植物克隆化基因功能分析及蛋白编码基因分离的研究

直到九十年代，以利用拟南芥菜和金鱼草突变体克隆到花器官发育基因为重要标志的植物发育研究取得了重大突破。但由于开花决定过程分子控制机理研究进展却较为缓慢。以mRNA人手利用差异筛选技术获得春化相关基因克隆(verc203，verc17)，Northem blot初步证明这些克隆对春化处理的特异性。根据反义RNA理论和技术对克隆化verc203基因功能进行研究。另外，从纯化蛋白人手，在金针菇中克隆到新的溶血性毒蛋白编码基因，并在细菌中得到表达，这一实验为利用此途径来克隆发育调控蛋白的编码基因进行了有益的技术路线探索。本研究的具体结果如下： 1． 春化相关基因(verc203)对冬小麦花发育调控 根据反义RNA的理论和技术进一步证明verc203在冬小麦开花启动和花发育过程中的可能控制作用。将verc203 DNA序列插入带有CaMV35S启动子和GUS基因的pBI221表达载体， 使之能在植物中高效表达反义RNA，并以转正义基因植株作为对照。通过花粉管途径转基因方法，得到326粒反义质粒冬小麦转化种子和1 98粒正义质粒冬小麦转化种子．所有转化处理种子和正常小麦种子萌发后，经春化处理，与未春化冬小麦植株一起培养直到对照植株达到成熟期。实验观察发现，转反义基因植株的抽穗受到明显的阻抑，开花过程大大推迟。其中表达较强的6株转基因植株甚至晚花五十多天。还发现一些表达较弱的植株尽管晚花20到30天，但花器官发育却受到影响，如，雄蕊缺失，穗发育异常等。Southemblot和PCR实验结果表明外源基因整合到转基因植株基因组。 Northrenblot以及报告基因(GUS)活性等实验证明反义基因在转反义基因植株RNA水平上得到高效表达。同时，GUS基因在蛋白质水平上得到表达。基于上述实验，有理由认为ver203基因可能是控制冬小麦春化诱导的成花启动和花发育过程中的主要基因之一。 2． 金针菇毒蛋白flammutoxin编码基因的克隆及其在细菌中的表达 利用色谱分离技术及SDS．PAGE电泳技术从金针菇(Flammuleina velutips)分离得到一种分子量为31 kDa、对人血红细胞具有溶血活性的蛋白质(flammutoxin)，通过蛋白质序列微量分析技术测得其N-端3 1个氨基酸顺序。利用氨基酸序列推测其编码基因序列信息，通过RT - PCR克隆到一个由973个核苷酸组成的编码基因，5’端93个核苷酸编码肽链序列与测得flammutoxin N-端氨基酸序列完全相同。基因序列同源性分析表明在GenBank (USA)，EMBL Database (Europe)和DDBJ (Japan)基因序列数据库中未找到与之同源的基因序列。这个基因与载体p-半乳糖苷酶部分基因编码的融合蛋白在E coli细胞中有效地得到表达。表达产物的SDS-PAGE，Western blot和溶血活性实验表明它是flammutoxin蛋白原编码基因。并对融合蛋白的溶血活性降低原因作了讨论。

