A novel family of antimicrobial peptides from the skin of Amolops loloensis

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Sichuan region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the rufous-spotted torrent frog, Amolops loloensis. Members of the new peptide family named amolopins are composed of 18 amino acids with a unique sequence, for example, NILSSIVNGINRALSFFG. By BLAST search, amolopins did no show similarity to any known peptides. Among the tested microorganisms, native and synthetic peptides only showed antimicrobial activities against Staphylococcus aureus ATCC2592 and Bacillus pumilus, no effects on other microorganisms. The CD spectroscopy showed that it adopted a structure of random combined with beta-sheet in water, Tris-HCl or Tris-HCl-SDS. Several cDNAs encoding amolopins were cloned from the skin cDNA library of A. loloensis. The precursors of amolopin are composed of 62 amino acid residues including predicted signal peptides, acidic propieces, and mature antimicrobial peptides. The preproregion of amolopin precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature amolopins are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. (C) 2008 Elsevier Masson SAS. All rights reserved.

Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates

Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemosensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantly with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.

青稞B组醇溶蛋白基因与上游调控区的克隆及基因表达

高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源，也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白（hordeins），占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点，大麦醇溶蛋白被划分为三种类型：富硫蛋白亚类(B，γ-hordeins)、贫硫蛋白亚类（C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白，它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明，大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H（5）上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白（B-hordein）。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织，探明Hor2位点基因表达的发育调控机制，最终达到改良禾谷类作物籽粒品质的目的，本研究以青藏高原青稞为材料，采用同源克隆法，分别克隆B-hordein基因和启动子，通过原核生物表达验证B-hordein基因功能，并利用实时定量PCR探索B-hordein基因表达时空关系，取得如下研究结果： 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料，根据GenBank中三个B-hordein基因序列（GenBank No. X03103, X53690和X53691）设计一对引物，通过PCR扩增，获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明，所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子，推测这11个克隆可能是假基因。推测的氨基酸序列分析表明，所有大麦B-hordein具有相似的蛋白质基本结构，均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构，更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析，结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族，来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族，表明这两个亚家族的成员存在显著差异。此外，我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征：即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组（除BXQ053，BZ09-1，BZ26-5分别单独聚为一类外）。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类，避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物，以青稞Z09和Z26的基因组DNA为模板，采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列（命名为Z09P和Z26P）。序列分析表明，推测的TATA box位于–80 bp，CAAT–like box位于–140 bp处。此外，Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box，EB)，在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构，预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节，推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中，将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高；3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物，同时，选择大麦α-微管蛋白基因（GenBank no. U40042）为看家基因并设计特异引物，利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达，荧光定量结果显示：两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录，而Z26开花4天后就有低水平B-hordein的表达，这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外，在4个不同的胚乳发育时期中，Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中，Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期，Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053，BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.

