瘤背石磺肌球蛋白重链(MyHC)基因的克隆与表达分析

肌球蛋白是动物体内重要的功能性马达蛋白,调控机体的信号传导、肌肉收缩和细胞器运动,为探究肌肉在瘤背石磺(Onchidium struma)由海洋向陆地进化过程中发挥作用的分子机制,实验以石磺科4种贝类转录组数据为基础,采用RACE方法从瘤背石磺肌肉中首次克隆到肌球蛋白重链(Myosin heavy chain,MyHC)基因cDNA的全长并进行组织表达分析。研究结果显示,瘤背石磺MyHC基因cDNA全长为7566 bp,包括5895 bp的开放阅读框,228 bp的5'端非翻译区,1443 bp的3'端非翻译区,共编码1964个氨基酸。预测该基因编码的蛋白质由31713个原子组成,分子式为C_(9765)H_(15897)N_(2849)O_(3150)S_(52),分子量约为225.28 kDa,理论等电点为5.56,N端信号肽具有29个氨基酸长度。瘤背石磺MyHC具有2个保守结构域MYSc_class_Ⅱ和Myosin_tail_l,且亲水性氨基酸集中在Myosin_tail_l区。系统进化树分析显示,瘤背石磺MyHC与光滑双脐螺(Biomphalaria glabrata)MyHC的亲缘关系最近。RT-PCR结果显示,MyHC基因在各个组织中均有表达,腹足和背部皮肤表达量最高,腹部皮肤、口球、肺囊中高表达,肝胰腺、蛋白腺、两性腺中微量表达(P0.05)。MyHC基因在瘤背石磺的主要运动器官腹足中高表达,说明肌肉运动对瘤背石磺湿地环境的两栖适应性具有至关重要的作用。实验结果为今后进一步研究瘤背石磺肌球蛋白重链基因进行原核表达及多克隆抗体的制备奠定了良好基础,更为深入探讨海洋无脊椎动物从海洋向陆地进化的研究提供有参考意义的分子依据。     

基于转录组测序对翘嘴鳜(Siniperca chuatsi) 2种肌球蛋白重链基因的克隆与分析

肌球蛋白重链(Myosin heavy chain,MYH)是骨骼肌粗肌丝的重要组成单位,其表达量高低影响肌纤维的组成和肌肉生长.为了解其在翘嘴鳜(Siniperca chuatsi)早期生长过程中的作用,本研究从前期生长差异显著的2组(快长组和慢长组)翘嘴鳜幼鱼转录组差异表达Unigene中筛选出2个MYH基因,RACE (Rapid amplification of cDNA ends)克隆到其全长cDNA(MYH-7a,MYH-7b).MYH-7a全长为6071bp,开放阅读框为5820 bp;MYH-7b全长为5896 bp,开放阅读框为5745 bp.序列分析显示,2个MYH均有Loopl、Loop2环、ATP结合位点等关键结构域;进化树聚类分析显示,MYH-7a与MYH-7b均属于慢肌球蛋白.实时荧光定量PCR验证发现,其在快长组样本中表达量显著高于慢长组,与转录组测序结果一致;检测其在翘嘴鳜心肌、红白肌和皮肤等14种组织的表达水平,结果显示,MYH-7a主要在心肌中表达,而MYH-7b主要在红肌中表达;在胚胎发育不同阶段,二者随着胚胎发育的进行,表达量不断增加;在幼鱼早期生长过程(孵化出膜后15 dph、30 dph和60 dph)的翘嘴鳜白肌中,在15 dph和30 dph快长组的表达量显著高于慢长组,而到60 dph时快长组的表达量均显著低于慢长组.翘嘴鳜MYH-7a、MYH-7b在快长组与慢长组鱼中的差异表达提示它们在翘嘴鳜胚胎及其早期生长发育过程中发挥重要作用.     

cDNA cloning and complete primary structures of myosin heavy chains from brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso fast skeletal muscles

