两个水稻Ⅰ型金属硫蛋白基因启动子功能初步分析
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Cu~(2+),Zn~(2+),Cd~(2+)诱导表达。诱导倍数为对照转基因植株的1.7~2.3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。
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 Cd~(2+), Cu~(2+) and Zn~(2+), and the GUS activity after treatment is 1.7~2.3 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.
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 Cd~(2+), Cu~(2+) and Zn~(2+), and the GUS activity after treatment is 1.7~2.3 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.
引文
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[2] 常团结, 朱祯. 植物金属硫蛋白研究进展(二). 生物技术通报, 2002, 18 (5): 1～6.
[3] Laplaze L, Gherbi H, Duhoux E, et al. Symbiotic and non–symbiotic expression of cgMT1, a metallothionein-like gene from the actinorhizal tree Casuarina glauca. Plant Mol Biol, 2002, 49 (1): 81～92.
[4] Chatthai M, Kaukinen K H, Tranbarger T J, et al. The isolation of a novel metallothionein-related cDNA expressed in somatic and zygotic embryos of Douglas-fir: regulation by ABA, osmoticum, and metal ions. Plant Mol Biol, 1997, 34 (2): 243～54.
[5] Foley RC, Liang ZM, Singh KB. Analysis of type 1 metallothionein cDNAs in Vicia faba. Plant Mol Biol, 1997, 33 (4): 583～591.
[6] 余荔华, 刘进元, 梅田正明, 等. 水稻金属硫蛋白核基因的克隆及其序列特征. 科学通报, 1999, 44 (15): 1645～1647.
[7] Winge DR, et al. Yeast metallothionein, sequence and metal-binding properties. J Biol Chem. 1985.260:14464-14470.
[8] 周妍娇, 熊焱, 李令媛, 等. 金属硫蛋白α和β结构域的结构功能比较研究. Vol.15, No.5, 1999: 772-778.
[9] Kagi JH, Schaffer A. Biochemistry of metallothionein. Biochemistry. 1988. 27: 8509.
[10] Cherian GM, Chan HM. Biological functions of metallothioneins. Metallothionein III. 1993. 87-109.
[11] Robinson NJ, Tommey AM, Kuske C, et al. Plant metallothioneins. BiochemJ. 1993. 295: 1-10.
[12] Binz PA, Kagi JHR. Metallothionein. http://www.unizh.ch/~mtpage/MT.html
[13] Liu JY, Lu T, Zhao NM. Classification and nomenclature of plant metallothionein-like proteins based on their cysteine arrangement patterns. Acta Bot. Sin. 2000. 42: 649-652.
[14] Rauser WE. Structure and function of metal chelators produced by plants: the case for organic acids, amino acids, phytin, and metallothioneins. Cell Biochem Biophys. 1999. 31: 19-48.
[15] Cobbett C, Goldsbrough PB. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual Review of Plant Biology. 2002. 53: 159–182.
[16] Buchanan-Wollaston V. Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Plant Physiol. 1994. 105: 839-846.
[17] Zhou J, Goldsbrough PB. Functional homologs of fungal metallothionein genes from Arabidopsis. Plant Cell, 1994, 6: 875-884.
[18] Matsumura H, Nirasawa S, Terauchi R. Technical advance: transcript profiling in rice (Oryza sativa L.) seedlings using serial analysis of gene expression (SAGE) Plant J. 1999. 20: 719-726.
[19] TIGR Gene Indices. 2001. https://www.tigr.org/tdb/tgi.shtml
[20] Yu LH, Umeda M, Liu JY et al. A novel MT gene of rice plants is strongly expressed in the nod portion of the stem. Gene. 1998. 206: 29-35.
[21] Clendennen SK, May GD. Differential gene expression in ripening banana fruit. Plant Physiol. 1997. 115: 463-469.
[22] Reid. SJ, Ross. GS, Up-regulation of two cDNA clones encoding metallothionein-like proteins in apple fruit during cool storage. Physiol. Plant.1997. 100: 183-189.
[23] Ledger SE, Gardner RC. Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var. deliciosa). Plant Mol Biol. 1994. 25: 877-886.
[24] Bhalerao R, Keskitalo J, Sterky F, et al. Gene expression in autumn leaves. Plant Physiol, 2003, 131: 430~442
[25] Liu P, Goh C J, Loh C S, et al. Differential expression and characterization of three metallothionein-like genes in Cavendish banana. Physiol Plant, 2002, 114: 241~250.
[26] Gong-Ke Zhou, Yu-Feng Xu, Jin-Yuan Liu, Characterization of a rice class II metallothionein gene: Tissue expression patterns and induction in response to abiotic factors, J Plant Physiol, 2005, 162: 686-696.
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