受脱落酸调节的拟南芥F-box基因的差异表达分析

脱落酸(Abscisic acid,ABA)是一种重要的植物激素,在种子休眠的建立、种子萌发、根发育和非生物胁迫反应过程中发挥作用。F-box蛋白是E3泛素连接酶SCF复合体的组成部分,通过特异识别和调节底物蛋白水平而调控植物生长发育过程。通过分析GEO基因芯片,筛选到38个受ABA调节的拟南芥候选F-box基因。选择其中6个F-box基因,进行实时荧光定量PCR分析。研究结果与基因芯片结果基本一致。分析启动子,发现候选基因含有大量ABA、干旱和胁迫相关的顺式作用元件。分析基因表达谱,发现部分基因在保卫细胞、种皮、花粉和衰老叶片中呈现高表达;大部分基因在ABA处理、胁迫和种子吸胀过程中表达量改变显著。这些分析结果为深入研究ABA调节植物生长发育和抗逆的分子机制提供了线索。     

葡萄F-box基因VvF-box5的基因结构与表达分析

F-box蛋白广泛存在于真核生物中,主要参与细胞周期调控、凋亡及多种激素信号转导等过程;近几年发现,F-box蛋白还介导了植物对逆境胁迫的应答响应,对维持植物正常生长发育至关重要。干旱等非生物逆境胁迫严重影响了葡萄正常生长发育和果实品质,克隆并分析干旱响应基因对改良葡萄的抗性有重要意义。本研究根据葡萄干旱转录组分析结果,发现11个F-box基因在干旱胁迫下表达量明显上调;其中,Vv F-box5基因位于葡萄第19条染色体上,对干旱胁迫的响应明显高于其他F-box成员;Vv F-box5基因含有5个外显子和4个内含子,内含子均含有保守的GT…AG序列;Vv F-box5基因包含1824 bp的开放阅读框,编码607个氨基酸,氨基酸序列的N端含有1个保守的F-box结构域,C端包含1个FBD和2个LRR结构域。启动子元件分析表明,Vv F-box5基因含有多种逆境应答元件,包括GA响应元件GARE-motif、Me JA响应元件CGTCAmotif、干旱胁迫相关元件ERE、HSE和LTR、光应答顺式作用元件ACE、Box4和Sp1以及与细胞周期调节和发育相关的元件等。实时荧光定量PCR结果显示,在干旱、高盐、ABA和Me JA处理下,Vv F-box5基因的表达量明显升高;亚细胞定位结果显示,Vv F-box5蛋白主要定位于洋葱表皮细胞的细胞核中;Vv F-box5的过表达明显提高了转基因拟南芥在干旱处理下的成活率。另外,本研究利用原核表达系统诱导6×His-Vv F-box5融合蛋白的表达,并使用蛋白标记亲和层析柱纯化获得了6×HisVv F-box5融合蛋白,为下一步深入研究Vv F-box5的功能奠定基础。     

葡萄F-box基因VvF-box5基因结构与表达分析

F-box蛋白广泛存在于真核生物中,主要参与细胞周期调控、凋亡及多种激素信号转导等过程;近几年发现,F-box蛋白还介导了植物对逆境胁迫的应答响应,对维持植物正常生长发育至关重要。干旱等非生物逆境胁迫严重影响了葡萄正常生长发育和果实品质,克隆并分析干旱响应基因对改良葡萄的抗性有重要意义。本研究根据葡萄干旱转录组分析结果,发现11个F-box基因在干旱胁迫下表达量明显上调;其中,VvF-box5基因位于葡萄第19条染色体上,对干旱胁迫的响应明显高于其他F-box成员;VvF-box5基因含有5个外显子和4个内含子,内含子均含有保守的GT…AG序列;VvF-box5基因包含1824 bp的开放阅读框,编码607个氨基酸,氨基酸序列的N端含有1个保守的F-box结构域,C端包含1个FBD和2个LRR结构域。启动子元件分析表明,VvF-box5基因含有多种逆境应答元件,包括GA响应元件GARE-motif、MeJA响应元件CGTCA-motif、干旱胁迫相关元件ERE、HSE和LTR、光应答顺式作用元件ACE、Box4和Sp1以及与细胞周期调节和发育相关的原件等。实时荧光定量PCR结果显示,在干旱、高盐、ABA和MeJA处理下,VvF-box5基因的表达量明显升高;亚细胞定位结果显示,VvF-box5蛋白主要定位于圆葱表皮细胞的细胞核中;VvF-box5的过表达明显提高了转基因拟南芥在干旱处理下的成活率。另外,本研究利用原核表达系统诱导6×His-VvF-box5融合蛋白的表达,并使用蛋白标记亲和层析柱纯化获得了6×His-VvF-box5融合蛋白,为下一步深入研究VvF-box5的功能鉴定基础。     

