大熊猫生长激素受体(GHR) cDNA 的克隆与序列分析

根据已报道的若干物种GHR 基因cDNA 序列设计引物, 利用RT- PCR 技术首次从大熊猫肝脏组织总RNA中扩增出GHR 基因编码区全长cDNA 序列, 克隆于pGEM®-T 载体后进行测序和序列分析。结果表明,大熊猫GHR 的ORF为1 917 bp , 编码638 个氨基酸的前体蛋白, 由18 个氨基酸的信号肽和620 个氨基酸的成熟肽组成,与人、狗、猪GHR 结构相似, 大熊猫GHR 成熟肽由246 个氨基酸的胞外区、24 个氨基酸的跨膜区和350 个氨基酸的胞内区组成, 并具GHR 的特征性结构。序列相似性比较显示, 大熊猫GHR 与哺乳类GHR 具有69 %～93 %的高序列相似性, 与爬行类和鸟类的序列相似性也达到60 % , 而与鱼类的序列相似性较低, 仅为30 %左右。与其它哺乳动物GHR 相比, 大熊猫GHR 在氨基酸序列上也存在明显的特异性。     

三角帆蚌GPX基因cDNA全序列的克隆及其编码蛋白的结构分析

根据本实验室构建的三角帆蚌cDNA文库中已标注的EST序列, 利用cDNA末端快速扩增法(RACE)克隆了三角帆蚌(Hyriopsis cumingii)谷胱甘肽过氧化物酶(Glutathione peroxidase, GPX)基因cDNA全序列。序列分析表明, 该基因cDNA序列全长1286 bp, 包括5′端非翻译区(Untranslated Region) 39 bp、3′端非翻译区659 bp和开放阅读框(Open reading frame, ORF)588bp, 共编码195个氨基酸, 分子量约为22.2 kDa, 理论等电点为8.44, 属于含硒类GPX。该氨基酸序列具有GPX所有亚型中均高度保守的3个环状结构, 对酶的三级结构起稳定作用。在线分析结果表明: GPX的氨基酸序列不存在明显的疏水区, 也不存在信号肽序列。氨基酸相似性对比结果显示, 三角帆蚌GPX氨基酸序列与脊椎动物GPX-2及GPX-1的序列相似度较高, 为73.1%-80.8%, 与其他型GPX相似度较小, 相似度低于60%。构建的系统进化树显示三角帆蚌GPX与其他几种鱼类GPX聚为一类, 与其他已发表的几种软体动物GPX相距较远, 推测本实验克隆的三角帆蚌GPX基因和已发表的软体动物不属于同一种GPX类型。     

3种不同食性淡水鱼类谷胱甘肽S-转移酶Pi、Mu、Theta型cDNA序列的克隆分析及表达

鱼类谷胱甘肽S-转移酶(glutathione S-transferase,GST)是鱼类一种重要的Ⅱ相去毒酶,在催化毒素与还原谷胱甘肽(GSH)加合去毒代谢过程中具有关键作用。采用RT-PCR及RACE法,分离、克隆得到草鱼、尼罗罗非鱼pi、mu、theta型GST(GSTpi、GSTmu、GSTtheta)基因、鲢鱼GSTmu、GSTtheta基因的cDNA部分序列并推测各自对应的氨基酸序列。氨基酸序列同源性比较和系统进化分析均表明,鲢鱼、草鱼、尼罗罗非鱼与鱼类GST同源性较高,与哺乳类、鸟类、两栖类GST同源性较低,可能与鱼类GST基因在水环境毒素去毒代谢中承担的特殊功能有关。而不同种鱼类GSTtheta的同源性明显要较GSTpi、GSTmu的同源性低,可能与不同淡水鱼类食性及对毒素耐受性不同有关。用实时荧光定量PCR(RT-PCR)检测三种鱼肝脏中三型GST基因组成型表达水平,发现三种鱼各型之间皆有一定差异,尼罗罗非鱼肝脏整体GSTs基因表达很低,GSTtheta显著低于草鱼(P<0.05),GSTmu显著低于鲢鱼(P<0.05)。本研究为从分子水平上研究不同型谷胱甘肽S-转移酶基因在不同食性淡水鱼类体内代谢去毒过程中的作用提供了基础。     