ABSTRACT:   The complete cDNA sequences encoding predominant types of myosin heavy chain (MYH) in the fast skeletal muscle were determined for brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso , which are used as materials for preparing high-quality surimi-based products. The cDNA consisted of 5973 and 5987 bp, respectively, and both encompassed an open reading frame encoding a polypeptide of 1936 amino acid residues. Brushtooth and wanieso lizardfish MYH showed the amino acid sequence identity of 92–93% to white croaker MYH, which was higher than that of 90% to walleye pollack MYH. The putative binding sites for ATP, actin, and regulatory and essential light chains in the subfragment-1 region of brushtooth lizardfish MYH exhibited a high identity with white croaker counterparts as well as the sequences of subfragment-2 and light meromyosin. In contrast, phylogenetic tree, constructed by the neighbor-joining method based on mitochondrial 16S rRNA gene, revealed that the two lizardfish species formed a cluster with walleye pollack, which was paraphyletic with white croaker. Therefore, a good reputation for lizardfish and white croaker to have a high thermal-gel forming ability seemed to be reflected by MYH rather than biological similarity as revealed by the mitochondrial 16S rRNA gene.     

Cloning,characterization and expression of the pepsinogen C from the golden mandarin fish <Emphasis Type="Italic">Siniperca scherzeri</Emphasis> (Teleostei: Perciformes)

Pepsinogens are precursors of pepsins, which are gastric digestive proteinases that degrade food proteins into peptides. In the study reported here, the cDNA and its corresponding genomic DNA of the golden mandarin fish (Siniperca scherzeri, Perciformes) pepsinogen C (PGC) were cloned and sequenced. The golden mandarin fish PGC gene was deduced to have nine exons and eight introns, a structure similar to the PGCs of other vertebrates. The full-length cDNA was found to contain a 37-bp 5′-untranslated region, a 1,164-bp open reading frame, and a 304-bp 3′-untranslated region and the PGC protein to consist of a signal peptide, an activation segment, and a pepsin moiety. A sequence analysis revealed that pairwise sequence similarities of PGC proteins are around 70% between golden mandarin fish and other vertebrate groups, and around 90% within the fish group. A comparison of vertebrate PGC protein sequences revealed two motifs. One was in the activation segment that occurred only in the mammal and avian PGCs, suggesting that PGCs active in homeotherms (mammal and avian) have different activation mechanisms than those in poikilotherms (amphibian and fish). The second was in the pepsin moiety that occurred only in fish, suggesting the primitive position of fish among vertebrates. PGC mRNA is mainly expressed in the stomach and esophagus and at much lower levels in the skin and muscle. Taken together with results reported from other studies, the results reported here will lead to a better understanding of the molecular mechanisms of fish digestive physiology and the evolution of fish pepsinogen genes.     

鳜IRAK4基因的克隆、组织表达及病毒感染后表达分析

为了研究鳜IRAK4生物学特性及其在抗病毒免疫应答中的作用,根据鳜转录组数据中筛选出的IRAK4 unigene序列设计引物,利用SMART-RACE技术克隆得到CDS全长为1389 bp的c DNA(命名为Sc IRAK),编码462个氨基酸,含有1个N端死亡结构域和1个保守的中央蛋白激酶结构域。采用荧光定量RT-PCR方法分析了Sc IRAK4在健康鳜各组织中的表达差异及病毒感染后在脾脏中的表达变化,结果显示,健康鳜中Sc IRAK4在肝脏中表达量最大,与其他组织差异显著,而在血液、脑和胃中表达量最低;传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)感染鳜后Sc IRAK4的表达量呈现下调趋势,24 h脾脏中的表达量达到最低,为对照组的45%;而鳜弹状病毒(siniperca chuatsi rhabdovirus,SCRV)感染鳜后Sc IRAK4的表达量呈现上调趋势,12 h脾脏中Sc IRAK4的表达量达到最高,为对照组的8.17倍,表明Sc IRAK4在抗ISKNV和SCRV的免疫应答中可能发挥不同的作用。本研究为进一步揭示Sc IRAK4的抗病毒免疫反应机制提供了依据。     

