苋菜IRT1基因克隆、序列及表达分析

通过对镉超积累苋菜品种天星米铁转运蛋白基因( IRT1)的克隆、序列及表达分析,旨在为植物修复镉污染土壤奠定基础.依据同源克隆原理,通过RACE技术克隆苋菜IRT1基因及生物信息学方法分析基因序列结构和功能,Northern杂交研究基因表达.苋菜IRT1基因cDNA全长1135 bp,包含完整的阅读框,编码322个氨基酸.苋菜IRT1蛋白与已知铁转运蛋白相似性在53.70％-63.04％,具有铁转运蛋白典型的功能结构特征,即N端含有1个信号肽、氨基酸序列上具有完整的ZIP家族功能结构域( Pfam:Zip)和7个跨膜结构域(TMs).苋菜IRT1蛋白还具有1个COG0428超级家族(转运二价金属离子功能)、2个蛋白激酶C磷酸化位点和2个酪蛋白Ⅱ磷酸化位点.低铁胁迫时苋菜根中IRT1基因表达量增加,加镉处理没有改变IRT1基因表达量.因此,推断苋菜IRT1基因是ZIP家族的一员,具有转运二价金属离子功能,将基因在GenBank中注册,序列号为:GU363501,命名为AmIRT1.     

利用基因芯片对MxIRT1蛋白膜泡在铁胁迫条件下外排途径的研究

本文以研究缺铁条件下Mx IRT1蛋白膜泡在细胞中的外排途径机制为目的,采用基因芯片方法,发现在铁胁迫环境中,膜泡运输通路相关基因上调表达,推测Mx IRT1蛋白膜泡通过粗面内质网运送至高尔基体,然后通过内体再运送至质膜。该研究有助于进一步研究高等植物中Mx IRT1蛋白相关的铁转运机制。     

紫花苜蓿MsMATE1基因克隆及其抵御铁胁迫功能的鉴定

多药和有毒化合物外排转运蛋白MATE能转运金属、激素、次生代谢物等多种底物,因而在植物的生长发育中发挥重要作用。本研究基于紫花苜蓿(Medicago sativa L.)基因组数据和缺铁胁迫的转录组数据,克隆获得紫花苜蓿MsMATE1及pMsMATE1启动子(GenBank登录号分别为MN547958和MT505313)。MsMATE1基因长1470 bp,编码489个氨基酸。系统进化分析表明,紫花苜蓿MsMATE1蛋白属于mate家族,与蒺藜苜蓿MtMATE(XP013453190.1)亲缘关系最近。生物信息学分析表明,MsMATE1具有典型的MATE_like超家族结构域,属于H+势驱动的真核亚类;MsMATE1为跨膜蛋白,二级结构的主要构成元件是α螺旋。pMsMATE1启动子长1598 bp,序列分析显示内含多个植物激素和逆境胁迫响应元件。MsMATE1在紫花苜蓿幼苗的根茎叶中都有表达,茎中的表达量最高。高铁和缺铁胁迫下,MsMATE1基因在紫花苜蓿幼苗各部位的表达显著上调,茎中上调最明显。高铁和缺铁胁迫,转MsMATE1基因烟草的3种抗氧化酶活性、叶绿素和可溶性蛋白含量显著增加,丙二醛含量显著降低。以上结果表明,MsMATE1在烟草中的异源表达提高了植物抵御高铁和缺铁胁迫的能力。MsMATE1可以作为应用基因工程方法改良植物铁胁迫耐受的重要候选基因,本研究为深入了解MsMATE1蛋白在植物响应铁胁迫中的分子机制奠定基础,MsMATE1基因应对其他金属胁迫或环境胁迫的功能有待于进一步鉴定。     

ZIP蛋白在植物中的功能分析

锌和铁是植物生长发育所必需的微量营养元素,在植物的光合作用、呼吸作用以及许多生化反应中起着非常重要的作用。植物体内锌铁处于平衡状态才能保证其正常的生长发育,而锌铁调控转运体ZIP对于锌铁吸收、转运及体内平衡的调节有重要作用。目前,对于植物中ZIP家族基因的研究有一定进展。对植物ZIP基因的表达、蛋白定位、酵母互补实验、过表达及基因敲除等研究结果进行综述,揭示了ZIP蛋白在植物发育过程中的作用。了解ZIP对于锌铁吸收、转运及体内平衡中的作用有助于通过转基因改良及常规育种将ZIP蛋白应用于农业生产上。     