青蒿开花与青蒿素生物合成相关性的研究-金丝桃和百金花二苯甲酮合酶基因的克隆，异源表达及功能分析

第一部分：青蒿开花与青蒿素生物合成相关性的研究 青蒿素是从中药青蒿中分离出的倍半萜内酯化合物，目前是世界上唯一有效的治疗脑型疟疾和抗氯喹恶性疟疾的药物。青蒿植株中青蒿素含量在开花期最高，但是目前尚不清楚开花与青蒿素生物合成的关系。为此，我们用光周期（短日照）诱导青蒿提前开花，不仅同时获得了开花与不开花的青蒿植株，而且还成功地在同一植株上诱导部分分枝开花，另一部分分枝保持营养生长状态。这一实验体系为研究青蒿开花与青蒿素生物合成的相关性奠定了基础。实验结果表明，开花与不开花青蒿植株青蒿素含量有明显差异。开花植株的青蒿素含量在前2周内逐渐提高，第三周（开花期）达到最高，并保持一周左右，在随后的2周内下降。青蒿植株开花后，叶片便开始老化变黄，逐渐死亡。未开花青蒿植株的青蒿素含量动态在前三周内与开花植株类似，但是这种高青蒿素含量状态能保持较长时间，至少在随后的2周内没有下降。未开花植株的叶片依然保持绿色。这一结果表明，开花不是导致青蒿素含量提高的直接原因。 扫描电镜观察结果表明，幼嫩叶片上的毛状腺体( trichrome)结构是完整的，而在老化的叶片上，则观察到了相当比例(40-50%)破损的腺体。这可能是导致青蒿素含量下降的直接原因。 不同生态型青蒿对光周期的反应是不同的。在北京地区，本地青蒿在8月初便开始开花，而来自四川武陵的青蒿则要到9月份才能开花。根据这一特性，采用“南蒿北栽”的方法，能够使青蒿保持较长时间的营养生长状态，延长适于采收的时间。 第二部分：金丝桃和百金花二苯甲酮合酶基因的克隆，异源表达及功能分析 植物次生代谢物山屯酮( Xanthones)仅存在于龙胆科和藤黄科植物中。它们具有抑制单胺氧化酶，细胞毒素及抗肿瘤活性。 含有1 3个碳原子的二苯甲酮是山屯酮生物合成的中间产物，是由二苯甲酮合酶催化合成的，这一反应是山屯酮生物合成的关键步骤。二苯甲酮合酶已经在金丝桃和百金花细胞悬浮培养系统中检测到，并进行了细致的生化水平上的研究。本研究是在上述研究的基础上，进一步克隆该酶的基因，并进行异源表达及功能分析工作，以便更好地了解和调控山屯酮的生物合成。 用PCR和RT-PCR技术，从金丝桃cDNA文库和逆转录产物中分别克隆到一个基因HBPS1和HBPS2，从百金花cDNA文库中克隆到一个基因CBPS1。HBPS1含有1402个碱基，其开放阅读框架编码390个氨基酸，分子量为42.7 kDa，等电点为6.55。HBPS2含有1398个碱基，其开放阅读框架编码395个氨基酸，分子量为42.8 kDa，等电点为5.78。CBPS1含有1383个碱基，其开放阅读框架编码389个氨基酸，分子量为42.7 kDa，等电点为7.88。与GenBank中序列同源性比较结果表明：在氨基酸水平上，HBPS1与茶（Camellia sinensis）查尔酮合酶的同源性高达92%，HBPS2与萝卜（Raphanus sativus）查尔酮合酶的同源性为64%，CBPS1与茶（Camellia sinensis）查尔酮合酶的同源性为71%。HBPS1与HBPS2的同源性仅为62%。 将三个新克隆的基因的ORF整合到载体pGEX-G上的谷胱甘肽还原酶S基因下游，构建成转化质粒，并在大肠杆菌中诱导表达。结果表明，这三个基因的ORF片段均能被表达成约68 kDa的产物，这与期望的结果一致。 活性检测结果表明，HBPS1是查尔酮合成酶，其底物为香豆酰辅酶A和丙二酸单酰辅酶A，对这两种底物的亲和性KM分别为：香豆酰辅酶A 2.8μM，丙二酸单酰辅酶A，11.2μM。最适反应条件是350C，pH7.0，DTT浓度10 μM。 HBPS2是二苯甲酮合酶，其底物是苯甲丙氨酰辅酶A，和丙二酸单酰辅酶A，对这两种底物的亲和性KM分别为：苯甲丙氨酰辅酶A 2.4 μM，丙二酸单酰辅酶A 9.6μM。最适反应条件是350C，pH 6.5，DTT浓度50 μM。而CBPS1则没有检测到任何活性。从同一种植物中同时获得了查尔酮合酶和二苯甲酮合酶，对研究这两种十分相近的酶的差异表达，酶促反应机制等问题将非常有利。