中华鲟阿黑皮素原cDNA 序列克隆及其序列分析

通过构建中华鲟(Acipenser sinensis)垂体的SMART cDNA 质粒文库, 首次从文库中筛选得到阿黑皮素原(Proopiomelanocortin, POMC) cDNA 全长序列, 分别为阿黑皮素原A 型基因 (Proopiomelanocortin I,AsPOMC-A, EV824935) 和阿黑皮素原B 型基因(Proopiomelanocortin II, AsPOMC-B, EV825368)。 其中,AsPOMC-A 和AsPOMC-B 分别在文库中出现128 次和19 次。AsPOMC-A 全长1122 bp, 有792 bp 的开放阅读框, 共编码264 个氨基酸。 AsPOMC-B 全长1216 bp, 有792 bp 的开放阅读框, 共编码264 个氨基酸。以其氨基酸序列为分子标记, 比较了3 种鲟鱼的共6 种POMC 的氨基酸同源性, 并利用已报道的鱼类的POMC氨基酸序列构建了系统发育树, 可识别2 个大的单系类群, 即类群I: 非新鳍亚纲类群(仅包含辐鳍亚纲的长吻雀鳝), 类群II: 新鳍亚纲类群。AsPOMC 可能是一种进化上更原始的基因。       

版纳微型猪近交系CATSPER3基因克隆、序列及发育进化分析

获得版纳微型猪近交系(BMI)CATSPER3基因部分编码区序列,通过生物信息学分析其氨基酸序列和进行不同物种间的同源性比较。以版纳微型猪近交系的公猪睾丸为材料提取RNA,RT-PCR方法扩增CATSPER3基因部分编码区序列,利用在线分析软件进行生物信息学分析。结果显示,扩增出CATSPER3基因部分编码区序列。生物信息学分析表明,推导氨基酸序列,编码蛋白分子量为19.397 3 k D,理论等电点为5.05;在氨基酸组成上,酸性氨基酸(Asp+Glu)有24个、碱性氨基酸(Lys+Arg)有16个,其中以亮氨酸(Leu)占13.3%、缬氨酸(Val)占9.6%、苏氨酸(Thr)占9.0%、苯丙氨酸(Phe)占8.4%、谷氨酸(Glu)占7.2%,天冬氨酸(Asp)占7.2%等含量较高。在核苷酸相似度上与普通猪相似度最高,与山羊核苷酸的相似度较低;分子系统进化树表明与非近交系猪同处于一个分支中,亲缘关系较近,与山羊和绵羊亲缘关系较远。     

人参euFUL基因的克隆与生物信息学分析

旨在研究人参(Panax ginseng C.A.Meyer)花的形成,根据GenBank数据库中基因片段设计特异引物,利用cDNA快速末端扩增方法(RACE),克隆了人参花中一个5'端部分缺失euFUL(命名为PgFu L)的编码区序列。获得的Pg FUL基因由867个核苷酸组成,编码212个氨基酸。生物信息学分析结果显示,PgFUL编码蛋白分子质量为24.64 k D,等电点p I5.80,不含信号肽,无跨膜区。二级结构中α-螺旋结构占60.19%、延伸链占5.21%、无规则卷曲占34.60%。序列比对和系统进化分析表明,PgFUL人参PgMADS protein3(BAK20018.1)的序列同源性为100%,与加拿大蝙蝠葛(AGX01589.1)相似度达95%,属于A功能基因中的eu FUL进化系。     