农杆菌介导番茄铁载体蛋白基因(LeIRT2)转化烟草的研究

构建了含有目的基因(LeIRT2)的双元载体pYF840,并成功地将植物性内含子构建到筛选标记基因新霉素磷酸转移酶基因(nptⅡ)和gus报告基因中。利用农杆菌介导法,将铁载体蛋白(LeIRT2)基因导入革新一号烟草,PCR、PCR-Southern blotting及Southern blottlng检测结果显示,有3个烟草株系的基因组中整合了完整的铁载体基因(LeIRT2)。水培试验结果显示转基因株系1、3不但提高了植株的铁吸收能力,而且还促进了植株的生长,但转基因株系2却与对照无明显区别。     

冬小麦植物铁载体分泌的杂种效应

缺铁是石灰性土壤常见的植物营养问题之一.禾本科植物种或基因型的植物铁载体分泌能力与耐缺铁有关,提高植物铁载体分泌能力是改良缺铁的土壤上植物铁aestivum L.) 3个杂交种及其4个亲本在缺铁营养液中植物铁载体的分泌及杂种的效应.植物铁载体的分泌率通过根分泌物对新形成的Fe(OH)3的活化能力进行测定, 在缺铁症出现时每隔2、3天测定1次.在缺铁条件下,所有基因型都分泌较多的植物铁载体,并且随缺铁症状的发展分泌量增加.杂交种具有对缺铁更敏感的反馈系统,在缺铁条件下,杂交种比亲本分泌铁载体的速度更快、量更高.通过分析杂交种和亲本的关系,认为可以通过对亲本分泌植物铁载体能力和配合力的选择,利用杂种优势来提高小麦铁的利用效率.     

拟南芥bHLH Ib转录因子调控FIT的转录

FIT是调控拟南芥铁稳态的一个关键调控因子，它在转录水平上受到缺铁诱导，但其背后的调控机制还不甚清楚。该研究以拟南芥bHLH38和FIT的单、双过表达植物及bHLH Ib四突变体植物为材料，采用缺铁(-Fe)处理实验和定量RT-PCR的方法从RNA角度分析了FIT转录水平的变化。结果表明：(1)在铁充足时，bHLH38过表达植物中FIT的转录水平显著高于其在野生型中的水平。(2)在bHLH Ib四突变体植物中FIT的转录水平不受缺铁诱导。(3)FIT单过表达不能激活内源FIT的转录，而在加铁(+Fe)条件下bHLH38和FIT的双过表达则可以激活内源FIT的转录。(4)在缺铁条件下，所有植物中FIT的转录水平均与野生型中的FIT水平无明显差异。基于以上结果认为，bHLH Ib转录因子是缺铁诱导FIT转录的必要条件，而非充分条件。该研究结果为深入了解植物通过多种途径共同维持铁稳态提供了新的见解。     

高山离子芥铁转运蛋白基因编码序列的克隆及其抗逆性表达的实时定量分析

铁转运蛋白 (iron transport protein,IRT)是具有跨膜运输离子功能的特殊蛋白质,属于金属离子转运家族的成员．本研究运用RACE方法从高山离子芥（Chorispora bungeana）中克隆得到完整的铁转运蛋白cDNA,命名为CbIRT（基因登录号为EU330924）．该基因全长1 290 bp,包含1个1 035 bp的开放阅读框（ORF）,编码344个氨基酸的蛋白．系统进化树分析显示,该基因与拟南芥AtIRT1和遏蓝菜TcIRT-G的亲缘关系最近,同源性分别达到了87.4%和86.5%,而在氨基酸序列水平与拟南芥AtIRT1的同源性达到了89%,表明克隆得到的CbIRT属于金属离子转运体家族成员．实时荧光定量方法对高山离子芥CbIRT基因在不同温度和铁营养水平条件下的表达情况进行分析表明,CbIRT对零下低温和零上低温的表达水平呈截然不同的反应;正常铁营养状态下,CbIRT是微量表达的,而缺铁及低温处理都可以大幅度地促进该基因的表达,富铁可以抑制该基因的表达．显示了该蛋白在转运Fe离子方面的重要作用．     

