生长素结合蛋白基因在黄瓜果实发育中的功能研究

应用逆转录-聚合酶链式反应(RT—PCR),从黄瓜子房(幼果)中扩增出生长素结合蛋白ABP1)cDNA片段。该基因在开花前1天的子房中表达信号较弱,在授粉后2、4和6天的幼果中表达较强;在开花后2天有单性结实能力的子房中表达信号较强,不能形成果实的子房中信号较弱,所以ABP1基因可能参与黄瓜果实的生长发育过程。将拟南芥ABP1基因转入黄瓜中,转基因黄瓜的单性结实率平均为31.7％,高于对照(19．9％)。由于黄瓜的单性结实主要与生长素有关,所以,转基因植株单性结实率的提高可能是由于子房增强了对自身所含生长素的敏感性所致,说明生长素结合蛋白参与生长素在黄瓜果实生长发育中的生理作用。     

甘蔗类异黄酮还原酶(IRL)基因的克隆与表达分析

采用RT-PCR技术从甘蔗中克隆So IRL基因,用生物信息学方法对获得的氨基酸序列进行分析,利用荧光定量PCR技术研究So IRL基因在甘蔗不同组织和不同胁迫条件下的表达特性。结果表明,克隆获得甘蔗So IRL,Gen Bank登录号为KF808324。该c DNA全长1 169 bp,含有1个927 bp的完整开放阅读框(ORF),编码309个氨基酸。系统进化树分析显示,甘蔗So IRL与玉米的IRL蛋白亲缘关系较近。q RT-PCR分析表明So IRL在甘蔗根、茎、叶中均有表达;在RSD病菌及低温(4℃)、聚乙二醇(PEG)、Na Cl和脱落酸(ABA)4种非生物胁迫下均被诱导表达,但表达模式不同。说明该基因可能参与甘蔗应答RSD过程,并可能在非生物胁迫中也发挥了作用。     

生长素结合蛋白基因在黄瓜果实发育中的功能研究

应用逆转录-聚合酶链式反应(RT-PCR),从黄瓜子房(幼果)中扩增出生长素结合蛋白(ABP1)cDNA片段.该基因在开花前1天的子房中表达信号较弱,在授粉后2、4和6天的幼果中表达较强;在开花后2天有单性结实能力的子房中表达信号较强,不能形成果实的子房中信号较弱,所以ABP1基因可能参与黄瓜果实的生长发育过程.将拟南芥ABP1基因转入黄瓜中,转基因黄瓜的单性结实率平均为31.7%,高于对照(19.9%).由于黄瓜的单性结实主要与生长素有关,所以,转基因植株单性结实率的提高可能是由于子房增强了对自身所含生长素的敏感性所致,说明生长素结合蛋白参与生长素在黄瓜果实生长发育中的生理作用.     

葡萄CBF4基因生物信息学及其对低温和硅酸钾响应分析

为探究葡萄CBF4基因的结构和表达特征,该研究以葡萄为材料,对葡萄 CBF4基因进行生物信息学及低温和硅酸钾响应分析。结果表明:(1)CBF4蛋白定位在细胞核,有5个磷酸化位点和14个糖基化位点,无信号肽,是一个亲水的、脂溶性较差的膜外蛋白。二级结构以无规则卷曲为主,比例为56.88%。该蛋白包含一个AP2/EREBP结构域。(2)CBF4蛋白的多序列和系统进化分析表明酿酒葡萄与美洲葡萄的同源性最高、亲缘关系最近。(3)荧光定量 PCR 分析显示,低温胁迫后CBF4基因在葡萄叶片中表达水平上调,说明CBF4基因可能参与了葡萄叶片低温胁迫的响应。低温条件下,施加硅酸钾CBF4基因表达具有差异性,说明该基因在不同的葡萄组织中对硅酸钾的响应机制可能不同。以上结果为进一步研究葡萄CBF4基因的功能和机理奠定了基础。     