黄颡鱼MHC class II基因全长的克隆及饲料维生素D3对其组织表达的影响

为进一步丰富鱼类MHC class II基因的研究, 同时也为进一步探讨低磷饲料中添加维生素D₃对鱼类免疫功能可能的影响, 实验利用RACE (Rapid-amplification of cDNA ends) 即cDNA末端快速扩增技术, 成功克隆出黄颡鱼(Pelteobagrus fulvidraco)主要组织相容性复合体(Major histocompatibility complex, MHC) class II基因, 全长1074 bp, 其中ORF (Open reading frame)708 bp, 编码236个氨基酸, 5′UTR (5′端非翻译区)78 bp, 3′UTR (3′端非翻译区)259 bp。进行氨基酸序列比对分析得到: 黄颡鱼MHC class II基因ORF氨基酸序列与长吻逘(Leiocassis longirostris)的氨基酸序列相似度最高为69.5%, 与锦鲤(Cyprinus carpio)的氨基酸序列相似度最低为50.4%。利用qPCR对黄颡鱼MHC class II基因进行组织表达分析, 结果表明MHC class II在小肠、肝脏、鳃中表达较高; 在肌肉、鳍条中表达较低; 而在肾、脾脏、脑、头肾中表达量极低(几乎检测不到)。在低磷饲料中添加维生素D₃显著诱导了该基因的上调表达。研究结果展示了黄颡鱼MHC class II基因的分子结构、组织表达以及维生素D₃的作用, 在降低磷排放的同时, 为今后黄颡鱼免疫抗病及分子选育等方向的深入研究及免疫型饲料的使用奠定了基础。     

一种新的甘露糖结合凝集素--朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列.该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517 bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白.成熟蛋白由109个氨基酸残基组成,分子量为11.9kD.成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4%、85.3%、80.7%和83.5%的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY).     

葡萄风信子谷胱甘肽S转移酶基因克隆与表达分析

以亚美尼亚葡萄风信子为材料,利用本课题组前期获得的葡萄风信子转录组数据库,根据已经获得的葡萄风信子GST基因片段,通过PCR技术克隆得到葡萄风信子GST基因的c DNA序列,命名为Ma GST。Ma GST的c DNA全长为711 bp,开放阅读框为666 bp,编码221个氨基酸,推测蛋白质分子量为54.1 k D,理论等电点p I为5.13。利用生物信息分析软件对Ma GST基因进行同源性比对和系统进化分析表明,结果显示该基因编码的氨基酸具有谷胱甘肽S转移酶典型的C端与N端双结构域,属于GST Tau家族蛋白;与洋葱和小麦GST基因的一致性分别为76.72%和62.50%。实时定量PCR结果显示Ma GST在葡萄风信子各组织器官表达强度相似,属于组成型表达;水杨酸(SA)可以明显诱导Ma GST表达,Ma GST对氯化钠(Na Cl)应答不是很明显,甚至表达量有稍微的下调。     

大眼长蝽卵黄原蛋白基因克隆、序列分析及表达研究

旨在为研究大眼长蝽(Geoco ris pallidipennis)卵黄原蛋白(Vitellogenin,Vg)分子特性及其基因生理功能。利用RT-PCR、RACE及ELISA方法对大眼长蝽Vg基因进行克隆、序列分析和表达研究。该基因c DNA全长5 667 bp(Gen Bank登录号:KP688587),编码1 848个氨基酸残基,N-末端的前19个氨基酸为信号肽。氨基酸序列中有两个保守的多聚丝氨酸区域和RXXR酶切位点,接近C-末端有GLAG基序,其后有5个保守的半胱氨酸残基,DGYR基序位于GLAG上游18个氨基酸残基处。该基因编码氨基酸序列与其它半翅目昆虫Vg氨基酸序列相似度较高,氨基酸序列分析显示它有Vg的典型特征,表明克隆的c DNA序列是大眼长蝽的Vg基因序列。ELISA检测发现随着发育时间的延长,卵黄原蛋白表达量逐渐增加,羽化后22 d达到高峰,随后开始下降,结果表明大眼长蝽雌虫卵黄原蛋白的表达量与大眼长蝽产卵量紧密相关。     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

Molecular and comparative analyses of type IV antifreeze proteins (AFPIVs) from two Antarctic fishes, Pleuragramma antarcticum and Notothenia coriiceps

