叶子花脱落酸生物合成关键酶基因NCED的克隆及调节开花功能初探

以叶子花品种‘大红宝巾’(Bougainvillea glabra‘Mrs Butt’)的2年生扦插苗为试验材料,克隆到了一个9-顺式-环氧类胡萝卜素双氧合酶(NCED)同源基因,并分析了内源脱落酸(ABA)含量及NCED活性等变化与该基因表达之间的内在联系,探讨ABA对促进叶子花开花的作用机理。结果表明:(1)外源50mg·L-1 ABA处理促进了叶子花开花,而与10μmol·L-1的去甲二氢愈创木酸(NDGA,ABA合成抑制剂)共处理可抑制这种效果。(2)外源ABA处理可诱导叶子花叶片中内源ABA含量和NCED含量与活性上升,这种诱导可被NDGA抑制。(3)克隆得到的NCED基因全长为2 380bp,其推定的编码蛋白包含618个氨基酸残基,与草莓中的FvNCED1同源性最高,命名为BgNCED1。(4)Real-time PCR结果显示,外源ABA处理显著诱导BgNCED1基因的表达,而10μmol·L-1的NDGA可显著抑制BgNCED1基因的诱导效果,这种表达模式与内源ABA含量及NCED活性等的变化趋势较为一致。研究认为,外源ABA可能通过诱导BgNCED1的表达,增强内源ABA的生物合成,进而促进叶子花从营养生长到生殖生长的转变,使其提前开花。     

模拟干旱胁迫下马铃薯StNCED1表达量及与ABA含量的相关性分析

干旱缺水是限制马铃薯产量和品质的关键因素之一。ABA是干旱胁迫应答基因调控网络中的重要组成;9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物ABA生物合成途径的关键限速酶,该基因的表达模式直接影响ABA代谢。但干旱胁迫下有关马铃薯NCED基因表达与激素、表型间的相关分析的研究尚少。本研究克隆了马铃薯NCED1基因的全长c DNA序列,检测了4个马铃薯种质材料在不同模拟干旱(PEG-6000)胁迫强度处理下NCED1基因表达量、ABA含量及根系长度间的相关性。结果表明,St NCED1全长2181 bp,包含一个1800 bp的完整开放读码框,编码599个氨基酸。5%PEG-6000和15%PEG-6000模拟干旱胁迫4周后,4个品种均表现出随胁迫强度的加重植株生长缓慢、矮小,根系长度显著降低。干旱敏感型材料早大白中根系长度变化最大,ABA含量显著高于其他3个种质材料。CIP478.9、star和米拉3份种质材料中St NCED1表达量与对照植株有显著差异,且随胁迫强度增加而增大;早大白中St NCED1表达量表现出先降低后升高趋势。模拟干旱胁迫下,ABA含量与St NCED1表达量之间呈现出正相关关系(R0.7)。研究结果为解析马铃薯响应干旱胁迫的调节机制及其在抗旱种质资源筛选中的应用提供了基础数据。     

茶树CsNCED2基因的克隆和表达分析

该研究以铁观音茶树品种叶片为材料,通过RT-PCR技术,克隆了茶树脱落酸(ABA)合成途径关键限速酶——9-顺式环氧类胡萝卜素裂解双加氧酶(9-cis-epoxycarotenoid dioxygenase,NCED)基因的全长cDNA序列。该基因cDNA全长1 931bp,包含1 821bp完整开放阅读框,共编码606个氨基酸残基。NCBI同源分析结果表明,与葡萄VvNCED2相似性最高(78%),命名为CsNCED2(NCBI登录号:MF765770)。氨基酸序列分析显示,其具有NCED家族的FLNO2258保守结构域,以及MIAHPKxDP和HDFAITE保守结构域序列;在保守区存在4个Fe2+活性组氨酸结合位点,N-端含有叶绿体转运肽。实时荧光定量PCR分析表明,CsNCED2基因在铁观音叶、茎和花中表达量较高;白茶萎凋和乌龙茶做青均可以诱导CsNCED2基因显著上调表达;除干旱胁迫抑制CsNCED2表达外,ABA和低温胁迫均能够诱导CsNCED2基因显著上调表达。表明CsNCED2基因在茶树ABA合成代谢以及胁迫响应中发挥重要作用。     

杧果LEAFY同源基因的分离及序列分析

分析在植物开花过程中起重要作用的LEAFY(LFY)基因的保守区序列,设计1对长度均为23bp的PCR引物,以杧果基因组DNA为模板,采用PCR方法扩增出长为822bp的DNA片段,克隆入pGEM-T Easy载体。测序和序列分析表明,获得了杧果LFY同源基因(miLFY)3’端的1个片段,该片段有1个415bp的内含子,编码区共编码135个氨基酸,其序列已经在GenBank中登记(登录号AY189684)。在GenBank中进行同源性检索,发现其氨基酸序列与其它植物LFY同源基因的氨基酸序列同源性高达74%~97%,推测它们具有相似的功能。     

湖北海棠MhPR1a基因的克隆与表达特性分析

以水杨酸诱导的湖北海棠[ Malus hupehensis (Pamp.) Rehd.]全长cDNA文库和基因组DNA为模板,克隆其PR1a基因(MhPR1a)的全编码区序列,并对该序列进行生物信息学分析;在此基础上利用荧光定量RT-PCR技术对湖北海棠根、茎和叶中该基因的表达特性及经过10μmol·L-1ABA、4℃低温处理及苹果蚜虫(Aphis citricola van der Goot)侵染后叶中该基因的表达特性进行了测定.结果表明:克隆获得的MhPR1a基因全长518 bp,最大开放阅读框为492 bp,编码162个氨基酸残基;编码的蛋白质为酸性蛋白,其相对分子质量为16 960,等电点pI 5.46;其基因组DNA序列与cDNA序列完全一致,说明MhPR1a基因内部没有内含子.湖北海棠MhPR1a基因与苹果(M.domestic Borkh.)和沙梨[Pyrus pyrifolia( Burm.f.)Nakai] PR1基因的cDNA序列及其编码的氨基酸序列同源性均较高,其中cDNA序列的同源性均为97％,氨基酸序列的同源性分别为95％和97％;系统树也显示MhPR1a基因编码的氨基酸序列与苹果和沙梨的亲缘关系最近,聚为一类.MhPR1a基因编码的氨基酸序列具有SCP保守结构域,含有1个信号肽和6个保守的半胱氨酸残基.在湖北海棠的叶、茎和根中MhPR1a基因均能表达,在根中的表达量最高.10 μmol·L-1ABA和4℃低温处理48 h后均可诱导MhPR1a基因的表达,且相对表达量明显高于对照(处理0h);苹果蚜虫也可诱导MhPR1a基因的表达,说明MhPR1a基因在湖北海棠抵抗植食昆虫和低温胁迫的过程中可能发挥着重要作用.     

糜子抗旱节水相关基因PmMYB的克隆及表达分析

根据在糜子抗旱节水分子基础研究中获得的一个糜子MYB基因的EST序列, 以其序列及水稻MYB18基因的序列为基础设计引物, 扩增得到1 739 bp的全长基因组序列。序列分析表明, 其包含121 bp(347~467 bp)和93 bp(599~691 bp)的两个内含子, 3个外显子; 全长cDNA序列为1 525 bp, 其中3′非翻译区为212 bp, 5′非翻译区为41 bp, 编码区为1 272 bp, 共编码424个氨基酸, C-端存在一个丝氨酸(Ser, S)丰富区。该基因具有两个典型的MYB类转录因子基因的DNA结合区(DNA-binding domain), 分别为13~63、66~114位氨基酸, 属于典型的R2R3-MYB转录因子。对其与水稻、玉米、火炬松、拟南芥、辣椒、陆地棉、大麦及茄子等9种植物的MYB基因的R2、R3重复区的氨基酸序列多重比较, 表明R2R3重复序列在植物中具有较高的保守性; 基于氨基酸序列的编码区系统进化树分析表明, 不同植物的MYB基因遗传分化很大, 序列相似性为32%~84%, 其中糜子MYB基因与水稻的MYB18相似程度最高(84%), 与大麦和玉米的相似性分别为46%和41%。通过半定量RT-PCR对其表达模式分析表明, 该基因在水分胁迫和干旱后复水条件下上调表达, 与糜子抗旱节水紧密相关。该基因的克隆为进一步探讨利用该基因改良其他植物的抗旱节水性奠定了良好的基础。     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT-PCR和PCR-RACE技术,从菊科植物甘菊(Dendranthema lavandulifolium)中克隆到2个甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase,BADH)基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152.DlBADH1的cDNA全长1821 bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918 bp,编码506个氨基酸的蛋白质.两个基因核苷酸序列的同源性为97%,推导的氨基酸序列的同源性为98%.与已发表的其它植物BADH基因氨基酸序列的同源性在64%以上.在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C).在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合.RT-PCR-Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员.     

