Isolation, characterization, and localization of AgaSGNH cDNA: a new SGNH-motif plant hydrolase specific to Agave americana L. leaf epidermis

GDSL and SGNH hydrolases are lipases involved in a wide range of functions, behaving in many cases as bifunctional enzymes. In this work, the isolation and characterization of AgaSGNH, a cDNA encoding a member of the SGNH-hydrolase superfamily from young leaf epidermis of the monocot Agave americana L., is reported. The protein possesses a typical signal peptide at its N-terminus that allows its secretion to the epidermis cell wall, as verified by immunolocalization experiments. In addition, the AgaSGNH sequence contains a His-Leu-Gly-Ala-Glu (HLGAE) motif which is similar to that observed in other plant acyltransferases. Expression levels by northern blot and in situ localization of the corresponding mRNA, as well as the immunolocalization of the protein in Agave young leaves indicate that the protein is specifically present in the epidermal cells. The detailed study performed in different parts of the Agave leaf confirms two aspects: first, the expression of AgaSGNH is limited to the epidermis, and second, the maximum mRNA levels are found in the epidermis of the youngest zones of the leaf which are especially active in cutin biosynthesis. These levels dramatically decrease in the oldest zone of the leaf, where the presence of AgaSGNH mRNA is undetectable, and the biosynthesis of different cuticle components is severely reduced. These data could be compatible with the hypothesis that AgaSGNH could carry out both the hydrolysis and the transfer, from an activated acyl-CoA to a crescent cutin in Agave americana leaves and, therefore, be involved in the still unknown mechanism of plant cutin biosynthesis.     

Molecular cloning of a cDNA fromBrassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis ofBrassica napus genomic DNA revealed the presence of 6 genes, 3 derived from theBrassica rapa parent and 3 fromBrassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.     

一种水稻酰基辅酶A结合蛋白cDNA的鉴定(英文)

对一水稻ｃＤＮＡ 克隆（Ｒ１９０８） 的分析表明, 其可能编码水稻酰基辅酶Ａ 结合蛋白（ａｃｙｌＣｏＡｂｉｎｄｉｎｇ ｐｒｏｔｅｉｎ,ＡＣＢＰ）。Ｓｏｕｔｈｅｒｎ 杂交显示水稻（ Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．） 基因组中仅有一个该基因的拷贝。Ｎｏｒｔｈｅｒｎ 分析表明水稻的ＡＣＢＰ基因在水稻的根、茎、叶、叶鞘、黄化苗和幼穗中皆表达,而以黄化苗的绿苗叶鞘中的表达强度高于绿苗叶片。     

Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product

Until recently, only cytosolic acyl-CoA binding proteins (ACBPs) have been characterized. The isolation of an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP that accumulates in developing seeds, designated ACBP1, has provided evidence for the existence of membrane-associated forms of ACBPs (Chye, 1998, Plant Mol. Biol. 38, 827-838). We now report on the isolation of its corresponding gene from an A. thaliana Columbia genomic library using the ACBP1 cDNA as a hybridization probe. Nucleotide sequence analysis of Arabidopsis ACBP1 showed that its promoter lacks a TATA box, resembling the promoters of rat, Drosophila and human genes encoding cytosolic ACBP and suggesting that it is a housekeeping gene. We show by Western blot analysis that ACBP1 expression in developing seeds coincides with lipid deposition and that homologues of membrane-associated ACBP1 exist in other plants. Using light microscopy, we show that ACBP1 is strongly expressed in the embryo at the cotyledons, hypocotyl, procambium of the axis and in most peripheral cells of the cotyledons and hypocotyl. Immunogold labelling localized ACBP1 to vesicles, to the plasma membrane especially at epidermal cells of heart, torpedo and cotyledonary stage embryos, and to the cell wall of the outer integument cells at the seed coat. Our results suggest that ACBP1 is involved in intermembrane lipid transport from the ER via vesicles to the plasma membrane where it could maintain a membrane-associated acyl pool; its immunolocalization to the cell wall of outer integument cells at the seed coat suggests a role in cuticle and cutin formation.     

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein in the silkworm,Bombyx mori

We have cloned the full length of a novel cDNA named Bombyx mori cuticle protein that contains an AlaAlaProAla/Val-repeat (BMCPA) from a cDNA library of integument in the larval silkworm. Both a typical tandem repeat (A-A-P-A/V) for cuticle protein and a unique tandem repeat with Ser, Ala, Gly, Pro, Val, Tyr and Thr were observed in the predicted amino acid sequence of the cDNA encoding BMCPA. Approximately 80% of the amino acids in BMCPA were composed of Ser, Ala, Gly, Pro, Val and Tyr. Northern-hybridization analysis indicated that BMCPA mRNA is expressed only in the larval epidermis and that the expression pattern of the BMCPA gene in the developmental stage was observed mainly at the larval stage. We propose BMCPA may be a novel component of cuticle, and may play an important role in the integument of the larval silkworm.     

Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain

Acyl-CoA binding proteins (ACBPs) are small (ca. 10 kDa) highly-conserved cytosolic proteins that bind long-chain acyl-CoAs. A novel cDNA encoding ACBP1, a predicted membrane protein of 24.1 kDa with an acyl-CoA binding protein domain at its carboxy terminus, was cloned from Arabidopsis thaliana. At this domain, ACBP1 showed 47% amino acid identity to Brassica ACBP and 35% to 40% amino acid identity to yeast, Drosophila, bovine and human ACBPs. Recombinant (His)6-ACBP1 fusion protein was expressed in Escherichia coli and was shown to bind 14[C]oleoyl-CoA. A hydrophobic domain, absent in the 10 kDa ACBPs, was located at the amino terminus of ACBP1. Using antipeptide polyclonal antibodies in western blot analysis, ACBP1 was shown to be a membrane-associated glycosylated protein with an apparent molecular mass of 33 kDa. The ACBP1 protein was also shown to accumulate predominantly in siliques and was localized to the seed within the silique. These results suggest that the biological role of ACBP1 is related to lipid metabolism in the seed, presumably in which acyl-CoA esters are involved. Northern blot analysis showed that the 1.4 kb ACBP1 mRNA was expressed in silique, root, stem, leaf and flower. Results from Southern blot analysis of genomic DNA suggest the presence of at least two genes encoding ACBPs in Arabidopsis.     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein from the Chinese oak Silkmoth, Antheraea pernyi.

In our research to identify gene involved in the cuticle protein, we cloned a novel cuticle protein gene, ApCP13, from the Chinese oak silkmoth, Antheraea pernyi, larvae cDNA library. The ApCP13 gene encodes a 120 amino acid polypeptide with a predicted molecular mass of 13 kDa and a pI of 4.01, and is intron-less gene. The ApCP13 contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the ApCP13 cDNA is most homologous to another wild silkmoth, A. yamamai CP12 (86% protein sequence identity), followed by Bombyx mori LCP18 (35% protein sequence identity). Northern blot analysis revealed that the ApCP13 showed the epidermis-specific expression. This is the first report of cuticle protein gene in the wild silkmoth, A. pernyi.     

Purification and sequence determination of a yellow protein from sexually mature males of the desert locust, Schistocerca gregaria

A yellow protein from abdominal cuticle of the desert locust, Schistocerca gregaria, has been purified and its amino acid sequence determined. The yellow color comes from bound carotene, the protein is only deposited in the epidermis and cuticle of male locusts during their sexual maturation, and the deposition is dependent upon a sufficiently high titer of juvenile hormone. The sequence of the protein is atypical for a cuticular protein, but it has some similarity to a putative juvenile hormone binding protein from Manduca sexta. It is suggested that the protein is involved in the transport of carotenes from internal tissues to epidermis and cuticle of the locust.     

A cDNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in Helicoverpa armigera: molecular characterization and expression analysis associated with pupal diapause

The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9-10 kDa highly conserved multifunctional protein that plays important roles in GABA(A) receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera.     

人类新基因ACBP5cDNA的克隆及其同源物在小鼠/鸡胚发育过程中的表达

利用电子克隆的方法寻找具有重要结构域的人类新基因ACBP5 ,根据得到的序列信息用RT PCR的方法获得全长基因 .通过生物信息学方法预测其结构 ,采用整体原位杂交和组织RT PCR的实验方法 ,在小鼠和鸡胚胎实验模型中研究该基因在发育过程中的表达情况 ,并对其功能进行初步的预测 ,获得一个含有乙酰辅酶A结合蛋白 (acyl CoAbindingprotein ,ACBP)结构域的人类新基因ACBP5 .ACBP5基因的cDNA长度为 10 83bp ,生物信息学方法预测其定位在人第 1号染色体上 ,包含 7个外显子 ,6个内含子 ,包含一个 35 4bp的完整阅读框架 ,编码一个 118个氨基酸残基的蛋白 .在以小鼠胚胎和鸡胚为模型的整体原位杂交中 ,以ACBP5基因全长编码区为探针的结果均显示该基因在胚胎头部特异表达 ,并且主要集中在中脑与间脑之间的峡部 .成体小鼠的组织RT PCR的结果显示 ,ACBP5的同源基因在各组织中均有表达 .这提示ACBP5基因在不同物种中的表达可能比较保守 ,并与头部发育有密切关系 ,同时也对维持细胞的正常功能起到重要的作用 .     

