also displayed the tolerance to high-tempreture or salt stress.4) Transgenic rice lines were achieved by co-cultivation of rice calli derived from mature seed scutella with A. tumefaciens containing p1301-35S::MTH1745. The results of RT-PCR indicated a high level of MTH1745mRNA in leaves and roots of these transgenic plants. When treated with different concentration of HgCl2, the transgenic rice seedlings displayed Hg tolerance with obvious phenotypes and more effective photosynthesis (net photosynthetic rate Pn, stomatal conductance Gs and transpiration rate Tr) compared to wild-type plants. Furthermore, antioxidant enzyme activities of superoxide dismutase and peroxidase were notably higher and increased content of non-protein thiols and reduced glutathione (GSH) were also observed. All these may indicate that the transgenic plants could enhance the detoxification of Hg by promoting the synthesis of protein, increasing the activities of compounds with-SH or-S-S, protecting the enzymes in the process of photosynthesis and then proventing the oxidative damage of membrane caused by mercury stress.更多还原