水稻受盐抑制基因OsZFP1的转基因分析

OsZFP1(水稻锌指蛋白1)基因编码的蛋白含有3个推测的Cys2/Cys2-型锌指结构域,它的表达受盐胁迫负调控。构建了以35S为启动子的OsZFP1基因的植物表达载体,并将其转入拟南芥(ArabidopsisthalianaL.)植物和水稻(OryzasativaL.)愈伤组织中以过量表达OsZFP1基因。转基因的拟南芥植株和水稻愈伤组织对盐处理的敏感性都比野生型要高。这一结果表明OsZFP1基因可能编码一种负调控蛋白,它可能抑制某些盐诱导基因的表达。在ABA处理下,转基因拟南芥植株比野生型植株抽苔晚,说明OsZFP1基因的作用可能受ABA调节。     

逆境相关植物锌指蛋白的研究进展

锌是植物必需的营养元素,锌指蛋白因其具有指状结构特征且能结合Zn2 而得名,植物锌指蛋白包含特有的QALGGH保守结构,可能涉及调控植物特有的生物学功能。人们已经从拟南芥(Arabidopsis thaliana)、矮牵牛(Petunia hybrida Vilm)、水稻(Oryza sativa)、大豆(Glycine max)、棉花(Gossypium hirsutum)等植物中克隆了许多编码锌指蛋白的基因,并对其结构及功能进行了研究。利用转基因技术,将一些与逆境胁迫相关的锌指蛋白基因在目标植物中过量表达后,能对植物起到增强抗逆性的作用,说明锌指蛋白在增强植物逆境抗性方面有着广阔的应用前景。     

水稻受盐抑制基因OsZFP1的转基因分析

OsZFP1(水稻锌指蛋白1)基因编码的蛋白含有3个推测的Cys2/Cys2-型锌指结构域,它的表达受盐胁迫负调控.构建了以35S为启动子的OsZFP1基因的植物表达载体,并将其转入拟南芥(Arabidopsis thaliana L.)植物和水稻(Oryza sativa L.)愈伤组织中以过量表达OsZFP1基因.转基因的拟南芥植株和水稻愈伤组织对盐处理的敏感性都比野生型要高.这一结果表明OsZFP1基因可能编码一种负调控蛋白,它可能抑制某些盐诱导基因的表达.在ABA处理下,转基因拟南芥植株比野生型植株抽苔晚,说明OsZFP1基因的作用可能受ABA调节.     

Mutations in the Dof zinc finger genes DAG2 and DAG1 influence with opposite effects the germination of Arabidopsis seeds

We describe the Arabidopsis gene DAG2 encoding a Dof zinc finger protein and show that it is involved in the control of seed germination. An Arabidopsis mutant line with a T-DNA insertion in DAG2 isolated by reverse genetics produces seeds that are substantially more dependent than the wild type on the physical stimuli-light and cold treatment-that promote germination. Mutant dag2 seeds also are less sensitive to the germination-promotive effect of gibberellins, because a 10-fold higher amount of gibberellins is needed to restore germination when endogenous gibberellin biosynthesis is blocked. The seed germination characteristics of the dag2 mutant are opposite to those of dag1, a knockout mutant of another Dof gene (DAG1) that we showed previously to be involved in the control of seed germination, and are similar to those of plants that overexpress DAG1. The promoter of the DAG2 gene is active specifically in the vascular system of the mother plant but not in the embryo, and segregation analysis indicates that the effect of the dag2 mutation is maternal. Both characteristics are in common with DAG1; additionally, the DAG1 and DAG2 proteins share high sequence homology and an identical zinc finger domain. These data suggest, and the germination phenotype of the double mutant is compatible with, a model whereby the zinc finger proteins DAG1 and DAG2 act on a maternal switch that controls seed germination, possibly by regulating the same gene(s).     

胡杨锌指蛋白基因克隆及其结构分析

锌指蛋白属于核转录因子家族,在原核生物与真核生物基因转录调控中发挥作用。分析了耐盐锌指蛋白Alfin-1基因在苜蓿与拟南芥中的保守性后,设计了一对引物。以胡杨水培叶片为材料,从总RNA中通过RT-PCR分离得到一个锌指蛋白基因,其cDNA长924bp。分析其氨基酸序列表明,存在一个典型的Cys2/His2锌指结构,从第556位开始有一个富含G的启动子结合位点GTGGGG。由于具有相同功能的转录因子在结构和DNA结合区的氨基酸序列上具有保守性,因此,从结构分析上可以推测该基因与Alfin-1在功能上是有一定的相关性。     

