Phytochrome-mediated control of COP1 gene expression in rice plants

We isolated a COP1 cDNA from rice and found that it could complement the Arabidopsis cop1-4 mutant. The putative rice COP1 protein has the Ring-finger, coiled-coil. and WD-40 repeat domains, which are also conserved in pea, tomato, and mammalian COP1 proteins. The degree of overall identity between rice COP1 and Arabidopsis COP1 is 73%, and the similarity value is 83%. Expression of rice COP1 was detected in almost all plant tissues, with the level being relatively higher in calli and very low in etiolated leaves. The expression level was positively controlled by light in etiolated and green leaves. At the end of the light period, expression of the gene in green leaves could be down-regulated by far-red light. This far-red light effect could be prevented by subsequent irradiation with red light. These results indicate that phytochrome regulates rice COP1 expression.     

植物COP1蛋白的结构与功能

组成型光形态建成 1 (constitutivelyphotomorphogenic 1 ,COP1 )蛋白是一个分子量为 76kD的核蛋白 ,它由 3个特殊的结构域组成即环形锌指结合域、卷曲螺旋形结构域和WD_40重复序列 ,并含有一个核定位信号和一个新型细胞质定位信号 ,它是一个光形态建成的抑制子 ,是一个光调控植物发育的分子开关。当植物在暗环境下生长时 ,COP1蛋白聚集在细胞核内 ,抑制光形态的建成 ,而在光环境下 ,COP1蛋白则分散到细胞质中 ,解除其抑制作用 ,恢复光形态建成。COP1蛋白在细胞内的核质分布受多个因素的影响 ,核内COP1通过与特异转录因子相互作用来调节光形态建成。继从植物中分离鉴定出COP1蛋白之后 ,动物体内也发现有COP1蛋白的存在 ,提示COP1蛋白可能在调节动物和植物的发育及信号转导等方面具有共同作用模式。     

甘蓝型油菜BnCOP1基因编码区全长cDNA的克隆与功能研究

通过分析拟南芥、豌豆、番茄和水稻的COP1 (constitutively photomorphogenic 1) 的cDNA序列, 运用RT-PCR和改进的基因组步行 (genome walking) 技术相结合的方法, 首次从甘蓝型油菜中克隆到油菜 BnCOP1编码区cDNA的全长序列, 其全长2 034 bp, 编码677个氨基酸. 同源 性分析表明, 其编码的氨基酸序列与拟南芥的同源性高达94%. 对BnCOP1编 码序列（cDNA）演绎出的氨基酸序列分析表明, 其编码的蛋白包含有N端的 环形锌指结合域(ring finger zinc binding domain, RING)、中间的卷曲 螺旋形结构域(coiled-coil domain, coiled-coil ), 7个C端的WD-40重复 序列(WD-40 repeats, WD-40)的功能域. 半定量RT-PCR和实时荧光定量PCR 分析该基因在油菜中的表达模式,结果显示, BnCOP1在甘蓝型油菜的各个组 织器官中均有表达,其中在花中的表达明显高于在根、叶、茎、果荚及子叶 和胚轴中,暗示该蛋白可能与开花途径相关. 过表达BnCOP1的转基因拟南芥 植株在高度、主茎的直径和叶片大小上都呈现出比野生型弱小的表型, 表 明BnCOP1抑制了拟南芥光形态建成从而影响了植物的生长发育.     

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。     

水稻中大麦Mlo和玉米Hm1抗病基因同源序列的分析和定位

大麦抗病基因Mlo和玉米抗病基因Hm1编码的产物不具有绝大多数植物抗病基因产物所含有的保守结构域。这两个抗病基因的作用机理也不符合基因对基因学说。从水稻中分离克隆了Mlo基因的同源序列OsMlo-1和玉米Hm1基因的同源序列DFR－1。利用水稻分子标记遗传连锁图,将OsMlo-1定位于水稻第六染色体的两俱RZ667和RG424之间;Osmlo-1距离这两个分子标记分别为20．6和6．0cM(centi-Morgan)。将DFR－1定位于水稻第一染色体两个分子标记R2635和RG462之间;DFR－1距离这两个分子标记分别为11．3和23．9cM。参照已发表的水稻分子标记连锁图,发现OsMlo-1和DFR－1的染色体位点分别与两个报道的水稻抗稻瘟病数量性状位点（QTL）有较好的对应关系。结果提示,水稻中与大麦Mlo 和玉米Hml同源的基因可能也参于抗病反应的调控。     