建兰Actin基因cDNA全长的克隆及其表达分析

根据单子叶植物的肌动蛋白基因(Actin)的保守区序列设计引物,采用RT-PCR和RACE技术从建兰(Cymbidium ensifolium)中分离出Actin基因cDNA全长.序列分析结果表明,建兰Actin基因长度为1 434 bp,编码区长度为1 134 bp,编码377个氨基酸,将其命名为CeActin,GenBank登录号为JN613147.CeActin推导的氨基酸序列与其他植物的同源性都较高,具有高度的保守性.采用半定量RT-PCR技术分析CeActin在建兰各组织及花不同发育时期的表达情况,结果表明,表达量没有明显差异,表明CeActin基因可作为内参基因.     

转基因枸杞表达HIV-1壳体蛋白病毒样颗粒疫苗表达载体的构建

利用PCR技术扩增出人免疫缺陷病毒HIV-1MA₄-CA融合基因,将其克隆到pGEM-T载体中,测定其核苷酸序列,并推导其氨基酸序列。该基因全长为450bp,与已发表的HIV-1的全序列基因(AF324493)完全同源,编码一个含150个氨基酸残基的蛋白质。将该基因与分泌型表达载体pCAMBIA1305.2连接,同时将水稻中富含甘氨酸蛋白的信号肽序列(GRP)引入MA₄-CA融合基因,构建了含MA₄-CA基因的植物分泌表达载体pCAMBIA1305.2-MA₄-CA。     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT-PCR和PCR-RACE技术,从菊科植物甘菊(Dendranthema lavandulifolium)中克隆到2个甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase,BADH)基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152。DlBADH1的cDNA全长1821 bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918 bp,编码506个氨基酸的蛋白质。两个基因核苷酸序列的同源性为97%,推导的氨基酸序列的同源性为98%。与已发表的其它植物BADH基因氨基酸序列的同源性在64%以上。在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C)。在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合。RT-PCR-Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员。     

Cloning and characterization of two 9-cis-epoxycarotenoid dioxygenase genes, differentially regulated during fruit maturation and under stress conditions, from orange (Citrus sinensis L. Osbeck)

There is now biochemical and genetic evidence that oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. The peel of Citrus fruit accumulates large amounts of ABA during maturation. To understand the regulation of ABA biosynthesis in Citrus, two full-length cDNAs (CsNCED1 and CsNCED2) encoding NCEDs were isolated and characterized from the epicarp of orange fruits (Citrus sinensis L. Osbeck). Expression of the CsNCED1 gene increased in the epicarp during natural and ethylene-induced fruit maturation, and in water-stressed leaves, in a pattern consistent with the accumulation of ABA. The second gene, CsNCED2, was not detected in dehydrated leaves and, in fruits, exhibited a differential expression to that of CsNCED1. Taken together, these results suggests that CsNCED1 is likely to play a primary role in the biosynthesis of ABA in both leaves and fruits, while CsNCED2 appears to play a subsidiary role restricted to chromoplast-containing tissue. Furthermore, analysis of 9-cis-violaxanthin and 9'-cis-neoxanthin, as the two possible substrates for NCEDs, revealed that the former was the main carotenoid in the outer coloured part of the fruit peel as the fruit ripened or after ethylene treatment, whereas 9'-cis-neoxanthin was not detected or was in trace amounts. By contrast, turgid and dehydrated leaves contained 9'-cis-neoxanthin but 9-cis-violaxanthin was absent. Based on these results, it is suggested that 9-cis-violaxanthin may be the predominant substrate for NCED in the peel of Citrus fruits, whereas 9'-cis-neoxanthin would be the precursor of ABA in photosynthetic tissues.     

Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles,water stress and abscisic acid

Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.     

Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid

The tomato mutant notabilis has a wilty phenotype as a result of abscisic acid (ABA) deficiency. The wild-type allele of notabilis, LeNCED1, encodes a putative 9-cis-epoxycarotenoid dioxygenase (NCED) with a potential regulatory role in ABA biosynthesis. We have created transgenic tobacco plants in which expression of the LeNCED1 coding region is under tetracycline-inducible control. When leaf explants from these plants were treated with tetracycline, NCED mRNA was induced and bulk leaf ABA content increased by up to 10-fold. Transgenic tomato plants were also produced containing the LeNCED1 coding region under the control of one of two strong constitutive promoters, either the doubly enhanced CaMV 35S promoter or the chimaeric 'Super-Promoter'. Many of these plants were wilty, suggesting co-suppression of endogenous gene activity; however three transformants displayed a common, heritable phenotype that could be due to enhanced ABA biosynthesis, showing increased guttation and seed dormancy. Progeny from two of these transformants were further characterized, and it was shown that they also exhibited reduced stomatal conductance, increased NCED mRNA and elevated seed ABA content. Progeny of one transformant had significantly higher bulk leaf ABA content compared to the wild type. The increased seed dormancy was reversed by addition of the carotenoid biosynthesis inhibitor norflurazon. These data provide strong evidence that NCED is indeed a key regulatory enzyme in ABA biosynthesis in leaves, and demonstrate for the first time that plant ABA content can be increased through manipulating NCED.     

Cloning of a 9-<Emphasis Type="Italic">cis</Emphasis>-epoxycarotenoid dioxygenase gene (<Emphasis Type="Italic">SgNCED1</Emphasis>) from <Emphasis Type="Italic">Stylosanthes guianensis </Emphasis>and its expression in response to abiotic stresses

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. Oxidative cleavage of cis-epoxycarotenoids catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is the main regulatory step in the biosynthesis of ABA in higher plants. A NCED gene, SgNCED1, was cloned from the dehydrated leaves of Stylosanthes guianensis. The 2,241-bp full-length SgNCED1 had a 1,809-bp ORF, which encodes a peptide of 602 amino acids. The deduced amino acid sequence of SgNCED1 protein shared high identity with other NCEDs. At the N-terminus of the SgNCED1 located a chloroplast transit peptide sequence. DNA blot analysis revealed that SgNCED1 was a single copy gene in the genome of S. guianensis. The relationship between expression of SgNCED1 and endogenous ABA level was investigated. The expression of SgNCED1 was induced in both leaves and roots of S. guianensis under drought stress. Dehydration and salt stress induced the expression of SgNCED1 strongly and rapidly. The ABA accumulation was coincidently induced with the SgNCED1 mRNA under drought, dehydration and salt stress. The expression of SgNCED1 and ABA accumulation were also induced under chilling condition.     

Regulation and manipulation of ABA biosynthesis in roots

Overexpression of 9-cis-epoxycarotenoid dioxygenase (NCED) is known to cause abscisic acid (ABA) accumulation in leaves, seeds and whole plants. Here we investigated the manipulation of ABA biosynthesis in roots. Roots from whole tomato plants that constitutively overexpress LeNCED1 had a higher ABA content than wild-type (WT) roots. This could be explained by enhanced in situ ABA biosynthesis, rather than import of ABA from the shoot, because root cultures also had higher ABA content, and because tetracycline (Tc)-induced LeNCED1 expression caused ABA accumulation in isolated tobacco roots. However, the Tc-induced expression led to greater accumulation of ABA in leaves than in roots. This demonstrates for the first time that NCED is rate-limiting in root tissues, but suggests that other steps were also restrictive to pathway flux, more so in roots than in leaves. Dehydration and NCED overexpression acted synergistically in enhancing ABA accumulation in tomato root cultures. One explanation is that xanthophyll synthesis was increased during root dehydration, and, in support of this, dehydration treatments increased beta-carotene hydroxylase mRNA levels. Whole plants overexpressing LeNCED1 exhibited greatly reduced stomatal conductance and grafting experiments from this study demonstrated that this was predominantly due to increased ABA biosynthesis in leaves rather than in roots. Genetic manipulation of both xanthophyll supply and epoxycarotenoid cleavage may be needed to enhance root ABA biosynthesis sufficiently to signal stomatal closure in the shoot.     