毛白杨（Populus tomentosa）木质素生物合成相关基因分离与木质素合成调节

木质素是植物体中具重要生物功能的次生代谢产物。然而纸浆生产主要是将原料中的木质素与用于造纸的纤维素分离，该工艺过程产生了造纸工业的主要污染废液，并且增加造纸成本。本研究目的在于利用反义RNA技术，在分子水平调节木质素的生物合成，降低中国特有造纸树种毛白杨的木质素含量，培育更适于我国造纸工业的原料树种。以下为本研究已取得的相关研究进展： 1.通过RT-PCR技术，从毛白杨中克隆了木质素生物合成的三个相关酶的cDNAs，它们分别为咖啡酸甲基转移酶(caffeic acid O-methyltransferase，COMT)、咖啡酰CoA甲基转移酶(caffeoyl Co-enzyme A O-methyltransferase，CCoAOMT)及香豆酸：辅酶A连接酶(4-coumarate: CoA ligase，4CL)。序列分析显示了毛白杨这三个基因与杨属中其它种的相应基因cDNA核苷酸序列高度同源。Northern点杂交分析表明，COMT、CCoAOMT及4CL基因在毛白杨正在生长的次生木质部中高水平表达，其表达高峰与树木的木质化进程同步;而在叶与叶柄中，这三个基因均不表达。COMT、CCoAOMT及4CL是木质素生物合成的相关酶，该表达特征与其基因功能相一致。本研究克隆的COMT、CCoAOMT及4CL基因的cDNAs已在GenBank注册登记，接受号分别为AF237777、AF240466、AF314180 (publish on Jan l,2002)。 2.通过一系列的DNA重组，构建了携带反义COMT、CCoAOMT或4CLcDNA的反义表达载体以及同时整合反义COMT与CCoAOMT cDNA的双价反义表达载体，PCR扩增与酶切检测确证构建无误。 3.以田间取材的速生三倍体毛白杨B19、B331及B304的茎尖、叶片与嫩茎为外殖体，首次获得了三倍体毛白杨的组培再生试管苗，并建立了速生三倍体毛白杨的组培再生系统，为通过基因工程改良其造纸性能奠定了基础。 4.农杆菌介导转化烟草，PCR与PCR-Southern检测表明我们获得了整合反义COMT、CCoAOMT cDNA及反义COMT及CCoAOMT cDNA共整合的转基因烟草。以Digoxigenin标记的对应于反义链的单链RNA为探针与转基因烟草的总RNA进行NoIthern点杂交，结果表明整合到其中的反义cDNA均已表达。转基因烟草的木质素分析将有助于对COMT及CCoAOMT两个甲基化酶功能的认识。 5.通过农杆菌介导，将反义CCoAOMT cDNA转入欧洲山杨与银白杨的杂交杨（P tremulaXP.alba）。经PCR，PCR-Southern及Southern检测，确认获得了转基因植株。以Digoxigenin标记的对应于CCoAOMT cDNA反义链的单链RNA为探针与转基因杂交杨总RNA进行Northern点杂交，结果表明整合到其中的反义cDNA已在转录水平表达。测定生长5-6个月的转基因杨树下部茎杆的Klason木质素含量，结果显示其中一个株系的Klason木质素含量比野生型对照下降17.9%，表明抑制杨树内源CCoAOMT基因表达可有效降低转基因植株的木质素含量。

