白桦锌指蛋白基因的分离及表达分析

植物锌指蛋白是成员众多的转录因子家族,在植物的生长发育和对非生物胁迫响应等过程中具有重要作用。根据Cys和His残基的数目和位置将锌指转录因子分为C_2H_2、C_8、C6、C_3HC_4、C_2HC、C_2HC5、C_4、CCCH和C_4HC_3九种类型。本实验利用其他物种中已报导的植物逆境胁迫相关的锌指蛋白转录因子基因序列作为信息探针,在白桦基因组数据库中筛选出10个基因,分别是4个C_2H_2型、5个C_3HC_4-RING型和一个CCCH型。通过q RT-PCR技术对筛选的基因在根中的表达模式进行了分析,结果显示Bp ZFP_2、Bp ZFP_3、Bp ZFP_4、Bp ZFP5和Bp ZFP_8的表达受盐和干旱胁迫的显著诱导,表明这些基因参与了逆境胁迫的应答。     

植物非生物逆境胁迫相关RING finger蛋白

RING finger蛋白是锌指蛋白类中一个庞大的蛋白家族,已成为拟南芥第三大蛋白家族,在水稻中也发现了488个。最典型的结构特点是序列内包含环指结构域(RING finger domain)。RING finger蛋白主要通过泛素化途径参与到植物细胞的生理生化过程。介绍了植物RING finger蛋白的结构特点和分类,综述它们的亚细胞定位,重点阐述它们在响应干旱胁迫、温度胁迫和盐胁迫等非生物逆境胁迫的调控作用。     

烟草C3HC4型锌指蛋白的原核表达及转基因植株功能研究

植物中C3HC4型RING锌指蛋白在泛素化、光形态建成、抗逆及生长发育等过程中均起到非常重要的作用。首次从本生烟中扩增获得一个锌指蛋白基因的完整阅读框,编码203个氨基酸,含有C3HC4型锌指结构域,命名为NbZFP1。将NbZFP1基因分别克隆到原核表达系统载体pGEX-6P-2以及pMAL-C2X中,成功构建了重组表达载体pGEX-6P-2-NbZFP1与pMALC2X-NbZFP1,经IPTG获得了大量可溶性表达的NbZFP1融合蛋白。将NbZFP1基因克隆于植物表达载体pBI121中,通过农杆菌介导法获得转基因烟草,PCR检测及Southern blot分析表明NbZFP1基因已整合到烟草基因组中,转NbZFP1基因烟草表现出株型紧凑,节间缩短,茎干粗壮和对TMV抗性增强。     

水稻锌指蛋白基因OsBBX6的克隆、表达及生物信息学分析

锌指蛋白作为植物体内一类重要的转录因子,对植物生长发育、基因调控以及响应外界环境变化方面发挥重要作用。Os BBX6基因属于水稻锌指蛋白B-Box基因家族成员,启动子元件分析发现其含有高温应答元件(HSE)、干旱应答元件(MBS)及非生物胁迫响应元件(TC-rich repeats)等逆境相关元件。组织特异性定量表达分析表明,Os BBX6在叶片中表达最高,根其次,茎和幼穗中表达最低。胁迫处理后的荧光定量PCR发现其受低温诱导上调,受高温、干旱、盐胁迫等抑制表达,表明其正向响应低温胁迫,负向响应高温、干旱、盐胁迫等。另外,本研究还克隆了OsBBX6基因,并对其进行了系统进化、蛋白跨膜、蛋白亚细胞定位及OsBBX6基因共表达等分析,为进一步研究其生物学功能奠定基础。     

大豆UBC基因家族鉴定及GmUBC46基因的功能初步分析

泛素/26S蛋白酶体途径在植物响应非生物胁迫反应中起着重要的作用。E2(泛素结合酶,UBC)是蛋白质泛素化中重要的泛素结合酶,与E1和E3共同参与蛋白降解途径。本研究通过构建隐马尔可夫模型,鉴定了54个大豆UBC基因,通过进化树分析将该家族成员分为11个亚家族(A-K)。蛋白保守结构域分析表明,GmUBC家族蛋白成员大部分含有保守Motif 1、Motif 2与Motif 3,且均属于泛素结合酶保守结构域。组织定位分析表明大部分GmUBC家族基因成员在大豆根、茎、叶、花等组织中有所表达。转录组数据表明有20个GmUBC基因在干旱、盐或冷胁迫下具有不同的表达模式,启动子顺式作用元件分析发现其胁迫响应过程可能与激素信号转导相关。进一步通过qRT-PCR发现GmUBC46基因能积极响应干旱、盐或冷胁迫诱导上调表达。通过酵母功能验证表明,GmUBC46基因降低了对干旱或盐胁迫的耐受性。综上,本研究初步阐明了大豆UBC基因家族的基本特性及GmUBC46基因的耐逆功能,为后续研究提供了重要依据和参考价值。     