冬小麦植物铁载体分泌的杂种效应

缺铁是石灰性土壤常见的植物营养问题之一。禾本科植物种或基因型的植物铁载体分泌能力与耐缺铁有关 ,提高植物铁载体分泌能力是改良缺铁的土壤上植物铁营养的关键措施之一。在水培条件下分析了冬小麦(TriticumaestivumL .) 3个杂交种及其 4个亲本在缺铁营养液中植物铁载体的分泌及杂种的效应。植物铁载体的分泌率通过根分泌物对新形成的Fe(OH) 3 的活化能力进行测定 ,在缺铁症出现时每隔 2、3天测定 1次。在缺铁条件下 ,所有基因型都分泌较多的植物铁载体 ,并且随缺铁症状的发展分泌量增加。杂交种具有对缺铁更敏感的反馈系统 ,在缺铁条件下 ,杂交种比亲本分泌铁载体的速度更快、量更高。通过分析杂交种和亲本的关系 ,认为可以通过对亲本分泌植物铁载体能力和配合力的选择 ,利用杂种优势来提高小麦铁的利用效率。     

缺铁水稻根调控因子和信号转导相关转录本微点阵分析

在《缺铁水稻根转录本微点阵分析》(见《生物信息学》2 0 0 4年第 3期 )一文中 ,介绍了缺铁诱导 5天的水稻根中四组上、下调控基因的转录本 ,他们是质膜蛋白和转运体相关的转录本 ,细胞骨架相关的 ,膜泡运输相关的 ,和物质能量代谢相关的转录本[1] 。本文对其余的上、下调基因的转录本作一介绍。在缺铁诱导的基因中还包括调控因子有关的 ,细胞周期与调控有关的 ,通讯与信息有关的和未被分类功能基因的转录本。本研究采用的水稻微点阵芯片是利用水稻的EST序列 ,既表达序列标签。EST可用于基因结构、表达和功能的分析 ;也可用于基因组研究 …     

The ZIP family of metal transporters

Members of the ZIP gene family, a novel metal transporter family first identified in plants, are capable of transporting a variety of cations, including cadmium, iron, manganese and zinc. Information on where in the plant each of the ZIP transporters functions and how each is controlled in response to nutrient availability may allow the manipulation of plant mineral status with an eye to (1) creating food crops with enhanced mineral content, and (2) developing crops that bioaccumulate or exclude toxic metals.     

Loading and bioavailability of iron in cereal grains

Cereal plants take up iron from the soil via a phytosiderophore-mediated chelation system. Following root absorption, iron is transported through the xylem and phloem of the plant with the help of a variety of efflux and influx transporters belonging to the Zrt Irt-like protein (ZIP) and yellow stripe-like (YSL) protein families. Iron-regulated transporter1, a member of the ZIP family, mobilises ferrous [Fe(II)] ions, while several YSL family members such as YSL2, YSL15 and YSL18 can transport both ferric [Fe(II)] and ferrous [F`III)] ions into developing grains via chelation with mugineic acid or its derivatives. The iron is accumulated largely in the outer aleurone layer and embryo of the grains, which are removed during milling, leaving behind consumable endosperm that contains a very low amount of iron. This review highlights the uptake, transport and loading mechanisms for iron in cereal grains and provides an overview of strategies adopted for developing highly iron-enriched grains.     