水稻中一个编码富含脯氨酸蛋白基因家族的表达和功能研究

OsPRP1是水稻中特有的，编码一类富含脯氨酸蛋白（proline-rich protein, PRP）的基因家族，该家族有4个成员。在GenBank中，仅找到了一个结构相对接近的玉米PRP蛋白，但是对它的研究仅限于序列上的某些描述，而且在序列上，仍然与这4个OsPRP1蛋白有明显的差别。OsPRP1蛋白明显有别于前人（1999）所描述的4类PRP蛋白。它们是植物中一类新的小分子量的PRP蛋白，仅有一个保守结构域而属于DUF1210蛋白超家族的成员。 四个OsPRP1基因在基因组中紧密串联排列，而且编码区高度保守，基因结构也高度一致，证明它们来源于同一个祖先基因，是通过基因复制的方式产生的。PAML分析也证明它们在进化时间上的相互距离并不远。而在进化过程中由于执行生理功能上的某种重要性，在编码区表现出很的保守性，因而很难在RNA水平和蛋白水平上研究四个OsPRP1基因的表达差异。但是它们在表达调节区（如启动子区）显示出明显的差异。在克隆启动子的基础上，用GUS报告基因的策略，没有检测到它们在拟南芥中的表达活性，说明它们具有一定的种属特异性。而在水稻中，这四个基因的表达显示出了明显的时序和空间差异，既表现出在组织器官及其发育阶段上的特异性和变化，又在一定程度上表现出交叉，出现了功能上的分化和重叠。根据启动子区上的顺式元件，检测了这些基因对6种植物生长调节物质和3种非生物胁迫因子的应答反应，证明它们的表达调节也表现出显著的差异和分化。因此，四个基因的进化出现了功能退化、亚功能化和产生新功能等多种命运，比理论模型预期的要复杂得多，可能在执行生理功能和对环境反应上具有各自的生物学意义。 植物细胞壁是多种碳水化合物和蛋白质相互交织在一起的亲水性网络。在保护和支撑原生质、细胞通信、细胞分化、细胞对生物的和非生物胁迫的抗性等方面发挥着重要作用。目前已知的大多数植物PRP蛋白被描述成细胞壁结构性蛋白。OsPRP1基因家族编码的蛋白质都有一个N-端信号肽，暗示它们可能是一类分泌蛋白。我们用OsPRP1.1::GFP融合蛋白进行了亚细胞定位，证明了OsPRP1蛋白的确也是细胞壁相关蛋白。用原核表达体系表达了GST-融合蛋白，制备抗体，通过免疫印迹证明这些蛋白不溶于温和的提取缓冲溶液中，但可以被高盐和强碱溶液溶解，且分子量增加了三倍。证明在体内可能出现修饰、与细胞壁其它组分相交联的现象。 在这四个基因中，只有OsPRP1.2能够在水稻根中特异表达。根的原位杂交实验证明，OsPRP1在分化成熟程度低的细胞中大量表达，而在分化成熟程度高的细胞中几乎不表达。GUS染色的结果同样发现，基因在维管柱特别是中柱鞘附近的薄壁细胞的表达比别的细胞要强得多，而且在根的生长方向上表现出与发育相关的表达特征。说明OsPRP1基因可能参与了这些细胞的分化、发育过程。而基因表达的组织器官特异性的差异和对不同刺激因素的不同反应意味着它们可能参与了多种生理过程。为此构建了一个RNAi的表达载体，转化水稻，Southern杂交实验得到9个单位点插入的T2代独立株系，其中有6个株系与野生型对照相比，主根的早期伸长受到显著抑制，主根的一级侧根地数量减少，根尖分生区的细胞在轴向上的伸长受到了抑制。结合表达定位和原位杂交的结果，我们对它们在植物生长发育中的功能进行了深入探讨。

