【作者】 李世鹏；

【导师】 李彦舫；

【作者基本信息】 吉林大学 ， 作物遗传育种， 2007， 硕士

【摘要】 金属硫蛋白（metallothionein,MT）是一种广泛存在于生物界、低分子量、富含巯基和高金属含量、功能独特的蛋白质。植物MT的功能目前尚无十分明确的结论,对其功能的研究还处于起步状态。本文从水稻基因组克隆了两个金属硫蛋白启动子,Genbank检索号分别为U43529、BE039194,分别命名为MT-1-1a和MT-1-4b。使用（PLACE）分析表明含有MRE以及多个金属效应元件及其它多个顺式作用元件。我们通过基因工程技术将其连接到pCAMBIA1381载体上,用农杆菌介导法转化拟南芥获得转基因植株。根据报告基因检测表明两个启动子主要驱动报告基因在拟南芥根叶部微管组织大量表达,且受伤诱导高效表达,其中MT-I-4b在表皮毛中优势表达。两个启动子均受Cu²⁺,Zn²⁺,Cd²⁺诱导表达。诱导倍数为对照转基因植株的1.7².3倍。表明该启动子表达具组织特异性、受发育时期调控及受非生物胁迫诱导表达的特性。更多还原

【Abstract】 Metallothioneins are a group of low molecular mass and cysteine-rich metal-binding proteins, ubiquitously found in most living organisms. They play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting against intracellular oxidative damages. Since being first purified as Cd-binding protein from horse kidney in 1957 by Margoshes and Vallee, MTs have been widely found in diverse organisms including mammals, plants, fungi as well as some prokaryotes.Recent studies in plants indicated that MTs prevalent in plants and participated in a range of physiological processes, including scavenging reactive oxidant species （ROS）, regulating cell growth, proliferation, embryogenesis, fruit development, and activity of metalloenzymes and transcription factors, involvement in metabolism of metallo-drug, response to stress conditions, as well as potentially involving in inflammation and cell apoptosis, and even acting as possible cellular redox sensors. All the studies suggested that MTs playes a substaintial role in plants. Researchers dedicated themselves to study on plant MTs in recent years because its organ specificity, phase specificity and high inducibility.It is an important subject on plant MTs’gene structure, upstream regulatory element, trans-factor and its regulation mechanism. It has been well characterized that in 5’upstream region of mammalian MT genes contains the MRE secquence TGCRCNC, where“R”represents a purine and“N”can be any base but“A”. Although there had several report about putative plant MRE, no one has had the core sequence of plant MRE practically identified.In this study, two promoters of rice MT genes MT-1-1a and MT-1-4b were selected by computer analysis, which may contain putative MRE sequence. A 1,065bp and a 1,094bp fragments just upstream of the ATG initiation site were obtained in diference from rice genomic DNA by PCR. Putative regulatory cis-element analysis of the target promoter fragment was carried using the software programs PLACE （ Plant Cis-acting Regulatory DNA Elements Database,http://www.dna.affrc.go.jp/）. A number of potential regulatory motifs corresponding to known cis-regulatory signals of eukaryotic genes were found in the two promoters. These motifs include TATA box and CAAT box, E-box involved in defense signaling and the W-box in response to wounding, as well as MYB binding site involved in drought-inducibility and ABRE cis-acting element involved in the abscisic acid responsiveness together with light-induced cis-element I-BOX. Moreover, some tissue-specific expression related cis-elements present in the two fragments such as pollen specific expression regulatory elements PSE, endosperm specific element ESE, seed germination element and root specific express element. In addition, element response for stress such as copper-response element CRE, metal response element MRE, etiolation related element and auxin responsive element ARE besides MT-1-4b contains a gibberellin-responsive element GARE. We could predict these two promoters might be induced by abiotic stresses.Then we connect these promoters with pCAMBIA1381 vector, a chimeric expression unit consisting of theβ-glucuronidase （GUSA） reporter gene were constructed. Then, using an Agrobacterium tumefaciens-mediated transformation method, we introduced the two promoters into Arabidopsis. The strongest histochemical staining for GUS activity was observed in the vascular tissue of root and rosette leaf, but no GUS signal was detected in vascular tissue of ageing leaf. For MT-1-4b, there exists an outstanding expressional character in trichome. A GUS fluorescence assay demonstrated the two promoters both in response to Cd²⁺, Cu²⁺ and Zn²⁺, and the GUS activity after treatment is 1.7².3 times higher than that of control. Spatial and temporal expression patterns and the expression of promoter-GUS responsive to cation indicated these promoters regulated by development stage, expressed in specific tissue and can be driven by abiotic stress.Our work provides an initial characterization on the plant cis-element MRE. The results presented here can be served as a useful foundation for further studies.更多还原