A distinctive sequence motif in the fourth transmembrane domain confers ZIP13 iron function in Drosophila melanogaster

The zinc/iron permease (ZIP/SLC39A) family plays an important role in metal ion transport and is essential for diverse physiological processes. Members of the ZIP family function primarily in the influx of transition metal ions zinc and iron, into cytoplasm from extracellular space or intracellular organelles. The molecular determinants defining metal ion selectivity among ZIP family members remain unclear. Specifically, we reported before that the Drosophila ZIP family member ZIP13 (dZIP13), functions as an iron exporter and was responsible for pumping iron into the secretory pathway. ZIP13 protein is unique in that it differs from the other LIV-1 subfamily members at transmembrane domain IV (TM4), wherein relative positions of the conserved H and D residues in the HNXXD sequence motif are switched, generating a DNXXH motif. In this study, we undertook an in vivo approach to explore the significance of this D/H exchange. Comparative functional analysis of mutants revealed that the relative positions of D and H are critical for the physiological roles of dZIP13 and its close homologue dZIP7. Swapping D/H position of this DNXXH sequence in dZIP13 resulted in loss of iron activity; normal dZIP13 could not complement dZIP7 loss, but swapping the two relative amino acid positions D and H in dZIP13 was sufficient to make it functionally analogous to its close homologue dZIP7. This work provides the first in vivo functional analysis of a structural motif required to differentiate different transporting functions of ZIPs.     

Effect of dietary iron deficiency and overload on the expression of ZIP metal-ion transporters in rat liver

The mammalian ZIP (Zrt-, Irt-like Protein) family of transmembrane transport proteins consists of 14 members that share considerable homology. ZIP proteins have been shown to mediate the cellular uptake of the essential trace elements zinc, iron, and manganese. The aim of the present study was to determine the effect of dietary iron deficiency and overload on the expression of all 14 ZIP transporters in the liver, the main site of iron storage. Weanling male rats (n = 6/group) were fed iron-deficient (FeD), iron-adequate (FeA), or iron-overloaded (FeO) diets in two independent feeding studies. In study 1, diets were based on the TestDiet 5755 formulation and contained iron at 9 ppm (FeD), 215 ppm (FeA), and 27,974 ppm (3% FeO). In study 2, diets were based on the AIN-93G formulation and contained iron at 9 ppm Fe (FeD), 50 ppm Fe (FeA), or 18916 ppm (2% FeO). After 3 weeks, the FeD diets depleted liver non-heme iron stores and induced anemia, whereas FeO diets resulted in hepatic iron overload. Quantitative RT-PCR revealed that ZIP5 mRNA levels were 3- and 8-fold higher in 2% FeO and 3% FeO livers, respectively, compared with FeA controls. In both studies, a consistent downregulation of ZIP6, ZIP7, and ZIP10 was also observed in FeO liver relative to FeA controls. Studies in H4IIE hepatoma cells further documented that iron loading affects the expression of these ZIP transporters. Overall, our data suggest that ZIP5, ZIP6, ZIP7, and ZIP10 are regulated by iron, indicating that they may play a role in hepatic iron/metal homeostasis during iron deficiency and overload.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

A ferroxidase encoded by FOX1 contributes to iron assimilation under conditions of poor iron nutrition in Chlamydomonas

When the abundance of the FOX1 gene product is reduced, Chlamydomonas cells grow poorly in iron-deficient medium, but not in iron-replete medium, suggesting that FOX1-dependent iron uptake is a high-affinity pathway. Alternative pathways for iron assimilation, such as those involving ZIP family transporters IRT1 and IRT2, may be operational.     

The prion-ZIP connection: From cousins to partners in iron uptake

ABSTRACT

Converging observations from disparate lines of inquiry are beginning to clarify the cause of brain iron dyshomeostasis in sporadic Creutzfeldt-Jakob disease (sCJD), a neurodegenerative condition associated with the conversion of prion protein (PrP^C), a plasma membrane glycoprotein, from α-helical to a β-sheet rich PrP-scrapie (PrP^Sc) isoform. Biochemical evidence indicates that PrP^C facilitates cellular iron uptake by functioning as a membrane-bound ferrireductase (FR), an activity necessary for the transport of iron across biological membranes through metal transporters. An entirely different experimental approach reveals an evolutionary link between PrP^C and the Zrt, Irt-like protein (ZIP) family, a group of proteins involved in the transport of zinc, iron, and manganese across the plasma membrane. Close physical proximity of PrP^C with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrP^C and ZIP14 suggest that PrP^C functions as a FR partner for certain members of this family. The connection between PrP^C and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrP^C in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains.