水稻小G蛋白OsPra2基因的克隆及功能分析

利用micro array 技术对水稻幼苗在营养胁迫条件下根部基因表达的研究中发现: 一个与豌豆Pra2(小G蛋白)基因有同源性的基因的RNA水平在营养胁迫后再补充营养时, 表达量下降。用RT-PCR和PCR方法分别获得该基因的cDNA克隆—OsPra2和该基因翻译起始位点上游1 kb的启动子序列。OsPra2基因编码的蛋白质具有结合GTP/GDP的4个保守结构域和构成小G蛋白Rab家族的特有的结构域。该基因cDNA与GST蛋白基因融合表达载体在洋葱表皮细胞中的瞬间表达结果显示该蛋白定位在在细胞膜和细胞核上, OsPra2基因启动子与GUS报告基因融合表达转基因水稻显示该基因启动子驱动GUS在胚芽鞘和根中表达, 35S启动子驱动OsPra2基因过表达转基因水稻与野生水稻株型相比明显矮化, 类似BR缺陷型植物株型。本实验还对OsPra2和P450蛋白的相互作用及在BR代谢途径中的可能作用进行了分析。     

一种转录受病原物接种和机械创伤诱导的水稻WRKY基因的鉴定

WRKY基因编码一类植物特异性转录因子,该类基因目前在双子叶植物中得到了较好的研究.为了研究WRKY基因在单子叶植物中的生物学功能,从水稻(Oryza sativa L.)中分离了OsiWRKY基因的cDNA.该cDNA可编码一个含482个氨基酸残基的蛋白质,该蛋白质预测的一级结构中含有一个WRKY结构域,在该结构域中发现了一个典型的C2-H2锌指结构模块.OsiWRKY预测的一级结构中还可能含有一个细胞核定位因子;利用瞬时表达体系,发现OsiWRKY与GFP绿色荧光蛋白(GFP)的融合蛋白的确定位在水稻细胞核中.在凝胶阻滞实验中,体外表达的OsiWRKY蛋白可特异地结合W-box顺式因子.在水稻白叶枯病原细菌接种实验中,OsiWRKy基因的转录本在抗病品种(IR-26)中的诱导增加强度明显高于感病品种(Jingang 30).在模拟接种的水稻中,OsiWRKy基因的转录本也表现诱导增加,但其诱导增加的起始时间和幅度要显著低于水稻白叶枯病原细菌接种的水稻植株.这些结果表明,OsiWRKY基因可能编码了一个含有WRKY结构域的转录因子,该转录因子可能参与了水稻对病原物接种和机械创伤的反应,但OsiWRKY蛋白调节两种反应的机制可能有所不同.     

一种转录受病原物接种和机械创伤诱导的水稻WRKY基因的鉴定

WRKY基因编码一类植物特异性转录因子,该类基因目前在双子叶植物中得到了较好的研究。为了研究WRKY基因在单子叶植物中的生物学功能,从水稻(Oryza sativa L.)中分离了OsiWRKY基因的cDNA。该cDNA可编码一个含482个氨基酸残基的蛋白质,该蛋白质预测的一级结构中含有一个WRKY结构域,在该结构域中发现了一个典型的C2-H2锌指结构模块。OsiWRKY预测的一级结构中还可能含有一个细胞核定位因子;利用瞬时表达体系,发现OsiWRKY与GFP绿色荧光蛋白(GFP)的融合蛋白的确定位在水稻细胞核中。在凝胶阻滞实验中,体外表达的OsiWRKY蛋白可特异地结合W-box顺式因子。在水稻白叶枯病原细菌接种实验中,OsiWRKY基因的转录本在抗病品种(IR-26)中的诱导增加强度明显高于感病品种(Jingang 30)。在模拟接种的水稻中,OsiWRKY基因的转录本也表现诱导增加,但其诱导增加的起始时间和幅度要显著低于水稻白叶枯病原细菌接种的水稻植株。这些结果表明,OsiWRKY基因可能编码了一个含有WRKY结构域的转录因子,该转录因子可能参与了水稻对病原物接种和机械创伤的反应,但OsiWRKY蛋白调节两种反应的机制可能有所不同。     

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.     