Novel potato C2H2‐type zinc finger protein gene,StZFP1, which responds to biotic and abiotic stress,plays a role in salt tolerance

Many TFIIIA‐type zinc finger proteins (ZFPs) play important roles in stress responses in plants. In the present study, a novel zinc finger protein gene, StZFP1, was cloned from potato. StZFP1 is a typical TFIIIA‐type two‐finger zinc finger gene with one B‐box domain, one L‐box domain and a DLN‐box/EAR motif. The StZFP1 genes belong to a small gene family with an estimated copy number of four or five, located on chromosome I. StZFP1 is constitutively expressed in leaves, stems, roots, tubers and flowers of adult plants. Expression of StZFP1 can be induced by salt, dehydration and exogenously applied ABA. StZFP1 expression is also responsive to infection by the late blight pathogen Phytophthora infestans. Transient expression analysis of StZFP1:GFP fusion protein revealed that StZFP1 is preferentially localised in the nucleus. Ectopic expression of StZFP1, driven by the Arabidopsis rd29A promoter in transgenic tobacco, increased plant tolerance to salt stress. These results demonstrate that StZFP1 might be involved in potato responses to salt and dehydration stresses through an ABA‐dependent pathway.     

The RING finger motif of photomorphogenic repressor COP1 specifically interacts with the RING-H2 motif of a novel Arabidopsis protein.

The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness. The COP1 protein contains a cysteine-rich zinc-binding RING finger motif found in diverse groups of regulatory proteins. To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger. This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay. In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster. The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other both in vitro and in yeast, whereas neither motif displayed significant self-association. Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction. Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain. The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.     

Photoperiod-regulated expression of the PpCOL1 gene encoding a homolog of CO/COL proteins in the moss Physcomitrella patens

The CONSTANS (CO) protein is a critical regulator of the photoperiodic control of flowering in Arabidopsis thaliana and Oryza sativa. We isolated a cDNA PpCOL1 encoding a homolog of the CO/CO-LIKE (COL) family proteins from a cryptogam Physcomitrella patens. The predicted PpCOL1 protein has N-terminal zinc finger and C-terminal CCT domains, which are conserved in the angiosperm CO/COL proteins. Structurally, PpCOL1 is the most closely related to the Group Ia or Ic proteins, which include AtCO and AtCOL1/2, among diverged members of the family. A transient expression assay using GFP showed that the CCT domain of PpCOL1 contains a nuclear-localizing signal. Northern blotting analyses revealed that the PpCOL1 expression is controlled by the circadian clock, and moreover, it is photoperiodically regulated at a gametophore stage when the rate of sporophyte formation is affected by day length. These observations indicate a possible involvement of PpCOL1 as a nuclear factor in the photoperiodic regulation of reproduction of Physcomitrella.     

小麦TaLSD1锌指蛋白基因的电子克隆及序列分析

采用电子克隆与RT-PCR相结合的技术,在条锈菌(Pucciniastriiformis f.sp.tritici)侵染的小麦中克隆了一个LSD1型锌指蛋白基因,命名为TaLSD1(GenBank登录号为EF553327)。序列分析表明,该基因全长1024bp,编码生成1个包含3个保守LSD1型锌指结构(CxxCxRxxLMYxxGASxVxCxxC)且长度为146个氨基酸的多肽。进化树分析表明,TaLSD1与水稻(Oryza sativa)、拟南芥(Arabidopsis thaliana)和芜菁(Brassica rapa)中部分含有3个保守LSD1型锌指结构的同源基因亲缘关系较近,而与其它包含不同数目的LSD1型锌指结构基因亲缘关系较远。推测TaLSD1在进化中丢失了部分序列,进而执行新的功能。半定量RT-PCR结果显示,该基因在亲和以及非亲和组合中的表达模式很相似,均表现在前期基因表达被抑制而后期恢复正常。初步推测TaLSD1在转录水平上的表达受光诱导,同时,作为一个细胞程序性死亡的负调控因子在小麦与条锈菌互作过程中起作用。     

TcZFP1: a CCCH zinc finger protein of Trypanosoma cruzi that binds poly-C oligoribonucleotides in vitro

We have identified two zinc finger proteins of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease in humans. These proteins, named tcZFP1 and tcZFP2, share the unusual zinc finger motif (CCCH) found in a diverse range of RNA-binding proteins involved in various aspects of the control of cell homeostasis and differentiation. We report here the functional expression of a recombinant tcZFP1, and the relative affinity and stability of the specific complexes formed between the protein and synthetic oligoribonucleotides containing C-rich sequences.