大豆C2H2型锌指蛋白基因SCTF-1的克隆及功能分析

锌指蛋白是一类具有手指型结构的蛋白质,其中一些锌指蛋白是转录因子,对真核生物的生长发育及非生物逆境胁迫的耐受能力都有着重要作用。文章从大豆(Glycine max(L.)Merr.)中克隆了一个新的C2H2型锌指蛋白基因SCTF-1(GenBank登录号:JQ692081),该基因包含一个699 bp的开放阅读框,编码233个氨基酸,无内含子,有两个典型的C2H2型锌指结构。锌指结构中有植物锌指蛋白特有的保守氨基酸序列QALGGH。经软件预测分析,其等电点pI=8.33,分子量24.9 kDa。农杆菌介导的洋葱表皮细胞GFP瞬时表达实验结果表明,SCTF-1蛋白能够定位到细胞核中。通过RT-PCR检测发现该基因在大豆叶和花中的表达量较高,在茎和根的表达量相对较低。在对大豆幼苗的低温胁迫中,SCTF-1基因的表达量明显增加。将SCTF-1基因转入烟草(Nicotiana tabacum L.)中,发现SCTF-1基因的过量表达能够明显提高转基因烟草的耐冷能力。     

植物C_2H_2型锌指蛋白的研究进展

锌指蛋白是转录因子的一种,对真核生物的生长发育及逆境胁迫的耐受能力都有着重要关系,而植物C2H2型锌指蛋白是研究较多、较为明确的一种锌指蛋白,该蛋白大部分锌指结构具有一段高度保守的氨基酸序列QALGGH,这是植物中独有的特征,且据报道该C2H2型锌指蛋白与逆境胁迫是相关的。本文主要综述了植物C2H2型锌指蛋白的分类、结构和功能,植物C2H2型锌指蛋白与DNA、RNA和蛋白质的相互作用,以及概述了与盐胁迫、低温胁迫、干旱胁迫、氧胁迫和光胁迫等逆境胁迫相关的植物C2H2型锌指蛋白,最后还对其进一步的深入研究进行了展望,这就为日后利用基因工程技术改良作物品质、提高作物的抗逆性提供了有利条件。     

小麦TaWRKY47基因的克隆与表达分析

为了探究小麦WRKY基因的功能,该研究采用RT PCR方法,在小麦叶片组织中克隆WRKY基因,并对其进行生物信息学和不同逆境胁迫下的表达分析。结果表明：(1)成功克隆得到1个小麦WRKY基因,命名为TaWRKY47。(2)TaWRKY47基因开放阅读框长度为900 bp,编码299个氨基酸,含有一个WRKY保守结构域和一个C₂HC锌指结构域,属于WRKY基因家族的第Ⅲ类成员。(3)亚细胞定位分析结果显示,TaWRKY47蛋白定位于细胞核。(4)荧光定量PCR结果表明,TaWRKY47基因在小麦根、茎、叶、雄蕊和雌蕊中均有表达,其中在雌蕊中表达量最高,且受低温、干旱、盐、ABA和H₂O₂等胁迫表达增强,推测TaWRKY47基因参与了小麦的逆境胁迫过程。该研究结果为进一步研究TaWRKY47基因功能与抗逆机制奠定了理论基础。     

小鼠睾丸和卵巢特异表达基因Zfp474的克隆与特征分析

运用NCBI中的数据库消减杂交(Digital Differential Display,DDD)分析方法,从小鼠睾丸组织中分离了一个含有C2HC／C3H结构的新型锌指蛋白基因——ZIM74(GenBank登录号：AY350709)。通过推导和进一步的RT-PCR实验证实：该基因含4个外显子,gDNA在染色体上跨度29869bp,定位于小鼠染色体18D1。cDNA编码一个含347个氨基酸的新蛋白,带有C2HC／C3H结构域。Northern杂交结果显示：该基因含有2．37kb大小的唯一转录本,主要在睾丸中强表达,卵巢中有表达,而在其他组织中该基因无表达。结果提示：Zfp474基因对精于发生和卵母细胞的发育可能起重要作用。     