水稻NACA基因家族的鉴定及NACA2功能研究

该研究从水稻中鉴定了5个编码NACA蛋白的基因,并对其理化性质、结构、定位及表达进行分析,并针对NACA2的亚细胞定位及其在抗旱过程中的生物学功能进行了初步研究。结果表明：(1) 5个水稻NACA蛋白的氨基酸序列中含有多个保守的基序,NACA1-NACA4均含有NAC结构域和UBA结构域,但NACA5序列较短且不含UBA结构域;不同植物NACA蛋白的氨基酸序列进化关系与物种之间的进化关系高度统一。(2)组织表达模式分析发现,NACA1、NACA2和NACA3在不同水稻组织中具有较高的表达水平,尤其是在生殖器官中,但NACA4和NACA5在不同组织中的表达水平均很低;NACA基因的表达受到甘露醇、脱落酸(ABA)、茉莉酸(JA)、氯化钠(NaCl)、水杨酸(SA)及低温胁迫的诱导,其中NACA2和NACA5的表达变化最明显。(3)亚细胞定位表明,NACA2蛋白定位于细胞核和细胞质中。(4)与野生型相比,过表达NACA2拟南芥株系的萎蔫程度较轻,颜色较绿,显著降低了叶片的失水速率,且复水速度明显更快,并显著增强了对干旱和渗透胁迫的抗性。研究表明,NACA2正调控植物对干旱胁迫的抗性,为进一...     

水稻种子发育期间特异锌指蛋白基因的筛选与分析

.锌指蛋白基因是植物基因组中最大最复杂的基因家族之一.大部分的锌指模体存在于转录因子中,它们在转录水平上参与植物生长发育及植物对生物和非生物胁迫的反应.为了解锌指蛋白基因在水稻种子发育中的作用,本研究通过多种数据库搜索获得了878个水稻锌指蛋白基因.从中选取311个利用RT-PCR技术分析它们在水稻成熟期根、茎、叶、花及不同发育阶段种子中的表达特征.结果发现,共有196个基因能在至少1个水稻器官中表达,其中10个为种子特异性表达基因.进一步分析发现,10个特异表达基因在水稻种子不同发育阶段中的表达具有种子阶段表达特异性.同时分析它们的基因及蛋白结构特点,结果显示它们的结构较简单,其中3个蛋白含有线粒体靶肽,5个蛋白含有CCCH锌指结构域.另外,分析种子特异性表达基因上游调控区的顺式作用元件,结果表明它们都含有TATA-box、CAAT-box和种子特异调控元件,除此之外还发现了光、激素和胁迫反应相关调控元件.这些结果为进一步研究它们在种子发育过程中的生物学功能提供了有用的线索.     

水稻含有B-box锌指结构域的OsBBX25蛋白参与植物对非生物胁迫的响应

锌指蛋白在调控植物生长发育和应对逆境过程中发挥着重要作用.为进一步研究锌指类蛋白参与植物非生物胁迫响应的分子机制,对水稻(Oryza sativa)中一个编码含有B-box锌指结构域蛋白的OsBBX25基因进行了功能分析.OsBBX25受盐、干旱和ABA诱导表达.异源表达OsBBX25的转基因拟南芥(Arabidopsis thaliana)与野生型相比对盐和干旱的耐受性增强,且盐胁迫条件下转基因植物中KIN1、RD29A和COR15的表达上调,干旱胁迫下KIN1、RD29A和RD22的表达上调.外源施加ABA时,转基因植物的萌发率与野生型之间没有明显差异.OsBBX25可能作为转录调控的辅助因子调节胁迫应答相关基因的表达,进而参与植物对非生物胁迫的响应.     