拟南芥GA2ox基因家族启动子分析

赤霉素是一类重要的植物激素,它参与调节植物生长发育的各个阶段。真核基因的表达受多种因素的调控,其中启动子在转录水平上的调节作用至关重要,但是目前对拟南芥GA2ox基因家族上游顺式元件的详细分析较少。本研究分析了AtGA2ox基因家族染色体定位、进化关系、蛋白模体以及顺式元件。分析结果表明：AtGA2ox基因家族分为2个亚类;具有相对集中的染色体分布：具有相似的内含子和外显子结构;AtGA2ox基因家族成员进化关系越相近,模体和顺式元件则越保守且分布位置更接近。PLACE和Plant CARE以及DMB分析显示,AtGA2ox基因家族存在许多保守元件,受多因素的调控,在此家族的上游调控区域普遍存在组织和器官特异性表达元件、光响应元停、激素响应元件以及其他环境响应元件。基因表达谱分析结果表明,在激素诱导下,AtGA2ox基因家族均有响应。这些元件不仅作为正调控元件,诱导部分AtGA2ox基因的表达,而且还能作为负调控元件．抑制其他AtGA2ox基因的表达。     

拟南芥转录因子GRAS家族基因群响应渗透和干旱胁迫的初步探索

GRAS家族是一类植物特有的转录调控因子,已有报道表明该家族基因在植物生长发育和光信号转导过程中具有重要作用.目前在拟南芥(Arabidopsis thaliana)基因组中已鉴定了33个GRAS家族基因.利用功能基因组学和生物信息学手段,通过基因芯片数据挖掘和基因功能预测,对拟南芥GRAS家族基因在渗透和干旱胁迫过程中的应答模式进行了初步探索,提出了一类响应渗透胁迫和干旱胁迫的拟南芥GRAS家族基因.以SCL13为例,利用基因芯片相关性和GO分析,对其在渗透胁迫信号转导过程中可能的调控机制进行了预测和分析.这一研究将为阐明GRAS家族基因参与水分胁迫的分子机制提供新的思路,同时也为植物抗逆分子育种提供候选基因.     

拟南芥转录因子GRAS 家族基因群响应渗透和干旱胁迫的初步探索

GRAS家族是一类植物特有的转录调控因子, 已有报道表明该家族基因在植物生长发育和光信号转导过程中具有重要作用。目前在拟南芥(Arabidopsis thaliana)基因组中已鉴定了33个GRAS家族基因。利用功能基因组学和生物信息学手段,通过基因芯片数据挖掘和基因功能预测, 对拟南芥GRAS家族基因在渗透和干旱胁迫过程中的应答模式进行了初步探索, 提出了一类响应渗透胁迫和干旱胁迫的拟南芥GRAS家族基因。以SCL13为例, 利用基因芯片相关性和GO分析, 对其在渗透胁迫信号转导过程中可能的调控机制进行了预测和分析。这一研究将为阐明GRAS家族基因参与水分胁迫的分子机制提供新的思路, 同时也为植物抗逆分子育种提供候选基因。     

赤霉素调节植物对非生物逆境的耐性

赤霉素（GAs）是一类重要的植物激素,调控植物生长发育的诸多方面．最近的研究表明,GA也参与对生物与非生物胁迫的响应,然而GA参与非生物胁迫响应的遗传学证据及其机制有待于进一步研究．本实验室前期研究证明,水稻EullfELONGATEDUPPERMOSTINTERNODE）通过一个新的生化途径降解体内的活性赤霉素分子,并参与调控水稻对病原菌的基础抗病性．本研究发现,euil突变体对盐胁迫能力降低,而超表达EUll基因的水稻和拟南芥耐盐性显著提高．进一步研究发现,积累高含量赤霉素的水稻euil突变体对脱落酸（ABA）的敏感性下降,而赤霉素缺失的EUll超表达转基因水稻和拟南芥均改变了对于ABA的敏感性．EUll基因的转录受逆境诱导,其功能缺失与超表达调控了逆境标志基因的表达．综上推测,GA可能是通过影响ABA的信号途径从而改变了植物对非生物胁迫的响应．     