鳜免疫球蛋白 D 重链基因的克隆与表达分析

用RACE-PCR和RT-PCR方法获得鳜(Siniperca chuatsi)膜结合型免疫球蛋白D(Membrane-bounded IgD,mIgD)重链基因的全长cDNA序列。鳜mIgD的cDNA全长为3358bp,其5l非编码区包含30bp,3l非编码区包含337bp;开放阅读框包含2991bp,编码996个氨基酸,基因结构为VDJ-μ1-δ1-δ2-δ3-δ4-δ5-δ6-δ7-TM。鱼类IgD恒定区氨基酸序列比对结果显示,鳜mIgD存在半胱氨酸和色氨酸保守位点,与其他鱼类IgD的相似性在37%～72%之间。用邻接法(Neighbor Joining)构建的鱼类免疫球蛋白基因的系统发育树表明,鱼类IgD形成独立的一支,鳜mIgD与牙鲆和庸鲽IgD聚为一支。荧光定量PCR结果显示,鳜mIgD的mRNA主要分布于外周血白细胞、胸腺、头肾、中肾和脾脏中。     

红螯螯虾冷休克蛋白Y-box编码基因的克隆及表达分析

采用RT-PCR和RACE技术,从红螯螯虾(Cherax quadricarinatus)血细胞中分离到冷休克蛋白Y-box编码基因的cDNA序列。结果显示,冷休克蛋白Y-box编码基因的长度为1 733 bp,其中包括82 bp的5'非翻译区、676 bp的3'非翻译区和975 bp的编码序列,可编码324个氨基酸。理论相对分子质量和等电点分别为35 800和9.81。结构域分析和序列比对显示,红螯螯虾冷休克Y-box蛋白由3部分组成:富含丙氨酸或脯氨酸区域、冷休克结构域和带电荷区域,其中冷休克结构域高度保守。系统进化树分析表明,红螯螯虾冷休克Y-box蛋白与水蚤等节肢动物冷休克Y-box蛋白聚类,且三级结构非常相似。最适温度养殖条件下,冷休克蛋白Y-box的虾组织分布特异性研究表明,红螯螯虾神经组织的冷休克蛋白Y-box转录活性最强,而后依次为肝脏、血淋巴和鳃,在肠和心脏中也有活性,在肌肉组织中转录保持较低水平,揭示神经组织是重要的冷休克Y-box蛋白表达区。     

Myofibrillar proteolysis by myofibril-bound serine protease from white croaker <Emphasis Type="Italic">Argyrosomus argentatus</Emphasis>

ABSTRACT:   The modori phenomenon is defined as heat-induced myofibrillar degradation caused by endogenous serine protease(s) of fish muscle during Kamaboko fish meat gel production. This study was undertaken to analyze myofibrillar proteolysis of white croaker Argyrosomus argentatus muscle, which is an ingredient of high quality Kamaboko, by myofibril-bound serine protease (MBSP) under conditions corresponding to the modori phenomenon. White croaker MBSP was stable between pH 2–11 and below 65°C, and about 60% of its initial activity remained after incubation for 2 h under the conditions at 65°C and pH 7.5. About 60% of the enzyme activity was suppressed by 0.5 M NaCl. White croaker MBSP degraded various myofibrillar proteins between 40 and 70°C and pH 6.0–9.0, and preferentially degraded myosin heavy chain rather than other myofibrillar proteins. The enzyme degraded the myosin heavy chain most strongly at 55°C and pH 7.0, and a major part of the bands of myosin heavy chain and its degradation products disappeared for a period of 2 h. These degradation characteristics are very similar to those observed during the modori phenomenon, indicating that MBSP could be a modori-inducing protease involved in the modori phenomenon of white croaker Kamaboko production.     

Myofibrillar proteins in white muscle of the developing African catfish Heterobranchus longifilis (Siluriforms, Clariidae)