A novel inhibitor of 9-cis-epoxycarotenoid dioxygenase in abscisic acid biosynthesis in higher plants

Abscisic acid (ABA) is a major regulator in the adaptation of plants to environmental stresses, plant growth, and development. In higher plants, the ABA biosynthesis pathway involves the oxidative cleavage of 9-cis-epoxycarotenoids, which may be the key regulatory step in the pathway catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). We developed a new inhibitor of ABA biosynthesis targeting NCED and named it abamine (ABA biosynthesis inhibitor with an amine moiety). Abamine is a competitive inhibitor of NCED, with a Ki of 38.8 microm. In 0.4 m mannitol solution, which mimics the effects of osmotic stress, abamine both inhibited stomatal closure in spinach (Spinacia oleracea) leaves, which was restored by coapplication of ABA, and increased luminescence intensity in transgenic Arabidopsis containing the RD29B promoter-luciferase fusion. The ABA content of plants in 0.4 m mannitol was increased approximately 16-fold as compared with that of controls, whereas 50 to 100 microm abamine inhibited about 50% of this ABA accumulation in both spinach leaves and Arabidopsis. Abamine-treated Arabidopsis was more sensitive to drought stress and showed a significant decrease in drought tolerance than untreated Arabidopsis. These results suggest that abamine is a novel ABA biosynthesis inhibitor that targets the enzyme catalyzing oxidative cleavage of 9-cis-epoxycarotenoids. To test the effect of abamine on plants other than Arabidopsis, it was applied to cress (Lepidium sativum) plants. Abamine enhanced radicle elongation in cress seeds, which could be due to a decrease in the ABA content of abamine-treated plants. Thus, it is possible to think that abamine should enable us to elucidate the functions of ABA in cells or plants and to find new mutants involved in ABA signaling.     

Overexpressing <Emphasis Type="Italic">SgNCED1</Emphasis> in Tobacco Increases ABA Level,Antioxidant Enzyme Activities,and Stress Tolerance

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme of ABA biosynthesis in higher plants. A NCED gene, SgNCED1, was overexpressed in transgenic tobacco plants which resulted in 51–77% more accumulation of ABA in leaves. Transgenic tobacco plants decreased stomatal conductance, transpiration rate, and photosynthetic rate but induced activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX). Hydrogen peroxide (H₂O₂) and nitric oxide (NO) in leaves were also induced in the transgenic plants. Compared to the wild-type control, the transgenic plants improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl-induced salinity stress. It is suggested that the ABA-induced H₂O₂ and NO generation upregulates the stomatal closure and antioxidant enzymes, and therefore increases drought and salinity tolerance in the transgenic plants.     

Molecular cloning and characterization of a dehydration-inducible cDNA encoding a putative 9-cis-epoxycarotenoid dioxygenase in Arachis hygogaea L.

A rate-limiting step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). Here we present the cloning, characterization of a cDNA from dehydrated peanut (Arachis hygogaea L.) leaves that encodes a putative NCED. The 2486-bp full-length cDNA (designated as AhNCED1), obtained by rapid amplification of cDNA ends (RACE), has an open reading frame of 601 amino acid residues and encodes a protein with a calculated molecular weight of 66.86 kDa and an isoelectric point of 8.39. Sequence analysis shows that the deduced amino acid sequence of AhNCED1 shares high identity with the reported NCED protein sequences. There is a 30-amino-acid chloroplast-targeting peptide at the N-terminus of the AhNCED1 protein predicted by iPSORT algorithm. Semi-quantification by duplex RT-PCR reveals that the expression of AhNCED1 is up-regulated by dehydration and that rehydration represses its expression. The organ specific expression pattern of AhNCED1 has been examined, which indicates its dominant expression in leaves and stems. Molecular analysis of the drought-inducible gene of peanut may be useful to investigate the response of agricultural crops to drought stress.