水稻C2H2型锌指蛋白基因RZF71的克隆与表达分析

利用生物信息学和RT-PCR方法从水稻幼苗组织中分离了1个新的C2H2型锌指蛋白基因RZF71, 该基因编码一条250个氨基酸残基的多肽, 含有两个典型的C2H2型锌指结构。半定量RT-PCR分析表明: RZF71在根、茎、叶和幼穗中呈组成性表达, 在根中的表达丰度略高; 在高盐和PEG6000胁迫的水稻幼苗组织中, RZF71的表达显著增强, 但低温和ABA处理对该基因的表达量影响不大。农杆菌介导的洋葱表皮细胞GFP瞬时表达实验表明: RZF71定位于细胞核内。讨论了RZF71可能作为一个转录调控因子在水稻耐高盐和渗透胁迫中的作用。     

普通小麦祖先种类TaNAC2a基因的生物信息和表达分析

采用生物信息学方法,利用核酸、蛋白数据库对普通小麦祖先种乌拉尔图小麦(Triticum urartu L.)和粗山羊草(Aegilops tauschii L.)NAC转录因子基因家族进行分析,分别鉴定出107、126个NAC蛋白家族成员。根据拟南芥、水稻NAC基因家族分类系统,将其分为15个亚族。通过与抗逆相关基因TaNAC2a进行同源进化树分析,发现5个TuNAC、6个AetNAC基因与其高度同源,对这些基因的蛋白结构域、基因结构、启动子顺式作用元件及组织表达特性进行分析。结果表明,11个NAC蛋白具有典型的NAC结构域。进化关系较近的基因具有相似基因结构;启动子区域预测发现其均含有逆境胁迫响应作用元件。实时荧光定量PCR结果显示,TuNAC、AetNAC基因分别在乌拉尔图小麦和粗山羊草根、胚芽鞘、叶组织中均有表达,并呈现出明显的组织表达特异性。通过芯片表达数据和逆境胁迫基因表达试验,推测AetNAC2c基因可能参与植物干旱胁迫响应,AetNAC2b可能参与调控植物的耐旱、耐低温胁迫反应。上述分析结果为普通小麦祖先种基因家族的系统研究,优异候选功能基因的预测、筛选提供了试验依据。     

Functional analysis reveals pleiotropic effects of rice RING-H2 finger protein gene OsBIRF1 on regulation of growth and defense responses against abiotic and biotic stresses

RING finger proteins comprise a large family and play key roles in regulating growth/developmental processes, hormone signaling and responses to biotic and abiotic stresses in plants. A rice gene, OsBIRF1, encoding a putative RING-H2 finger protein, was cloned and identified. OsBIRF1 encodes a 396 amino acid protein belonging to the ATL family characterized by a conserved RING-H2 finger domain (C-X2-C-X15-C-X1-H-X2-H-X2-C-X10-C-X2-C), a transmembrane domain at the N-terminal, a basic amino acid rich region and a characteristic GLD region. Expression of OsBIRF1 was up-regulated in rice seedlings after treatment with benzothaidiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid and jasmonic acid, and was induced differentially in incompatible but not compatible interactions between rice and Magnaporthe grisea, the causal agent of blast disease. Transgenic tobacco plants that constitutively express OsBIRF1 exhibit enhanced disease resistance against tobacco mosaic virus and Pseudomonas syringae pv. tabaci and elevated expression levels of defense-related genes, e.g. PR-1, PR-2, PR-3 and PR-5. The OsBIRF1-overexpressing transgenic tobacco plants show increased oxidative stress tolerance to exogenous treatment with methyl viologen and H2O2, and up-regulate expression of oxidative stress-related genes. Reduced ABA sensitivity in root elongation and increased drought tolerance in seed germination were also observed in OsBIRF1 transgenic tobacco plants. Furthermore, the transgenic tobacco plants show longer roots and higher plant heights as compared with the wild-type plants, suggesting that overexpression of OsBIRF1 promote plant growth. These results demonstrate that OsBIRF1 has pleiotropic effects on growth and defense response against multiple abiotic and biotic stresses.     