Expression of an N-terminal fragment of COP1 confers a dominant-negative effect on light-regulated seedling development in Arabidopsis.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an essential regulatory gene that plays a role in light control of seedling development in Arabidopsis. The COP1 protein possesses three recognizable structural domains: a RING finger zinc binding domain near the N terminus, followed by a coiled-coll domain and a domain with WD-40 repeats in the C-terminal half. To determine whether COP1 acts specifically as a light-inactivable repressor of photomorphogenic development and to elucidate the functional roles of the specific structural domains, mutant cDNAs encoding the N-terminal 282 amino acids (N282) of COP1 were expressed and analyzed in transgenic plants. High-level expression of the N282 fragment caused a dominant-negative phenotype similar to that of the loss-of-function cop1 mutants. The phenotypic characteristics include hypersensitivity of hypocotyl elongation to inhibition by white, blue, red, and far-red light stimuli. In the dark, N282 expression led to pleiotropic photomorphogenic cotyledon development, including cellular differentiation, plastid development, and gene expression, although it has no significant effect on the hypocotyl elongation. However, N282 expression had a minimal effect on the expression of stress- and pathogen-inducible genes. These observations support the hypothesis that COP1 is directly involved in the light control of seedling development and that it acts as a repressor of photomorphogenesis. Further, the results imply that the N282 COP1 fragment, which contains the zinc binding and colled-coil domains, is capable of interacting with either downstream targets or with the endogenous wild-type COP1, thus interfering with normal regulatory processes. The fact the N282 is able to interact with N282 and full-length COP1 in yeast provided evidence for the latter possibility.     

水稻蛋白激酶基因Pti1的克隆与分析

植物Pti1基因编码丝氨酸/苏氨酸蛋白激酶,是重要的抗病相关基因。在水稻抗褐飞虱基因Qbp1所在的染色体区段存在一个与番茄Pti1基因高度同源的序列片段。从抗虫水稻B5中分离了Pti1基因的全长cDNA克隆,测定了基因组序列。分析发现,水稻中Pti1基因长度有5644bp,含有7个内含子,编码368个氨基酸的激酶蛋白。其蛋白质的C末端在不同植物之间具有高度的保守性,而N末端的变异则相对较大。对不同水稻材料Pti1基因的序列进行了比较,发现药用野生稻与栽培稻之间存在较大的差异,而栽培稻各品种之间的差异较小。讨论了Pti1基因在抗虫防卫反应中可能的作用。     

新基因水稻 OsLSD1 的克隆及拟南芥和水稻类 LSD1 基因家族的生物信息学分析

类 LSD1 (LSD1-like) 基因家族是一类特殊的 C2C2 型锌指蛋白基因,编码植物特有的转录因子 . 目前已经研究的 2 个成员拟南芥 LSD1 (lesions stimulating disease resistance 1) 和 LOL1 (LSD-One-Like 1) 基因均参与植物细胞程序化死亡 (programmed cell death, PCD) 的调控 . 从水稻 cDNA 文库中克隆到 1 个类 LSD1 基因,命名为 OsLSD1. 该基因长 988 bp ,包含一个 432 bp 的开放阅读框,推导的氨基酸序列 (143 个氨基酸 ) 含有 3 个内部保守的锌指结构域 . DNA 印迹结果表明 OsLSD1 基因在水稻基因组中为单拷贝,且在根、茎和叶中表达 . 借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出 5 个和 7 个 ( 包括 OsLSD1) 类 LSD1 基因 . 分析了这些类 LSD1 基因的结构,蛋白质结构域组成 . 系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类 LSD1 基因分为 2 类 . 虽然不存在拟南芥或水稻特有的类 LSD1 蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件 .     

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.     