Antifreeze protein type IV (AFPIV) cDNAs and genomic DNAs from the Antarctic fishes Pleuragramma antarcticum (Pa) and Notothenia coriiceps (Nc) were cloned and sequenced, respectively. Each cDNA encoded 128 amino acids, with 94% similarity between the two and 83% similarity with AFPIV of the longhorn sculpin, Myoxocephalus octodecemspinosus. The genome structures of both genes consisted of four exons and three introns, and were highly conserved in terms of sequences and positions. In contrast, the third intron of PaAFPIV had additional nucleotides with inverted repeats at each end, which appeared to be a MITE-like transposable element. Comparative analysis revealed that fish AFPIVs were widely distributed across teleost fishes, well conserved in their intron positions, but more variable in intron sequences and sizes. However, the intron sequences of two Antarctic fishes were highly conserved, indicating recent radiation of notothenioids in the evolutionary lineage. The recombinant PaAFPIV and NcAFPIV were expressed in E. coli, and examined antifreeze activity. PaAFPIV and NcAFPIV gave ice crystals with star-shaped morphology, and thermal hysteresis (TH) values were 0.08°C at the concentration of 0.5mg/ml.     

Alignment of protein sequences using secondary structure: a modified dynamic programming method

A method for comparison of protein sequences based on their primary and secondary structure is described. Protein sequences are annotated with predicted secondary structures (using a modified Chou and Fasman method). Two lettered code sequences are generated (Xx, where X is the amino acid and x is its annotated secondary structure). Sequences are compared with a dynamic programming method (STRALIGN) that includes a similarity matrix for both the amino acids and secondary structures. The similarity value for each paired two-lettered code is a linear combination of similarity values for the paired amino acids and their annotated secondary structures. The method has been applied to eight globin proteins (28 pairs) for which the X-ray structure is known. For protein pairs with high primary sequence similarity (greater than 45%), STRALIGN alignment is identical to that obtained by a dynamic programming method using only primary sequence information. However, alignment of protein pairs with lower primary sequence similarity improves significantly with the addition of secondary structure annotation. Alignment of the pair with the least primary sequence similarity of 16% was improved from 0 to 37% 'correct' alignment using this method. In addition, STRALIGN was successfully applied to seven pairs of distantly related cytochrome c proteins, and three pairs of distantly related picornavirus proteins.     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板, 采用PCR技术, 扩增气溶素基因(aerA)的DNA片段, 将其克隆到pMD18-T载体上。通过序列测定, 分析结果表明：所克隆的1393 bp片段为aerA部分序列, 编码产生464个氨基酸。BZ与NK之间aerA核苷酸同源性为97.6%, 氨基酸同源性为98.3%, 与其它分离物核苷酸同源性为71.6%~97.5%, 氨基酸同源性为68.0%~98.9%。利用邻接法构建了aerA分子树状图, 树状图分析表明：气单胞菌属各分离物聚为三支, 其中嗜水气单胞菌各菌株之间关系密切, 被聚类为同一支。     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板,采用PCR技术,扩增气溶素基因(aerA)的DNA片段,将其克隆到pMDl8-T载体上.通过序列测定,分析结果表明:所克隆的1393 bp片段为aerA部分序列,编码产生464个氨基酸.BZ与NK之间aerA核苷酸同源性为97.6%.氨基酸同源性为98.3%,与其它分离物核苷酸同源性为71.6%～97.5%,氨基酸同源性为68.0%～98.9%.利用邻接法构建了aerA分子树状图,树状图分析表明:气单胞菌属各分离物聚为三支,其中嗜水气单胞菌各菌株之间关系密切,被聚类为同一支.     

黄河裸裂尻鱼细胞色素C氧化酶Ⅰ、Ⅱ和Ⅲ亚基基因的克隆及序列特征分析

采用RT-PCR技术克隆获得了黄河裸裂尻鱼(Schizopygopsis pylzovi)CO Ⅰ、Ⅱ、Ⅲ基因的编码序列,并对此进行了初步分析.结果表明,黄河裸裂尻鱼CO I基因全长为1 551 bp,开放阅读框(ORF)由基因全长组成,编码516个氨基酸;COⅡ基因全长为691 bp,开放阅读框为690 bp,编码2...     