Distribution patterns of 104 kDa stress-associated protein in rice

A 104 kDa protein (SAP 104) accumulates in rice seedlings in response to several abiotic stress conditions and immunological homologues of rice SAP 104 have been detected in several monocot and dicot species, as also Neurospora crassa, a fungus. In this report, we show that the amino acid sequence of a tryptic peptide generated from purified SAP 104 bears significant homology with an ATP-binding domain of the HSP 100 family proteins of Arabidopsis thaliana and Glycine max. It is further shown that differential uninduced and induced (by high-temperature stress) levels of this protein are accumulated in various organs of the mature rice plant grown under field conditions. Significant uninduced levels of this protein were in particular found in developing and mature rice grains. Seeds/grains of several other plant genera (i.e. Triticum aestivum, Zea mays, Brassica juncea) were also found to contain high uninduced levels of SAP 104. Importantly, the levels of uninduced SAP 104 in rice grains were found to decline during the seed germination phase: after two days of germination, this protein was undetectable in tissues representing pooled sample of seeds and just-emerged seedlings. Tissue print-immunoblotting analysis has indicated that in seeds high levels of this protein are specifically present in the embryo portion.     

苹果DREB转录因子家族全基因组鉴定与分析

DREB转录因子属于AP2/ERF转录因子家族,能够与DRE/CRT顺式作用元件特异性结合,调控与逆境应答基因的表达,因而在植物应对低温、干旱、高盐等逆境胁迫中发挥重要作用。该研究利用苹果全基因组数据,通过生物信息学手段鉴定苹果DREB转录因子家族成员,并分析DREB转录因子家族保守域特点与功能及表达情况。结果表明:从苹果全基因组中共鉴定出60个DREB转录因子家族成员,与拟南芥和水稻相比基本一致,通过引入拟南芥DREB基因进行系统发生分析,进一步可以将其细分为6个亚组;结构域和保守元件分析表明,DREB基因家族含有一个AP2保守结构域;染色体定位表明,苹果DREB基因分布于11条染色体上,部分基因存在串联复制现象;基因结构分析显示,该亚家族基因不含内含子。利用同源拟南芥RNA-Seq数据分析结果表明,DREB转录因子家族对低温、ABA调节等非生物胁迫具有调控作用,同时在DREB亚家族中每个亚组响应不同的非生物胁迫;通过分析DREB基因在不同组织中的表达情况,结果显示DREB基因在植物根部中的表达量最强,其次是叶。     

Protein phosphatases: a genomic outlook to understand their function in plants

Protein phosphatases are the vital regulatory components of various signal transduction pathways in eukaryotes. Signaling pathways triggered during stress and development have been regulated by different classes of protein phosphatases in plants. Recently, genome-wide expressional analysis in Arabidopsis and crop plant such as rice revealed differential expression pattern for several protein phosphatases under different abiotic stresses, in various tissues and at different developmental stages. This expression pattern could be extrapolated to the possible function of protein phosphatases in abiotic stress signaling and tolerance, and during plant development. Here, we discuss organisation and expression patterns of members of the protein phosphatase gene family, and their potential functional role in plants.     

普通烟草ATⅢ氨基转移酶家族序列鉴定与表达分析

氨基转移酶是5'-磷酸吡哆醛依赖酶,在植物的生长发育和非生物胁迫的反应中起重要作用。ATⅢ氨基转移酶家族(classⅢ aminotransferase family)是转氨酶家族中一个非常重要的亚家族。本研究利用普通烟草(Nicotiana tabacum)基因组序列信息,鉴定出26个ATⅢ家族成员,对烟草ATⅢ家族进行理化性质分析表明,普通烟草ATⅢ家族成员之间的理化性质差异较大;系统进化和结构域分析显示,烟草ATⅢ家族成员可形成4个分支,同一分支内ATⅢ家族成员的保守结构域的种类和组织形式高度一致;将19个家族成员定位在12条染色体上;分析普通烟草转录组数据,结果显示大多数家族成员在不同组织中都有表达,主要集中在叶脉、打顶后茎和叶、离体叶片等组织。对NtATⅢ1和NtATⅢ2基因的qRT-PCR分析显示,这两个基因主要在植物地上组织中表达。本研究为普通烟草ATⅢ基因的功能研究提供依据。     

Comprehensive expression analysis of rice phospholipase D gene family during abiotic stresses and development

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.