High Level of COP1 Expression is Associated with Poor Prognosis in Primary Gastric Cancer

COP1 (constitutive photomorphogenic 1, also known as RFWD2) is a p53-targeting E3 ubiquitin ligase containing RING-finger, coiled-coil, and WD40-repeat domains. Recent studies have identified that COP1 is overexpressed in several cancer types and that increased COP1 expression promotes cell proliferation, cell transformation, and tumor progression. In the present study, we investigated the expression and prognostic value of COP1 in primary gastric cancer. To investigate the role of the COP1 gene in primary gastric cancer pathogenesis, real-time quantitative PCR and western blotting were performed to examine COP1 expression in paired cancerous and matched adjacent noncancerous gastric tissues. The results revealed high COP1 mRNA (P=0.030) and protein (P=0.008) expression in most tumor-bearing tissues compared with the matched adjacent non-tumor tissues. The correlated protein expression analysis revealed a negative correlation between COP1 and p53 in gastric cancer samples (P=0.005, r=-0.572). Immunohistochemical staining of gastric cancer tissues from the same patient showed a high COP1 expression and a low p53 expression. To further investigate the clinicopathological and prognostic roles of COP1 expression, we performed immunohistochemical analysis of 401 paraffin-embedded gastric cancer tissue blocks. The data revealed that high COP1 expression was significantly correlated with T stage (P=0.030), M stage (P=0.048) and TNM stage (P=0.022). Consistent with these results, we found that high expression of COP1 was significantly correlated with poor survival in gastric cancer patients (P<0.001). Cox regression analyses showed that COP1 expression was an independent predictor of overall survival (P<0.001). Our data suggest that COP1 could play an important role in gastric cancer and might serve as a valuable prognostic marker and potential target for gene therapy in the treatment of gastric cancer.     

植物COP1蛋白的结构与功能

组成型光形态建成1(constitutively photomorphogenic 1,COP1)蛋白是一个分子量为76 kD的核蛋白,它由3个特殊的结构域组成即环形锌指结合域、卷曲螺旋形结构域和WD_40 重复序列,并含有一个核定位信号和一个新型细胞质定位信号,它是一个光形态建成的抑制子,是一个光调控植物发育的分子开关。当植物在暗环境下生长时,COP1蛋白聚集在细胞核内,抑制光形态的建成,而在光环境下,COP1蛋白则分散到细胞质中,解除其抑制作用,恢复光形态建成。COP1蛋白在细胞内的核质分布受多个因素的影响,核内COP1通过与特异转录因子相互作用来调节光形态建成。继从植物中分离鉴定出COP1蛋白之后,动物体内也发现有COP1蛋白的存在,提示COP1蛋白可能在调节动物和植物的发育及信号转导等方面具有共同作用模式。     

Regulation of COP1 nuclear localization by the COP9 signalosome via direct interaction with CSN1

COP1 and COP9 signalosome (CSN) are key regulators of plant light responses and development. Deficiency in either COP1 or CSN causes a constitutive photomorphogenic phenotype. Through coordinated actions of nuclear- and cytoplasmic-localization signals, COP1 can respond to light signals by differentially partitions between nuclear and cytoplasmic compartments. Previous genetic analysis in Arabidopsis indicated that the nuclear localization of COP1 requires CSN, an eight-subunit heteromeric complex. However the mechanism underlying the functional relationship between COP1 and CSN is unknown. We report here that COP1 weakly associates with CSN in vivo . Furthermore, we report on the direct interaction involving the coiled-coil domain of COP1 and the N-terminal domain of the CSN1 subunit. In onion epidermal cells, expression of CSN1 can stimulate nuclear localization of GUS-COP1, and the N-terminal domain of CSN1 is necessary and sufficient for this function. Moreover, CSN1-induced COP1 nuclear localization requires the nuclear-localization sequences of COP1, as well as its coiled-coil domain, which contains both the cytoplasmic localization sequences and the CSN1 interacting domain. We also provide genetic evidence that the CSN1 N-terminal domain is specifically required for COP1 nuclear localization in Arabidopsis hypocotyl cells. This study advances our understanding of COP1 localization, and the molecular interactions between COP1 and CSN.     

Evidence for functional conservation of a mammalian homologue of the light-responsive plant protein COP1.