转Rubisco 活化酶大型同工酶基因(rca)水稻的光合作用特性研究

Rubisco 是催化光合暗反应第一步反应的酶，是唯一能将CO2 转变成碳水化合物的酶，由它固定和最后转化成的碳水化合物提供了植物、动物和微生物的食物和能量。但是，Rubisco 催化该反应的效率十分低，使之成为光合作用的限速步骤。由于Rubisco 的合成和催化过程十分复杂，人们很难通过直接改造Rubisco 提高植物固定CO2 的能力。而Rubisco 活化酶能活化Rubisco，使植物在生理CO2 浓度下具有最大的CO2 同化速率，因此研究活化酶有重要意义。水稻活化酶有2 个同工酶，大型同工酶比小型同工酶C 端多37 个氨基酸，其中包括两个Cys 残基。这两个Cys 残基的存在使活化酶大型同工酶对ADP 的存在更加敏感，其活性在硫氧还蛋白的介导下能被基质中氧化还原状态的变化所调节。由于活化酶大型同工酶对调节Rubisco 的活性具有的这种特殊作用，在本研究中，将活化酶大型同工酶rca基因用正义和反义引入水稻基因组，获得了过量表达活化酶大型同工酶基因和反义抑制活化酶基因表达的转基因植株，对其光合作用进行了生理和生化分析。 本研究的主要结果如下： Rubisco 活化酶大型同工酶基因的克隆：从水稻镇恢249 中克隆了1525 bp 的活化酶大型同工酶cDNA 序列。经过测序，它与报道的粳稻品种活化酶大型同工酶cDNA 序列（rca）完全相同。 构建了4 个植物表达载体：3 个为过量表达rca的载体，分别是pCBUbirca，pCBSrca 和 pCBSUbirca ，其中rca分别在水稻中高效表达的玉米Ubiquitin 启动子、受光调控的Rubisco 小亚基基因启动子和由这两个启动子构成的双启动子控制下表达; 1 个在Ubiquitin 启动子控制的反义rca载体，即 pCBUbi-antirca。 获得了转化rca的水稻再生植株：用日本晴，台北309，武育梗7 号和籼稻品种培矮64S 水稻成熟种子诱导愈伤组织。用改良的农杆菌浸染法将rca基因转化这些愈伤组织，在潮霉素筛选压力下获得抗性愈伤组织，经过2 天的干燥处理后，转入到含山梨醇的高渗分化培养基上培养，能迅速获得大量的芽和转化体再生植株。 获得了转rca基因的水稻植株：抗性愈伤组织和再生水稻幼苗的叶片经GUS 染色呈蓝黑色。PCR 扩增转基因水稻基因组内的潮霉素基因和rca，大部分转基因水稻中含有841 bp 的潮霉素基因片段和1525 bp 长的rca cDNA 片段。251 粒T1 代转基因水稻种子中189粒呈现潮霉素抗性，抗性种子/非抗性种子的比率约为3：1，接近孟德尔分离规律。Southern杂交表明rca序列已整合到水稻基因组，一般含1－2个拷贝。Western 杂交显示Rubisco 活化酶含量在转pCBUbi －antirca 的水稻中和对照比，几乎看不出，被反义抑制;转pCBUbirca 的水稻与对照含量相差无几;转pCBSUbirca，pCBSrca 载体的水稻中活化酶的含量比对照有极显著的增加。 T1 代转rca水稻的光合作用发生显著变化：转pCBSrca 和pCBSUbirca 的水稻在饱和光强下的Rubisco 初始活性、羧化效率、光合速率都明显高于对照，但是表观量子效率、色素含量和Rubisco 总活性与对照相似。两者相比，前者比后者更高;转反义rca（pCBUbi-antirca）基因的水稻饱和光强下的光合速率、表观量子效率、羧化效率、Rubisco 初始活性明显降低，色素含量和Rubisco 总活性基本不变;转pCBUbirca 的水稻中，光合作用的各项参数与对照基本相似。 T1 代转rca水稻的叶绿素荧光明显改变：转pCBSrca 和pCBSUbirca 的水稻ΦPSII 的值明显高于对照，而且前者qP 的值明显高于对照。两者相比，前者的ΦPSII 和qP 的值比后者高;转反义rca的水稻ΦPSII，F′v/F′m，qP 值和对照比都明显降低，但qN 的值升高;转pCBUbirca 载体的水稻中，叶绿素荧光的各项参数与对照基本相似。 转rca基因的水稻生长发育的变化：转pCBUbirca 载体的水稻整个生长发育过程与对照相似;转化pCBSrca 和pCBSUbirca 载体的水稻和对照比，植株高大，生长发育速度加快，抽穗、开花和结籽的时间提前。两者本身相比，前者比后者明显;转反义rca（pCBUbi-antirca）基因的水稻生长发育延迟，植株矮小，种子败育。 由上可见，Rubisco 活化酶大型同工酶rca基因在Rubisco 小亚基基因启动子、Ubiquitin 基因启动子和Rubisco 小亚基基因启动子共同控制下正义转入水稻的转基因植物光合作用的参数最好，光合效率提高，植物表型最好，生长发育加快，提前开花结籽。这一研究可能为获得高光合效率和高产量的水稻奠定了基础。

蓝藻磷脂磷酸酶基因的缺失对光合作用的影响

磷脂酰甘油(phosphatidylglycerol, PG)是类囊体膜(也叫光合膜)中唯一的一种磷脂。在蓝藻中，PG的合成途径为：磷脂酸(phosphatidic acid, PA)胞嘧啶双磷酸-二酰基甘油 (cytidine diphosphate diacylglycerol, CDP-DAG) 磷酸磷脂酰甘油 (phosphatidylglycerol phosphate, PGP)PG。其中最后一步反应是由PGP去磷酸化而生成PG，催化该反应的是PGP磷酸酶。然而迄今为止，PGP磷酸酶还没有在蓝藻和高等植物中得到克隆和鉴定。本工作在鱼腥藻Anabaena sp. PCC7120中通过将一个可能编码PGP磷酸酶的基因(alr1715)进行突变，获得缺失PG的突变体。与野生型相比，该突变体PG的含量降低了30%左右。突变后的蓝藻藻丝发黄、生长缓慢，叶绿素含量降低。整体细胞的光合作用活性、光系统II(photosystem II，PSII)的放氧活性以及PSII反应中心的光能转化效率显著下降，传递给PSII的激发能减少。