黄花棘豆脱水素OoY_2K_4的鉴定及表达分析

该研究以黄花棘豆cDNA为模板,采用同源克隆法,从黄花棘豆转录组数据库中克隆获得1个响应逆境胁迫的胚胎发育晚期丰富蛋白基因,命名为OoY_2K_4;OoY_2K_4基因ORF为786bp,编码261个氨基酸,含有2个保守的Y片段和4个K片段,为典型的Y_2K_4类脱水蛋白亚家族成员;OoY_2K_4蛋白不具有跨膜结构域,不存在信号肽,亲水性极强,含有1个糖基化位点和17个磷酸化位点;亚细胞定位显示,OoY_2K_4蛋白定位于细胞质中。多序列比对发现,OoY_2K_4蛋白与其他物种第二组LEA蛋白(脱水素)序列高度保守;进化树分析显示,该序列与三叶草、蒺藜苜蓿和紫花苜蓿相似度最高,亲缘关系最近。采用qRT-PCR对OoY_2K_4基因在干旱、高盐、低温以及脱落酸、乙烯、赤霉素处理下的表达分析显示,干旱和高盐胁迫可显著诱导OoY_2K_4基因表达,而低温胁迫下基本无变化;激素处理均可诱导OoY_2K_4基因高效表达,其中脱落酸诱导下OoY_2K_4基因表达最显著。研究推测,OoY_2K_4基因可能通过依赖ABA的信号途径参与黄花棘豆对干旱和高盐逆境胁迫的应答反应。     

木薯MeASR基因克隆及表达分析

脱落酸-胁迫-成熟诱导蛋白(Abscisic acid-stress-ripening,ASR)在植物对非生物逆境胁迫的应答过程中发挥着重要作用。利用PCR技术从木薯中克隆了第一个ASR基因Me ASR,序列分析表明该基因开放阅读框(ORF)330 bp,编码109个氨基酸。多序列比对和进化树分析表明该基因所编码的蛋白具有ASR家族蛋白的保守结构域,与番茄ASR家族蛋白Sl ASR4具有较近的亲缘关系。亚细胞定位分析表明Me ASR定位在细胞核,实时荧光定量PCR分析表明该基因的表达显著受渗透胁迫和ABA诱导。结果表明,Me ASR可能作为转录因子参与木薯对干旱逆境胁迫应答及ABA信号调节。     

黑毛雪兔子通气组织发育相关基因ShCTR1的克隆及表达

黑毛雪兔子(Saussurea inversa Raab-Straube)是一种典型的高山植物,具有发达的通气组织。该研究以黑毛雪兔子为材料,利用同源克隆和RACE技术克隆了通气组织形成相关基因(ShCTR1),并对其进行序列分析、系统进化分析、表达分析和亚细胞定位分析,以探讨该基因的功能及其与通气组织的关系。结果表明:(1)成功克隆获得ShCTR1基因cDNA全长为2891 bp(NCBI登录号为:ON081649),包含2550 bp的开放阅读框,编码849个氨基酸,理论等电点5.90,分子式C_(4066)H_(6417)N_(1159)O_(1268)S_(43),为疏水蛋白,无跨膜结构。(2)系统进化树分析显示,黑毛雪兔子ShCTR1与菜蓟(Cynara cardunculus L.)CcCTR1的氨基酸序列相似度最高;非编码区序列分析发现ShCTR1基因含有大量的光响应元件,表明ShCTR1基因可能参与对紫外胁迫的响应。(3)实时荧光定量分析显示,ShCTR1基因在根、茎和叶中均有表达,且在根中的表达量最高;在紫外、低温和低氧胁迫下,ShCTR1基因的表达量均上调,证明ShCTR1基因参与了对以上3种环境胁迫的响应。(4)亚细胞定位显示,ShCTR1主要分布于细胞核。研究推测,黑毛雪兔子ShCTR1基因可能在通气组织的形成以及对逆境胁迫的响应中具有重要作用。     

割手密醛脱氢酶ScALDH基因的克隆与表达分析

该研究从甘蔗细茎野生种(割手密Saccharum spontaneum)中克隆抗旱相关的基因Sc ALDH,并分析其在干旱处理条件下的表达情况和序列特征。利用RT-PCR技术克隆甘蔗的ALDH基因片段,并对其核苷酸和氨基酸序列进行分析。使用NCBI Blastx、ORF finder、Mega、NCBI Conserved Domain Search等程序对其分别进行不同物种氨基酸比较、开放阅读框(ORF)寻找、进化树及保守序列分析,并用Real Time-PCR分析所克隆基因在干旱胁迫前后的表达差异。结果表明:克隆出甘蔗的ALDH基因片段,总长度为1996bp,其中蛋白质编码区(CDS)全长1524bp,编码508个氨基酸;与其它物种ALDH类蛋白氨基酸序列有很高的同源性,有ALDH家族的保守序列,并含有完整的开放阅读框,系统进化树分析显示与玉米的蛋白质亲缘关系最近;Real timePCR数据表明,在干旱胁迫下,随着干旱时间的延长,该基因的表达量呈持续积累的表达模式。总体上来说,该基因对干旱胁迫显著表达。利用RT-PCR技术克隆的甘蔗ALDH基因,属于ALDH蛋白家族的一员,具有其典型的功能域,该基因在干旱胁迫过程中参与抗旱作用。该研究结果为野生种资源开发、优良抗旱亲本选择和培育抗旱性强甘蔗品种提供了参考依据。     