Identified in Arabidopsis as a repressor of light-regulated development, the COP1 (constitutively photomorphogenic 1) protein is characterized by a RING-finger motif and a WD40 repeat domain [1]. The subcellular localization of COP1 is light-dependent. COP1 acts within the nucleus to repress photomorphogenic development, but light inactivates COP1 and diminishes its nuclear abundance [2]. Here, we report the identification of a mammalian COP1 homologue that contains all the structural features present in Arabidopsis COP1 (AtCOP1). When expressed in plant cells, a fusion protein comprising mammalian COP1 and beta-glucuronidase (GUS) responded to light by changing its subcellular localization pattern in a manner similar to AtCOP1. Whereas the mammalian COP1 was unable to rescue the defects of Arabidopsis cop1 mutants, expression of the amino-terminal half of mammalian COP1 in Arabidopsis interfered with endogenous COP1 function, resulting in a hyperphotomorphogenic phenotype. Therefore, the regulatory modules in COP1 proteins that are responsible for the signal-dependent subcellular localization are functionally conserved between higher plants and mammals, suggesting that mammalian COP1 may share a common mode of action with its plant counterpart in regulating development and cellular signaling.     

Isolation and mapping of rFUS6, a rice orthologue of Arabidopsis thaliana FUS6.

COP9 complex is one of the most important components that act in repressing photomorphogenesis in Arabidopsis thaliana. FUS6 has been identified as one of eight subunits of the COP9 complex in Arabidopsis. Using Arabidopsis Fus6 cDNA as a probe, we screened a rice root cDNA library and a rice genomic library. A 1730-bp cDNA was obtained, which has an open reading frame corresponding to 441-amino-acid. This 441 amino acids putative protein has 67% identity with Arabidopsis COP11/FUS6 (AtFUS6) and 40% identity with human GPS1, an AtFUS6 orthologue. So we designated this novel gene as rFUS6. The 6.2-kb genomic sequence of rFUS6 was also obtained. Sequence comparison showed that the rFUS6 gene had six exons and five introns. Sequence inspection of the 5'-flanking region revealed the presence of some potential light-regulated cis-elements such as a G-box, GT-1 binding sites, and a TGACG motif. Southern hybridization with rice total DNA showed that rFUS6 was perhaps a single copy gene. The rFUS6 locus was mapped by hybridization with a rice BAC library membrane and the results showed that rFUS6 had a locus at 16.3 cM of chromosome 1.     

Overexpression of Arabidopsis COP1 results in partial suppression of light-mediated development: evidence for a light-inactivable repressor of photomorphogenesis.

Arabidopsis seedlings are genetically endowed with the capability to follow two distinct developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. The regulatory protein CONSTITUTIVE PHOTO-MORPHOGENIC1 (COP1) has been postulated to act as a repressor of photomorphogenesis in the dark because loss-of-function mutations of COP1 result in dark-grown seedlings phenocopying the light-grown wild-type seedlings. In this study, we tested this working model by overexpressing COP1 in the plant and examining its inhibitory effects on photomorphogenic development. Stable transgenic Arabidopsis lines overexpressing COP1 were generated through Agrobacterium-mediated transformation. Overexpression was achieved using either the strong cauliflower mosaic virus 35S RNA promoter or additional copies of the wild-type gene. Analysis of these transgenic lines demonstrated that higher levels of COP1 can inhibit aspects of photomorphogenic seedling development mediated by either phytochromes or a blue light receptor, and the extent of inhibition correlated quantitatively with the vivo COP1 levels. This result provides direct evidence that COP1 acts as a molecular repressor of photomorphogenic development and that multiple photoreceptors can independently mediate the light inactivation of COP1. It also suggests that a controlled inactivation of COP1 may provide a basis for the ability of plants to respond quantitatively to changing light signals, such as fluence rate and photoperiod.     

The role of COP1 in light control of Arabidopsis seedling development

The Arabidopsis seedling can follow two contrasting developmental programmes, photomorphogenesis in light and skotomorphogenesis in darkness. CONSTITUTIVE PHOTOMORPHOGENIC I (COP1) is an essential regulatory gene required for repression of seedling photomorphogenic development in darkness. Recent mutational and overexpression analyses of the COP1 gene suggest a central role for COP1 in light control of seedling development and point to functional implications of its structural domains. Cell biological studies of COP1 have provided a clue to how light regulates the repressive activity of COP1 and thus seedling development.