烟草C2H2锌指蛋白转录因子家族成员的鉴定与表达分析

C2H2锌指蛋白转录因子家族在真核生物中具有重要的生物学功能,广泛参与植物叶的发生、花器官的调控、侧枝的形成及逆境胁迫等生命过程。植物C2H2锌指蛋白不仅结合DNA和RNA,而且与蛋白质之间相互作用。本研究利用普通烟草(Nicotiana tabacum)基因组数据库,运用Blastp比对,结合Pfam和SMART分析,鉴定了118条普通烟草C2H2锌指蛋白家族成员;对烟草C2H2锌指蛋白家族进行了进化树分析、结构域分析、物理化学性质分析、染色体定位、基因结构分析、三维结构分析及组织表达分析等。结果表明：不同成员的氨基酸长度差异较大;系统进化及结构域分析显示,所有C2H2家族成员可以被分为5个亚家族,同一亚家族成员之间在结构域和理化性质上呈现较高一致性;每个成员都含有C2H2结构域,在数量上存在较大差异;将所有基因家族成员定位在22条染色体上;组织表达分析表明,每个C2H2亚家族都有成员在不同组织中表达,在叶及根中有些基因的表达量较高。     

Global Analysis of Ankyrin Repeat Domain C3HC4-Type RING Finger Gene Family in Plants

Ankyrin repeat (ANK) C3HC4-type RING finger (RF) genes comprise a large family in plants and play important roles in various physiological processes of plant life. In this study, we identified 187 ANK C3HC4-type RF proteins from 29 species with complete genomes and named the ANK C3HC4-type RF proteins the XB3-like proteins because they are structurally related to the rice (Oryza sativa) XB3. A phylogenetic relationship analysis suggested that the XB3-like genes originated from ferns, and the encoded proteins fell into 3 major groups. Among these groups, we found that the spacing between the metal ligand position 6 and 7, and the conserved residues, which was in addition to the metal ligand amino acids, in the C3HC4-type RF were different. Using a wide range of protein structural analyses, protein models were established, and all XB3-like proteins were found to contain two to seven ANKs and a C3HC4-type RF. The microarray data for the XB3-like genes of Arabidopsis, Oryza sative, Zea mays and Glycine max revealed that the expression of XB3-like genes was in different tissues and during different life stages. The preferential expression of XB3-like genes in specified tissues and the response to phytohormone and abiotic stress treatments of Arabidopsis and Zea mays not only confirmed the microarray analysis data but also demonstrated that the XB3-like proteins play roles in plant growth and development as well as in stress responses. Our data provide a very useful reference for the identification and functional analysis of members of this gene family and also provide a new method for the genome-wide analysis of gene families.     

T-cell regulator RNF125/TRAC-1 belongs to a novel family of ubiquitin ligases with zinc fingers and a ubiquitin-binding domain

The recently identified RNF125 [RING (really interesting new gene) finger protein 125], or TRAC-1 (T-cell RING protein in activation 1), is unique among ubiquitin ligases in being a positive regulator of T-cell activation. In addition, TRAC-1 has been shown to down-modulate HIV replication and to inhibit pathogen-induced cytokine production. However, apart from the presence of an N-terminal C3HC4 (Cys(3)-His-Cys(4)) RING domain, the TRAC-1 protein remains uncharacterized. In the present paper, we report novel interactions and modifications for TRAC-1, and elucidate its domain organization. Specifically, we determine that TRAC-1 associates with membranes and is excluded from the nucleus through myristoylation. Our data are further consistent with a crucial role for the C-terminus in TRAC-1 function. In this region, novel domains were recognized through the identification of three closely related proteins: RNF114, RNF138 and RNF166. TRAC-1 and its relatives were found to contain, apart from the RING domain, a C2HC (Cys(2)-His-Cys)- and two C2H2 (Cys(2)-His(2))-type zinc fingers, as well as a UIM (ubiquitin-interacting motif). The UIM of TRAC-1 binds Lys(48)-linked polyubiquitin chains and is, together with the RING domain, required for auto-ubiquitination. As a consequence of auto-ubiquitination, the half-life of TRAC-1 is shorter than 30 min. The identification of these novel modifications, interactions, domains and relatives significantly widens the contexts for investigating TRAC-1 activity and regulation.     

Isolation and characterization of a novel human gene (HFB30) which encodes a protein with a RING finger motif.

A human cDNA, HFB30, encoding a novel protein that contains a RING finger (C3HC4-type zinc finger) motif was isolated. This cDNA clone consists of 3056 nucleotides and encodes an open reading frame of a 474 amino acid protein. From RT-PCR analysis, the messenger RNA was ubiquitously expressed in various human tissues. The gene was located to the chromosome 5q23.3-q31.1 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. Furthermore, the gene consists of nine exons that span about 20 kb of genome DNA.     