苹果SRS基因家族的鉴定与功能分析北大核心CSCD

SHI-related sequence(SRS)基因家族通过介导激素变化以调控植物成花及生长发育,并且在适应环境胁迫中起重要调控作用。该研究基于苹果(Malus domestica Borkh.)基因组数据,通过生物信息学手段鉴定苹果SRS基因家族成员,并分析SRS基因家族特点与功能及表达情况。结果表明:(1)苹果MdSRS基因家族共包含11个成员,分别命名为MdSRS1-MdSRS11,不均匀地分布在苹果的9条染色体上。(2)MdSRS蛋白包含229~414个不等的氨基酸残基,等电点分布在6.38~9.36之间;亚细胞定位结果表明,MdSRS蛋白大多分布于细胞膜,在细胞核、叶绿体中也有分布。(3)通过引入拟南芥、水稻、番茄及杨树的SRS基因进行系统发育分析表明,将11个MdSRSs分成5个亚族(A-A),在A4中分布最多。(4)顺式作用元件分析表明,11个MdSRSs启动子上游2 000 bp序列分布有激素、环境适应性和逆境诱导等响应元件。(5)荧光定量PCR结果显示,苹果MdSRS基因家族在盐胁迫和干旱胁迫下总体呈下调表达,在ABA胁迫后大多呈上调表达,是具有很大潜力的抗性候选基因,说明SRS家族对ABA调节等非生物胁迫具有调控作用。研究认为,SRS家族的11个成员均参与了调控干旱、盐及ABA胁迫多种逆境的响应,推测在实际苹果生产中对抵御不良环境具有重要作用。     

植物体内干旱信号的传递与基因表达

干旱是严重影响植物生长发育的重要环境胁迫因子之一。干旱能影响植物的水分状态,使植物缺水遭受伤害。近年来,相继从拟南芥等植物中克隆出了一些受干旱诱导的基因,如蛋白激酶基因、光合基因、渗透调节基因、功能蛋白基因（如LEA基因）等。干旱等胁迫信号经历一系列的传递过程,最后诱导这些特定基因的表达。在植物体中,可能存在依赖ABA型和不依赖ABA型两条干旱信号的传递途径。近年来从高等植物中分离出一系列调控干旱相关基因表达的转录因子,通过转录因子之间以及与其它相关蛋白之间的相互作用,激活或抑制干旱等胁迫因子诱导的基因表达。     

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.     

Gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature interaction

Soybean is an important oilseed crop grown globally. However, two examples of environmental stresses that drastically regulate soybean growth are low light and high-temperature. Emerging evidence suggests a possible interconnection between these two environmental stimuli. Low light and high-temperature as individual factors have been reported to regulate plant hypocotyl elongation. However, their interactive signal effect on soybean growth and development remains largely unclear. Here, we report that gibberellins (GAs) and auxin are required for soybean hypocotyl elongation under low light and high-temperature interaction. Our analysis indicated that low light and high-temperature interaction enhanced the regulation of soybean hypocotyl elongation and that the endogenous GA₃, GA₇, indole-3-acetic acid (IAA), and indole-3-pyruvate (IPA) contents significantly increased. Again, analysis of the effect of exogenous phytohormones and biosynthesis inhibitors treatments showed that exogenous GA, IAA, and paclobutrazol (PAC), 2, 3, 5,-triiodobenzoic acid (TIBA) treatments significantly regulated soybean seedlings growth under low light and high-temperature interaction. Further qRT-PCR analysis showed that the expression level of GA biosynthesis pathway genes (GmGA3ox1, GmGA3ox2 and GmGA3) and auxin biosynthesis pathway genes (GmYUCCA3, GmYUCCA5 and GmYUCCA7) significantly increased under (i) low light and high-temperature interaction and (ii) exogenous GA and IAA treatments. Altogether, these observations support the hypothesis that gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature stress interaction.     