黄瓜生长素结合蛋白cDNA片段的克隆及其表达

以黄瓜子房 (幼果 )RNA为模板 ,应用逆转录 聚合酶链式反应 (RT PCR) ,首次扩增出黄瓜生长素结合蛋白基因 (ABP1)cDNA片段 ,并进行测序和同源性分析。对ABP1基因在黄瓜子房 (幼果 )中的mRNA表达水平作了初步探讨 ,结果表明 ,该基因在开花前 1d的子房中表达信号较弱 ,在授粉后 2、4和 6d的幼果中表达增强 ;开花后 2d未经授粉的子房中 ,绿而膨大、能形成单性结实果者信号较强 ,黄而萎蔫、不能形成果实者信号较弱。Southern杂交结果表明 ,黄瓜生长素结合蛋白为小基因家族编码     

独行菜LaAP2基因克隆、生物信息学分析及表达分析

AP2/ERF是广泛存在于植物中一类重要的转录因子,调控一些参与非生物胁迫相关基因的表达,帮助植物提高逆境胁迫能力。为了深入探讨LaAP2在独行菜耐受低温萌发及幼苗耐受低温生长中的功能,该研究基于前期对独行菜(Lepidium apetalum)转录组数据库分析,克隆获得一个显著上调表达的AP2/ERF家族序列LaAP2。该基因cDNA全长为1 005 bp,编码氨基酸序列包含一个AP2和一个B3结构域,属于AP2/ERF转录因子RAV亚家族。推定的LaAP2蛋白分子量为37.744 67 kD,等电点为9.49。该蛋白氨基酸序列同亚麻荠、拟南芥、油菜等物种显示出较高同源性,系统进化分析结果表明与拟南芥亲缘关系较近。氨基酸序列分析预测表明,LaAP2基因所编码的蛋白不具备信号肽区段,无跨膜区,不属于分泌蛋白,可能为亲水性蛋白;定位于细胞质的可能性为56.5%,定位于细胞核的可能性为21.7%;其主要二级结构元件为无规则卷曲、延伸链、α-螺旋。Real-time PCR分析独行菜幼苗中LaAP2在低温4℃处理下的表达,显示LaAP2表达受低温胁迫呈先下降后升高趋势。这表明LaAP2在独行菜幼苗抵抗低温胁迫中起调控作用。     

滇水金凤花距发育相关基因ABP的克隆及表达分析

为探究滇水金凤(Impatiens uliginosa)ABP基因的结构和表达特征,该研究以滇水金凤为材料,采用RT-PCR 技术对滇水金凤ABP基因进行克隆,运用DNAMAN和MEGA对其所编码的蛋白序列进行同源性分析和系统进化分析,并利用qRT-PCR分析ABP基因的时空表达模式。结果表明:(1)滇水金凤ABP基因的cDNA 全长为627 bp,编码208 aa,命名为IuABP基因,其蛋白具有Cupin超家族蛋白的典型结构。(2)同源性分析表明滇水金凤ABP基因的氨基酸序列与喜马拉雅凤仙花(I. glandulifera)、月季(Rose chinensis)、木薯(Manihot esculenta)等物种的同源性均达71%; 系统进化分析表明IuABP与喜马拉雅凤仙花(Impatiens glandulifera)聚为一支,亲缘关系最近。(3)qRT-PCR分析表明IuABP基因在滇水金凤花距发育的3个时期及2个部位均有表达。随着花距的发育,IuABP基因在滇水金凤花距檐部的表达量呈先下降后上升的趋势,在盛花期时达最高,而在花距距部的表达量逐渐下降。以上结果为进一步研究滇水金凤ABP基因在花距发育中的功能及其表达调控机制提供了一定的理论参考。     