海湾扇贝超氧化物歧化酶家族基因结构、表达和多态性分析

海湾扇贝Argopecten irradian Lamarck于1982年从美国引种到中国，由于具有较快的生长速度和很高的经济效益，海湾扇贝成为中国最主要的养殖贝类之一。近年来海湾扇贝养殖遇到了死亡率高等问题，深入开展海湾扇贝功能基因的研究，尤其是免疫相关基因及其机制研究并在此基础上寻找扇贝疾病防治的有效方法对海湾扇贝的健康养殖十分重要。 对于贝类免疫系统来说，其血细胞在先天性免疫防御中起着重要的作用。当受到外界病原侵染时，贝类血细胞的一个重要免疫反应就是吞噬作用。在吞噬病原过程中，受到病原侵染的贝类还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发与呼吸链相耦联的活性氧（ROS）的大量产生。这些活性氧具有极强的反应特性，能破坏病原微生物的结构和功能分子，实现对入侵病原的杀灭。利用活性氧对被吞噬的病原进行杀灭，这是吞噬作用消除病原抵御侵染的重要机制。但由于活性氧分子反应的非特异性，它们也会破坏宿主机体细胞内的功能蛋白分子、不饱和脂肪酸分子和核酸等，对细胞造成严重的伤害，进而导致机体生理机能的损伤和免疫系统的破坏。所以，及时消除病原感染机体内过量产生的ROS，维持相关细胞的正常代谢，对提高机体抵抗力和免疫力具有重要的作用。O2-是生物体内产生的第一种活性氧分子，其他的活性氧分子也是由它衍生而来，消除过量O2-是消除过量活性氧危害的第一步也是关键一步。生物体内，超氧化物歧化酶（SOD）是催化O2-发生歧化反应，消除O2-的关键酶。 首先，本文通过RACE方法获得了海湾扇贝SOD家族全部三种基因的cDNA全长并对其进行了序列的生物信息学分析，海湾扇贝AiCuZnSOD全长cDNA为1047个碱基，其中开放阅读框为459个碱基，编码152个氨基酸，与栉孔扇贝Chlamys farreri的CuZnSOD相似度为77.5%，与长牡蛎Crassostrea gigas的相似度为75%，与人的相似度为74.7%。AiMnSOD全长cDNA为1207个碱基，其中开放阅读框为678个碱基，编码226个氨基酸，序列比对结果发现AiMnSOD的氨基酸序列与虾夷扇贝Mizuhopecten yessoensis和皱纹盘鲍Haliotis discus hannai的相似度分别为85%和78.4%，与哺乳动物相似度也在68%~72%之间。AiECSOD全长cDNA为893个碱基，其中开放阅读框为657个碱基，编码218个氨基酸。AiECSOD与其它物种ECSOD相似度比较低。与线虫Brugia pahangi的相似度为27.9%，与疟蚊Anopheles gambiae的相似度为31.4%，与斑马鱼Danio rerio的相似度为27.8%，与人的相似度也只有28.6%，与同是贝类的长牡蛎ECSOD也只有28.1%的相似性。主要原因是AiECSOD的信号肽和肝磷脂结合区域在各物种中无同源性。 其次，采用qRT-PCR（quantitative real time PCR）方法分析三种SOD基因在不同组织中的表达情况，结果表明三种SOD基因的组织表达有所差异。AiCuZnSOD基因在鳃中表达水平最高，其次是血细胞和性腺，在外套膜、闭壳肌和肝胰脏表达水平较低。AiMnSOD基因在鳃中表达水平最高，其次是外套膜，在血细胞、性腺，而在肝胰脏和闭壳肌表达较弱。AiECSOD基因在血细胞中表达水平最高，其次是肝胰脏，在鳃、闭壳肌表达水平较低，而性腺和外套膜没有检测到。同时，采用qRT-PCR对鳗弧菌Vibrio angullarum感染后海湾扇贝血细胞中三种SOD基因mRNA表达变化进行了检测。AiCuZnSOD表达量在各个时间段没有显著差异(P > 0.05)。AiMnSOD的表达量在1.5 h时略有下降，在3 h时达到最高表达量，是空白组（0h）的3倍(P < 0.01)，从6 h到24 h表达量逐渐下降，24 h时表达量是空白组的1.6倍，24 h到48 h又稍有升高。AiECSOD的表达量在1.5 h时有所下降，是空白组的0.3倍（P < 0.05)，随后逐渐升高，在12 h时达到最高表达量，是空白组（0h）的4.5倍（P < 0.01），从24 h到48 h表达量逐渐下降并恢复到空白组的水平。在对照组，各个时间点没有显著差异（P > 0.05）。在鳗弧菌感染后，海湾扇贝三种SOD的表达并不一致，且差异比较显著。AiCuZnSOD被认为是构成性表达基因，其受外界刺激的影响最小，AiMnSOD和AiECSOD受刺激后表达上调比较明显。 第三，采用Genome-walking的方法得到了海湾扇贝三种SOD基因的基因组全长和近端启动子序列并对其进行了相关分析。AiCuZnSOD的基因组序列全长为4279bp，包含有4个外显子和3个内含子。AiMnSOD的基因组序列全长为10692bp，包含有4个外显子和3个内含子。AiECSOD的基因组序列全长为5276bp，包含有5个外显子和4个内含子。三种基因外显子和内含子的结合处序列遵循-AT/GT-原则。我们把海湾扇贝SOD家族的三个基因的近端启动子进行了比较分析。发现三种SOD在靠近起始密码子的位置都有Oct-1结合位点。三种SOD共有的转录位点有：Oct-1、C/EBPalp、Oct2.1、Sp-1和GATA-1。AiCuZnSOD和AiMnSOD共有的转录位点有：ICSBP、Ftz、TATA-box、C/EBPbeta和Antp。AiCuZnSOD和AiECSOD共有的转录位点有：AP-1和NFκB。AiMnSOD和AiECSOD共有的转录位点有：GR和ER。AiCuZnSOD独有的位点有：SRF、YY-1和NF-1。AiMnSOD独有的位点有：HNF-1、Hb、MEB、NF-muE1、Pit-1a和Eve。AiECSOD独有的位点有：CREB、RATA-alph、Kruppel-like和AP-3。 此外，通过构建原核表达载体，本研究对AiCuZnSOD和AiECSOD基因进行了体外重组表达，并对纯化的重组蛋白进行了酶活分析。酶活分析表明，重组AiCuZnSOD蛋白有较高的酶活和稳定性。 最后，我们对海湾扇贝三种SOD基因的部分区域，包括启动子、编码区，部分内含子区域进行了SNP检测，并对SOD基因部分SNP位点多态性和鳗弧菌敏感性进行了相关分析。三种SOD基因中，我们共发现了59个SNP位点，其中AiECSOD的SNP位点最多，特别是在启动子区，AiCuZnSOD和AiMnSOD多态性较低。其中AiCuZnSOD启动子区的-1739 T-C 位点的基因型和等位基因，AiECSOD启动子区的-498 A-T和-267 G-A等位基因频率，AiECSOD的第一个外显子38 Thr-Lys的多态性在敏感和抗菌群体中存在显著差异（P < 0.05）。