Molecular cloning and characterization of RNF26 on human chromosome 11q23 region, encoding a novel RING finger protein with leucine zipper

Genetic alterations of RING finger genes, encoding an ubiquitin-protein ligase, are implicated in several types of human cancer through dysregulation of growth regulators. Here, a novel RING finger gene, RNF26, was cloned and characterized. The RNF26 gene on human chromosome 11q23 region was found to encode a polypeptide of 433 amino acids with the N-terminal leucine zipper domain and the C-terminal RING finger domain. Among the RING finger protein family, RING finger domains of RNF26, CGR19, NEURL, KIAA0554, and AK022937 were found to constitute a novel C3HC5 subfamily, which is distinct from C3H2C3 or C3HC4 subfamilies. RING finger domain of RNF26 was most homologous to that of CGR19 (49% amino-acid identity). The 3.2-kb RNF26 mRNA was expressed ubiquitously in normal human tissues, but was upregulated in several human cancer cell lines, including HL-60 (promyelocytic leukemia), HeLa S3 (cervical uterus cancer), SW480 (colorectal cancer), and MKN7 (gastric cancer). In addition, RNF26 was upregulated in 50% of primary gastric cancer examined in this study. Although substrates of ubiquitination mediated by RNF26 remain to be elucidated, RNF26 upregulation in several types of human cancer might be implicated in carcinogenesis through dysregulation of its substrates.     

巴西橡胶树环锌指蛋白基因克隆及其分子特性

在先前的研究中通过抑制缩减杂交获得了一个在巴西橡胶树胶乳中特异表达的片段（HbSSH10）,该片段含有“RING finger”或“C3HC4”保守序列。根据HbSSH10的序列信息设计引物并通过3’-RACE和5＇-RACE的方法,获得了一个全长的cDNA（HbRZF）。该cDNA含有589个核苷酸,含有完整的阅读框架,编码156个氨基酸。从它推导出的氨基酸序列中含有“RING finger”或“C3HC4”保守区（氨基酸100～144）。该氨基酸序列与Poncirus trifoliata、Arabidopsis thaliana和Thellungiella halophila的环锌指蛋白的同源性分别为48％、52％和50％。Northern杂交分析表明HbRZF在胶乳中大量表达,在叶片中微量表达,而在根和花中几乎没有表达。茉莉酸处理可以诱导胶乳中HbRZF的表达,而乙烯对胶乳中HbRZF的表达基本上没有影响。     

The Arabidopsis RING E3 ubiquitin ligase AtAIRP2 plays combinatory roles with AtAIRP1 in abscisic acid-mediated drought stress responses

The ubiquitin (Ub)-26S proteasome pathway is implicated in various cellular processes in higher plants. AtAIRP1, a C3H2C3-type RING (for Really Interesting New Gene) E3 Ub ligase, is a positive regulator in the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)-dependent drought response. Here, the AtAIRP2 (for Arabidopsis ABA-insensitive RING protein 2) gene was identified and characterized. AtAIRP2 encodes a cytosolic C3HC4-type RING E3 Ub ligase whose expression was markedly induced by ABA and dehydration stress. Thus, AtAIRP2 belongs to a different RING subclass than AtAIRP1 with a limited sequence identity. AtAIRP2-overexpressing transgenic (35S:AtAIRP2-sGFP) and atairp2 loss-of-function mutant plants exhibited hypersensitive and hyposensitive phenotypes, respectively, to ABA in terms of seed germination, root growth, and stomatal movement. 35S:AtAIRP2-sGFP plants were highly tolerant to severe drought stress, and atairp2 alleles were more susceptible to water stress than were wild-type plants. Higher levels of drought-induced hydrogen peroxide production were detected in 35S:AtAIRP2-sGFP as compared with atairp2 plants. ABA-inducible drought-related genes were up-regulated in 35S:AtAIRP2-sGFP and down-regulated in atairp2 progeny. The positive effects of AtAIRP2 on ABA-induced stress genes were dependent on SNF1-related protein kinases, key components of the ABA signaling pathway. Therefore, AtAIRP2 is involved in positive regulation of ABA-dependent drought stress responses. To address the functional relationship between AtAIRP1 and AtAIRP2, FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2-2 and atairp1 plants, respectively. Constitutive expression of FLAG-AtAIRP1 and AtAIRP2-sGFP in atairp2-2 and atairp1 plants, respectively, reciprocally rescued the loss-of-function ABA-insensitive phenotypes during germination. Additionally, atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation lines were more tolerant to dehydration stress relative to atairp1 and atairp2-2 single knockout plants. Overall, these results suggest that AtAIRP2 plays combinatory roles with AtAIRP1 in Arabidopsis ABA-mediated drought stress responses.