Cytosolic glutathione S-transferases of Oesophagostomum dentatum

Oesophagostomum dentatum stages were investigated for glutathione S-transferase (GST) expression at the protein and mRNA levels. GST activity was detected in all stages (infectious and parasitic stages including third- and fourth-stage larvae of different ages as well as males and females) and could be dose-dependently inhibited with sulfobromophthalein (SBP). Addition of SBP to in vitro larval cultures reversibly inhibited development from third- to fourth-stage larvae. Two glutathione-affinity purified proteins (23 and 25 kDa) were detected in lysates of exsheathed third-stage larvae by SDS-PAGE. PCR-primers were designed based on peptide sequences and conserved GST sequences of other nematodes for complete cDNA sequences (621 and 624 nt) of 2 isoforms, Od-GST1 and Od-GST2, with 72% nucleotide similarity and 75% for the deduced proteins. Genomic sequences consisted of 7 exons and 6 introns spanning 1296 bp for Od-GST1 and 1579 and 1606 bp for Od-GST2. Quantitative real-time-PCR revealed considerably elevated levels of Od-GST1 in the early parasitic stages and slightly reduced levels of Od-GST2 in male worms. Both Od-GSTs were most similar to GST of Ancylostoma caninum (nucleotides: 73 and 70%; amino acids: 80 and 73%). The first three exons (75 amino acids) corresponded to a synthetic prostaglandin D2 synthase (53% similarity). O. dentatum GSTs might be involved in intrinsic metabolic pathways which could play a role both in nematode physiology and in host-parasite interactions.     

Insular Freshwater Fish Faunas of Micronesia: Patterns of Species Richness and Similarity

Freshwater fishes are an important but relatively little known component of the highly diverse fish fauna of Micronesia. Localities supporting communities of freshwater fishes include large high islands, with considerable habitat complexity, and smaller low islands, such as atolls and raised coral islands, with limited freshwater habitat. Both types of islands may support species with adult life history styles that are (a) amphidromous and catadromous, (b) euryhaline (often estuarine), or (c) marine species which enter freshwater from time to time. We compared patterns of species richness and similarity between Micronesian localities for amphidromous and catadromous, euryhaline and marine species (ACEM) pooled, and for amphidromous and catadromous species (ACFW). Species richness of both ACEM and ACFW fishes was greatest on larger high islands compared to smaller high and all low islands. Cluster analysis of similarity indices for ACEM species between localities revealed two faunal components: high islands and low islands. High islands were further partitioned into a Caroline Islands cluster and a separate Mariana Islands cluster. Cluster analysis of ACFW species was more complex. One cluster consisted of a low island and a small high island, both in the Carolines chain and with limited freshwater habitat. The second cluster was partitioned into high islands and low islands that reflected influences of both size and geographical location.     

Characterization of erythrocyte carbonic anhydrase in an ancient fish,the longnose gar ( Lepisosteus osseus)

This study investigates the evolutionary history of vertebrate red blood cell carbonic anhydrase (CA) by characterizing the isozyme properties and nucleotide sequence of an ancient fish, the longnose gar ( Lepisosteus osseus). The inhibitor sensitivities of gar rbc CA closely resembled those for mammalian CA II, as well as those for CAs from more recently evolved fishes. The kinetic properties of gar rbc CA were not closely aligned with either mammalian CA I and CA II, but fit well into an emerging phylogenetic pattern for early vertebrates. Gar rbc CA cDNA was also amplified from mRNA using 5' and 3'-RACE and the open reading frame consisted of 786 bp. This sequence shares approximately 65% identity with the nucleotide and amino acid sequences of both mammalian CA I and CA II. When the amino acid sequences within the active site are compared, gar rbc CA differs from mammalian CA I, CA II and CA VII by 9, 4 and 3 of the 36 amino acids, respectively. Phylogenetic analyses suggest that gar rbc CA diverged before the amniotic CAs (CA I, CA II and CA III), but after CA V and CA VII.