Assignment of the Auxin Binding Abilities of ABP44 in Gel

Several approaches were successfully performed to directly assignand characterize auxin binding of ABP₄₄ in gel. The 44 kDa highaffinity auxin binding protein ABP₄₄ from pea was tested forits ability to bind 5-azido-[7-³H]-IAA in photoaffinity labelingexperiments. Competition experiments with several auxin analoguesconfirm data published previously (Reinard and Jacobsen 1995).Critical reflections of the limitations of the method are alsodiscussed. Immunostaining using the antibody D16 (Napier andVenis 1992), which is directed against the putative bindingsite of ABP1, revealed that ABP₄₄'s auxin binding site is atleast partially related to the corresponding site of ABP1. Nevertheless,both proteins do not share any further immu-nological relationships.Our results with D16 recommend a careful reconsideration ofdata published by other authors. Furthermore, a 80 kDa, dimericglutathione dependent formaldehyde dehydrogenase (FDH) frommung bean, described recently, was found to be different fromABP₄₄. In contrast to the described FDH, ABP₄₄ exhibited noFDH activity. ³Recent address: Zentrum f. Molekularbiologie der Entziindung,Universitat Minister, v.-Esmarch-Str. 56, D-48149 Miinster,F.R.G.     

Cloning and characterization of a gene encoding ABP57, a soluble auxin-binding protein

Auxin-binding protein 57 (ABP₅₇), a soluble auxin-binding protein, acts as a receptor to activate plasma membrane (PM) H⁺-ATPase. Here, we report the cloning of abp57 and the biochemical characterization of its protein expressed in E. coli. The analysis of internal amino acid sequences of ABP₅₇ purified from rice shoots enabled us to search for the corresponding gene in protein DB of NCBI. Further BLAST analysis showed that rice has four abp57-like genes and maize has at least one homolog. Interestingly, Arabidopsis seems to have no homolog. Recombinant ABP₅₇ expressed in E. coli caused the activation of PM H⁺-ATPase regardless of the existence of IAA. Scatchard analysis showed that the recombinant protein has relatively low affinity to IAA as compared to natural ABP₅₇. These results collectively support the notion that the cloned gene is responsible for ABP₅₇.     

Indole-3-lactic acid is a weak auxin analogue but not an anti-auxin

Indole-3-lactic acid (ILA) is a naturally occurring indole derivative, preferably detected in soil bacteria and fungi and only in low amounts in plants. T-DNA gene 5 of Agrobacterium tumefaciens was found to be involved in the synthesis of ILA in transformed plant tissues, but the physiologic relevance for ILA production in plants is unclear. The related molecular structure of ILA to the natural auxin indole-3-acetic acid (IAA) makes ILA a good candidate for an auxin analogue. We examined the possible auxin activity of ILA on elongation, proliferation, and differentiation in Pisum sativum L. Results presented in this paper indicate that there are no or only weak effects of ILA toward the activity of auxins when used in the physiologic concentration range. Furthermore, no antagonistic effects of ILA were found. Biochemical analysis using the equilibrium dialysis binding system resulted in no high affinity ILA binding to an enriched protein fraction containing auxin-binding protein (ABP₄₄), whereas 1-naphthaleneacetic acid exhibited high affinity auxin binding.Abbreviations IAA indoleacetic acid - ILA indole-3-lactic acid - T-DNA transferred DNA - ABP auxin-binding protein - NAA naphthaleneacetic acid - MS Murashige and Skoog - MES 2-(N-morpholino)ethanesulfonic acid - BAP 6-benzylaminopurine     

ABP1: An auxin receptor for fast responses at the plasma membrane

Auxin-binding protein 1 (ABP1) is an auxin receptor for responses not primarily regulated by gene regulation. One fast response is protoplast swelling. By using immunological ABP1 tools we showed that the highly conserved box a is not alone important for auxin binding. Box c is another part of the auxin binding domain.¹ Here we present a novel method to analyze auxin-induced, ABP1-mediated effects at the plasma membrane on single cell level in vivo. The fluorescence of FM4-64 in the plasma membrane is reduced by auxin and this response is mediated by ABP1. This method indicates a functional role of ABP1 at the plasma membrane.Key words: Auxin-binding protein 1, auxin, receptor, protoplast, plasma membrane, FM4-64     

Altered Expression of Auxin-binding Protein 1 Affects Cell Expansion and Auxin Pool Size in Tobacco Cells

Auxin-binding protein 1 (ABP1) has an essential role in auxin-dependent cell expansion, but its mechanisms of action remain unknown. Our previous study showed that ABP1-mediated cell expansion is auxin concentration dependent. However, auxin distribution in plant tissue is heterogeneous, complicating the interpretation of ABP1 function. In this study, we used cells in culture that have altered expression of ABP1 to address the mechanism of ABP1 action at the cellular level, because cells in culture have homogeneous cell types and could potentially circumvent the heterogeneous auxin-distributions inherent in plant tissues. We found that cells overexpressing ABP1 had altered sensitivity to auxin and were larger, with nuclei that have undergone endoreduplication, a finding consistent with other data that support an auxin extracellular receptor role for ABP1. These cells also had a higher free auxin pool size, which cannot be explained by altered auxin transport. In cells lacking detectable ABP1, a higher rate of auxin metabolism was observed. The results suggest that ABP1 has, beyond its proposed role as an auxin extracellular receptor, a role in mediating auxin availability.     