Transcriptional regulation of gibberellin metabolism genes by auxin signaling in Arabidopsis

Auxin and gibberellins (GAs) overlap in the regulation of multiple aspects of plant development, such as root growth and organ expansion. This coincidence raises questions about whether these two hormones interact to regulate common targets and what type of interaction occurs in each case. Auxins induce GA biosynthesis in a range of plant species. We have undertaken a detailed analysis of the auxin regulation of expression of Arabidopsis (Arabidopsis thaliana) genes encoding GA 20-oxidases and GA 3-oxidases involved in GA biosynthesis, and GA 2-oxidases involved in GA inactivation. Our results show that auxin differentially up-regulates the expression of various genes involved in GA metabolism, in particular several AtGA20ox and AtGA2ox genes. Up-regulation occurred very quickly after auxin application; the response was mimicked by incubations with the protein synthesis inhibitor cycloheximide and was blocked by treatments with the proteasome inhibitor MG132. The effects of auxin treatment reflect endogenous regulation because equivalent changes in gene expression were observed in the auxin overproducer mutant yucca. The results suggest direct regulation of the expression of GA metabolism genes by Aux/IAA and ARF proteins. The physiological relevance of this regulation is supported by the observation that the phenotype of certain gain-of-function Aux/IAA alleles could be alleviated by GA application, which suggests that changes in GA metabolism mediate part of auxin action during development.     

能源植物小桐子赤霉素合成代谢及信号转导相关基因的鉴定及序列分析

赤霉素(gibberellin,GA)是一类非常重要的植物激素,在植物种子萌发、茎干伸长、叶片生长、腺毛发育、花粉成熟、开花诱导和果实成熟等生长发育过程中都发挥着重要的作用。GA在一年生草本植物中可以促进开花,而在大多数多年生木本植物中则抑制成花诱导。为了更好地研究赤霉素在木本油料能源植物小桐子(Jatropha curcas)开花调控方面的作用机理,我们对小桐子整个基因组中参与GA合成代谢和信号转导的全部基因进行了鉴定和序列分析。这些基因包括6个多基因家族编码的蛋白,即GA2氧化酶(GA2-oxidase,GA2ox)、GA3氧化酶(GA3-oxidase,GA3ox)、GA20氧化酶(GA20-oxidase,GA20ox)、GID1(GIBBERELLIN INSENSITIVE DWARF1)、DELLAs和F-box蛋白,以及2个单基因编码的蛋白,EL1(EARLY FLOWERING1)和SPY(SPINDLY)。采用拟南芥和水稻中已经鉴定的上述基因编码的蛋白序列在小桐子基因组序列数据库和本实验的小桐子转录组数据库中进行BLASTP分析,找到17个同源蛋白的全长序列,并将其与28个拟南芥的、16个水稻的、24个葡萄的和22个蓖麻的同源蛋白构建系统发育树进行比对分析。结果表明,小桐子中参与赤霉素合成代谢及信号转导的大多数基因与蓖麻和葡萄同源基因的相似度更高。     

AGF1, an AT-hook protein, is necessary for the negative feedback of AtGA3ox1 encoding GA 3-oxidase

Negative feedback is a fundamental mechanism of organisms to maintain the internal environment within tolerable limits. Gibberellins (GAs) are essential regulators of many aspects of plant development, including seed germination, stem elongation, and flowering. GA biosynthesis is regulated by the feedback mechanism in plants. GA 3-oxidase (GA3ox) catalyzes the final step of the biosynthetic pathway to produce the physiologically active GAs. Here, we found that only the AtGA3ox1 among the AtGA3ox family of Arabidopsis (Arabidopsis thaliana) is under the regulation of GA-negative feedback. We have identified a cis-acting sequence responsible for the GA-negative feedback of AtGA3ox1 using transgenic plants. Furthermore, we have identified an AT-hook protein, AGF1 (for the AT-hook protein of GA feedback regulation), as a DNA-binding protein for the cis-acting sequence of GA-negative feedback. The mutation in the cis-acting sequence abolished both GA-negative feedback and AGF1 binding. In addition, constitutive expression of AGF1 affected GA-negative feedback in Arabidopsis. Our results suggest that AGF1 plays a role in the homeostasis of GAs through binding to the cis-acting sequence of the GA-negative feedback of AtGA3ox1.     

Gibberellins are required for seed development and pollen tube growth in Arabidopsis

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.