An anionic antimicrobial peptide from toad Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide. named maximin H5. was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2,. Containing three aspartate residues and no basic amino acid residues. maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides. only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5. while the other bacteria] and fungal strains tested ere resistant to it. The presence of metal ions. like Zn2+ and Mg2+, did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians, The results provide the first evidence that. together kith cationic antimicrobial peptides. anionic antimicrobial peptides may also exist naturally as part of the innate defense system. (C), 2002 Elsevier Science (USA). All rights reserved.

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Identification and cloning of a trypsin inhibitor from skin secretions of Chinese red-belly toad Bombina maxima

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K-i value of 0.06 muM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K-i value of 1 muM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions. (C) 2002 Elsevier Science Inc. All rights reserved.

Cloning of novel bombesin precursor cDNAs from skin of Bombina maxima

Amphibian skin is a rich resource of bioactive peptides like proline-rich bombesin from frog Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises proline-rich bombesin and a novel peptide, designated as bombestatin, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of the novel peptide is WEVLLNVALIRLELLSCRSSKDQDQKESCGMHSW, in which two cysteines form a disulfide bond. A BLAST search of databases did not detect sequences with significant similarity. Bombestatin possesses dose-dependent contractile activity on rat stomach strips. The differences between cDNAs encoding PR-bombesin plus bombestatin and PR-bombesin alone are due to fragment insertions located in 3'-coding region and 3'-untranslational region, respectively. (c) 2005 Elsevier B.V. All rights reserved.

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B. maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1: 1 molar ratio. The equilibrium dissociation constants (K-D) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys(53)-Cys(62)), which comprises the scissile bond Arg(58)(P-1)-His(59)(P-1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Maximin 9, a novel free thiol containing antimicrobial peptide with antimycoplasma activity from frog Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides, like maximins and maximin Hs from frog Bombina maxima. Novel cDNA clones encoding a precursor protein, which comprises a novel maximin peptide (maximin 9) and reported maximin H3, were isolated from two constructed skin cDNA libraries of B. maxima. The predicted primary structure of maximin 9 is GIGRKFLGGVKTTFRCGVKDFASKHLY-NH2. A surprising substitution is at position 16, with a free cysteine in maximin 9 rather than usual conserved glycine in other reported maximins. Maximin 9, the homodimer form and its Cys(16) to Gly(16) mutant were synthesized and their antimicrobial activities were evaluated. Unlike previously reported maximin 3, the tested bacterial and fungal strains were resistant to maximin 9, its homodimer and the Cys(16) to Gly(16) mutant (with MICs > 100 mu M). On the other hand, interestingly, while eight clinical Mollicutes strains were generally resistant to maximin 9 homodimer and its Cys(16) to Gly(16) mutant, most of them are sensitive to maximin 9 at a peptide concentration of 30 mu M, especially in the presence of dithiothreitol. These results indicate that the presence of a reactive Cys residue in maximin 9 is important for its antimycoplasma activity. The diversity of antimicrobial peptide cDNA structures encountered in B. maxima skin cDNA libraries and the antimicrobial specificity differences of the peptides may reflect well the species' adaptation to the unique microbial environments. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile(1)-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar raito of 1:58, it augmented the activity of bradykinin up to two-fold. (C) 2004 Elsevier B.V. All rights reserved.

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.

A novel bradykinin-like peptide from skin secretions of the frog, Rana nigrovittata

A bradykinin-like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin-N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Ranakinin-N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration-dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin-N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in the absence of a dibasic site for trypsin-like proteinases and an unusual -AEVA- insertion in the N-terminal part of ranakinin-N. The -AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first reported bradykinin-like peptide with a neutral net charge at the N-terminus. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.