Molecular analysis of auxin-specific signal transduction

The auxin-binding protein (ABP1) of maize has been purified, cloned and sequenced. Homologues have been found in a wide range of plants and at least seven ABP sequences from four different species are now known. We have developed a range of anti-ABP antibodies and these have been applied to analysis of the structure, localization and receptor function of ABP. ABP1 is a glycoprotein with two identical subunits of apparent M _r =22 kDa. The regions recognised by our five monoclonal antibodies (MAC 256–260) and by polyclonal antisera from our own and other laboratories have been specified by epitope mapping and fragmentation studies. All polyclonal anti-ABP sera recognise two or three dominant epitopes around the single glycosylation site. Two monoclonals (MAC 256, 259) are directed at the endoplasmic reticulum (ER) retention sequence KDEL at the C-terminus. Early biochemical data pointed to six amino acids likely to be involved in the auxin binding site. Inspection of the deduced sequence of ABP1 showed a hexapeptide (HRHSCE) containing five of these residues. Antibodies were raised against a polypeptide embracing this region and recognised ABP homologs in many species, suggesting that the region is highly conserved. This is confirmed by more recent information showing that the selected polypeptide contains the longest stretch of wholly conserved sequence in ABP1. Most strikingly, the antibodies show auxin agonist activity against protoplasts in three different electrophysiological systems-hyperpolarization of tobacco transmembrane potential; stimulation of outward ATP-dependent H⁺ current in maize; modulation of anion channels in tobacco. The biological activity of these antibodies indicates that the selected peptide does form a functionally important part of the auxin binding site and strongly supports a role for ABP1 as an auxin receptor. Although ABP contains a KDEL sequence and is located mainly in the ER lumen, the electrophysiological evidence shows clearly that some ABP must reach the outer face of the plasma membrane. One possible mechanism is suggested by our earlier demonstration that the ABP C-terminus recognised by MAC 256 undergoes an auxin-induced conformational change, masking the KDEL epitope and it is of interest that this C-terminal region appears to be important in auxin signalling [22]. So far we have been unable to detect the secretion of ABP into the medium of maize cell (bms) cultures reported by Jones and Herman [7]. However, recent silver enhanced immunogold studies on maize protoplasts have succeeded in visualizing ABP at the cell surface, as well as auxin-specific clustering of the signal induced within 30 minutes. The function of ABP in the ER, as well as the mechanisms of auxin signal transduction both at plasma membrane and gene levels remain to be elucidated.     

Expression and Subcellular Localization of Arabidopsis thaliana Auxin-Binding Protein 1 (ABP1)

Expression of Arabidopsis thaliana ABP1 (AUXIN-BINDING PROTEIN 1) was studied using a promoter:GUS approach. Two promoter regions were analyzed. The 1585-bp promoter region upstream of the translation start site (P _ABP1 ) showed different activity compared to the promoter region that included, in addition, the first two introns and three exons of the transcribed ABP1 sequence (P _ABP1i1,2), indicating that cis elements were present downstream of the start codon. P _ABP1i1,2-driven β-glucuronidase activity was highest in growing leaves, in the root meristem, in vascular tissues, and in hydathodes. ABP1 promoter activities overlapped largely but not completely with that of DR5, which is a marker for the ARF-AuxRE-dependent auxin response. Subcellular ABP1 localization was studied using a constitutively overexpressed EGFP-ABP1 fusion protein. Results confirmed predominant localization to the endoplasmic reticulum as was concluded previously.     

Does Auxin Binding Protein 1 Control Both Cell Division and Cell Expansion?

The Auxin-Binding Protein 1 (ABP1) was identified over 30 years ago thanks to it''s high affinity for active auxins. ABP1 plays an essential role in plant life yet to this day, its function remains ‘enigmatic.’ A recent study by our laboratory shows that ABP1 is critical for regulation of the cell cycle, acting both in G₁ and at the G₂/M transition. We showed that ABP1 is likely to mediate the permissive auxin signal for entry into the cell cycle. These data were obtained by studying a conditional functional knock-out of ABP1 generated by cellular immunization in the model tobacco cell line, Bright Yellow 2.Key Words: auxin responses, auxin-binding protein 1, immunomodulation, cellular immunisation