A water-soluble protein specific to the adult cuticle in Tenebrio: Its use as a marker of a new programme expressed by epidermal cells

The water-soluble proteins of abdominal cuticle of larvae, pupae and adults subjected to electrophoresis, revealed a specific pattern for each stage. An antibody against an adult specific 18 kdaltons protein was produced. Electrophoresis analysis of SDS slab gel and Western-blots revealed that the 18 kdaltons adult protein is not present in the water-soluble fraction from haemolymph or fat body of pupae which secreted pre-ecdysial adult cuticle. Immuno-histochemical methods showed that the cross-reaction of the immune serum was limited to the apical part of the adult epidermis and to the cuticle lamellae which are not stabilized. Therefore, the 18 kdaltons protein is specific for the adult cuticle and originates in the epidermis.     

Ethylene- and pathogen-inducible Arabidopsis acyl-CoA-binding protein 4 interacts with an ethylene-responsive element binding protein

Six genes encode proteins with acyl-CoA-binding domains in Arabidopsis thaliana. They are the small 10-kDa cytosolic acyl-CoA-binding protein (ACBP), membrane-associated ACBP1 and ACBP2, extracellularly-targeted ACBP3, and kelch-motif containing ACBP4 and ACBP5. Here, the interaction of ACBP4 with an A. thaliana ethylene-responsive element binding protein (AtEBP), identified in a yeast two-hybrid screen, was confirmed by co-immunoprecipitation. The subcellular localization of ACBP4 and AtEBP, was addressed using an ACBP4:DsRed red fluorescent protein fusion and a green fluorescent protein (GFP):AtEBP fusion. Transient expression of these autofluoresence-tagged proteins in agroinfiltrated tobacco leaves, followed by confocal laser scanning microscopy, indicated their co-localization predominantly at the cytosol which was confirmed by FRET analysis. Immuno-electron microscopy on Arabidopsis sections not only localized ACBP4 to the cytosol but also to the periphery of the nucleus upon closer examination, perhaps as a result of its interaction with AtEBP. Furthermore, the expression of ACBP4 and AtEBP in Northern blot analyses was induced by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, methyl jasmonate treatments, and Botrytis cinerea infection, suggesting that the interaction of ACBP4 and AtEBP may be related to AtEBP-mediated defence possibly via ethylene and/or jasmonate signalling.     

Recombinant expression,purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae

Biotechnology Letters - To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA...     

The acyl-CoA binding protein is required for normal epidermal barrier function in mice

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ～50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.     

商陆抗病毒蛋白cDNA的克隆,测序及其植物表达载体的构建

商陆抗病毒重【'1（m）卜。。）（1。Ill'l－。l。。111。。。""。',NP）是从美洲商陆叶片。I。分或Utoot件如VR,分子量约30hi）L'。PAP属典型的中链核栩然大活主l'I,对动物病毒和植物病;每均计）'们的抗病;沙作用','－。在国外,PAP达国已被川于如N...加'1物从因?..     

Arabidopsis Acyl-CoA-Binding Protein ACBP2 Interacts With an Ethylene-Responsive Element-Binding Protein,AtEBP, via its Ankyrin Repeats

Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.     

藏东南色季拉山薄毛海绵杜鹃叶解剖结构特征与环境适应性

以藏东南色季拉山9个不同海拔梯度的薄毛海绵杜鹃(Rhododendron aganniphum var.schizopeplum)叶片为试验材料,采用石蜡切片技术,测定10项叶片解剖结构指标,应用叶片解剖结构指标的可塑性指数和相关性分析方法探索薄毛海绵杜鹃对藏东南色季拉山强紫外辐射和高寒环境的适应性。结果表明:(1)薄毛海绵杜鹃叶片为异面叶,上表皮有明显的角质层,下表皮有表皮毛,栅栏组织细胞2～3层。(2)随着海拔的升高,叶片角质层厚度、上下表皮厚度、栅栏组织厚度、海绵组织厚度、叶片厚度呈现明显增大趋势,而组织结构紧密度和疏松度变化不显著,主脉突起度呈现下降趋势。(3)各项叶片解剖结构指标的可塑性指数显示,薄毛海绵杜鹃在解剖结构上表现出较小的可塑性,对外界环境的适应能力较弱。(4)依据薄毛海绵杜鹃各项叶片解剖结构指标的相关性分析结果,除海绵组织厚度与栅栏组织厚度、下表皮厚度与上角质层厚度之间相关性不显著外,其余各指标之间均呈显著相关关系,且叶片的解剖结构指标方面也存在明显的协同进化现象。研究发现,藏东南色季拉山薄毛海绵杜鹃通过增加叶片角质层厚度、表皮厚度和叶肉厚度等解剖结构指标的方式增强对外界极端环境的适应能力,从而有利于其在恶劣的高山生境下生存繁衍,使该物种成为生态位理论中的广幅种。