A family of interesting exact solutions of the sine-Gordon equation

By using AKNS [Phys. Rev. Lett. 31 (1973) 125] system and introducing the wave function, a family of interesting exact solutions of the sine-Gordon equation are constructed. These solutions seem to be some soliton, kink, and anti-kink ones respectively for the different choice of the spectrum, whereas due to the interaction between two traveling-waves they have some properties different from usual soliton, kink, and anti-kink solutions.

A scheme for multiple sequences alignment optimization-an improvement based on family representative mechanics features

As a basic tool of modern biology, sequence alignment can provide us useful information in fold, function, and active site of protein. For many cases, the increased quality of sequence alignment means a better performance. The motivation of present work is to increase ability of the existing scoring scheme/algorithm by considering residue–residue correlations better. Based on a coarse-grained approach, the hydrophobic force between each pair of residues is written out from protein sequence. It results in the construction of an intramolecular hydrophobic force network that describes the whole residue–residue interactions of each protein molecule, and characterizes protein's biological properties in the hydrophobic aspect. A former work has suggested that such network can characterize the top weighted feature regarding hydrophobicity. Moreover, for each homologous protein of a family, the corresponding network shares some common and representative family characters that eventually govern the conservation of biological properties during protein evolution. In present work, we score such family representative characters of a protein by the deviation of its intramolecular hydrophobic force network from that of background. Such score can assist the existing scoring schemes/algorithms, and boost up the ability of multiple sequences alignment, e.g. achieving a prominent increase (50%) in searching the structurally alike residue segments at a low identity level. As the theoretical basis is different, the present scheme can assist most existing algorithms, and improve their efficiency remarkably.

Generating artificial homologous proteins according to the representative family character in molecular mechanics properties - an attempt in validating an underlying rule of protein evolution

The molecular mechanics property is the foundation of many characters of proteins. Based on intramolecular hydrophobic force network, the representative family character underlying a protein’s mechanics property is described by a simple two-letter scheme. The tendency of a sequence to become a member of a protein family is scored according to this mathematical representation. Remote homologs of the WW-domain family could be easily designed using such a mechanistic signature of protein homology. Experimental validation showed that nearly all artificial homologs have the representative folding and bioactivity of their assigned family. Since the molecular mechanics property is the only consideration in this study, the results indicate its possible role in the generation of new members of a protein family during evolution.