Developmental changes in myofibrillar protein composition were investigated in the myotomal muscle of the African catfish, Heterobranchus longifilis (Clariidae), by several electrophoretic techniques. The main muscle fibres of larvae and the fast-white muscle fibres of juvenile and adult fish were found to express distinct myosin heavy chain and myosin light chain 2 (LC2) isoforms. Three myosin LC2 chains were successively detected, differing by their isoelectric points. In contrast, the alkali light chains remained qualitatively and quantitatively unchanged during fish growth. Actin, -tropomyosin, and troponin-C (TN-C) were also similar in larval, juvenile, and adult white muscle, but an additional larval tropomyosin isoform was found in the first developmental stages. Two isoforms of troponin-T (TN-T) and troponin-I (TN-I) were synthesised in the course of fish growth. Transition from the larval to the adult isoform was much faster for TN-T than for TN-I. Slow-red muscle myofibrils from adult H. longifilis showed no common component (except actin) with larval, juvenile, or adult fast-white muscle myofibrils. Red myofibrils displayed a single TN-T and a single TN-I isoform, but two isoforms of TN-C. The myofibrillar protein isoforms synthesised at any given developmental stage almost certainly reflect changes in the functional requirements of swimming muscles in the course of fish development.     

斜带石斑鱼IgM、IgZ和IgD重链基因的克隆

应用RACE方法获得斜带石斑鱼膜结合型免疫球蛋白M(membrane-bound immu-noglobulin M,mIgM),膜结合型免疫球蛋白D(mIgD),分泌型免疫球蛋白Z(secretory immu-noglobulin Z,sIgZ)的重链基因。斜带石斑鱼膜结合型IgM重链恒定区包含3个恒定区结构域(μ1,μ2,μ3)以及两个跨膜外显子(TM1,TM2),TM1外显子与μ3结构域末端相连接。氨基酸序列相似性分析结果显示,斜带石斑鱼mIgM各恒定区与牙鲆mIgM恒定区相似性最高,为53%~78%。mIgD的cDNA全长为3 375 bp,开放阅读框包含3 006 bp,其恒定区由1个μ1外显子,7个δ外显子以及跨膜区组成。斜带石斑鱼IgD恒定区与鳜IgD各恒定区氨基酸序列相似性最高,δ1~δ7的相似性分别为75.5%、75.8%、65.4%、76.6%、88.1%、90.6%、82.8%,TM结构域为82.7%。sIgZ的基因结构与其他硬骨鱼类sIgZ的结构相似,包括4个外显子和3个内含子,内含子长度分别为222、129和458 bp。利用半定量PCR分别检测了这3种基因在斜带石斑鱼各器官/组织中的表达,发现mIgM在头肾、肾脏、脑、脾脏、肠、鳃、心脏和胸腺中均有表达;mIgD的mRNA在头肾、肾脏以及胸腺中有较高的表达,在肠中表达量较低;sIgZ mRNA主要分布于淋巴组织如头肾、肾及脾脏中,而在鳃、心脏和胸腺中的丰度较低。     

Complete nucleotide sequence of a cDNA encoding a myosin heavy chain from mantle tissue of scallop Patinopecten yessoensis

ABSTRACT: It has been reported that the amino acid sequences of striated and catch muscle myosin heavy chains from two scallop species ( Argopecten irradians and Placopecten magellanicus ) are almost identical, but that the ATPase activities between these myosins vary several-fold. These myosin sequences have been useful for identifying the region that modulates the ATPase activity of scallop myosin. In the present study, a cDNA encoding a myosin heavy chain was isolated from the mantle tissue of scallop Patinopecten yessoensis . The cDNA is composed of 6067 base pairs (bp) including an open-reading frame of 5841 p, which encodes an amino acid sequence of 1947 residues. The deduced amino acid sequence of P. yessoensis mantle myosin had a high identity of 90%, 92%, and 91% to P. magellanicus , A. irradians , and Pecten maximus striated muscle myosins, respectively. Interestingly, while the deduced amino acid sequences of around adenosine triphosphate-binding and actin-binding sites of the mantle myosin are homologous to those of A. irradians striated muscle myosin, the subfragment 2 hinge region and the non-helical tail region are similar to those of catch muscle myosin.     

杂交鳜与鳜鱼、斑鳜肌肉营养成分和氨基酸含量比较

通过测定鳜鱼、斑鳜和杂交鳜的肌肉营养成分和18种氨基酸含量，结果表明：杂交鳜、斑鳜和鳜鱼的肌肉水分、脂肪、灰分的含量无显著差异（P〉0．05），杂交鳜与斑鳜的肌肉蛋白质含量无显著差异（P〉0．05），但均显著高于鳜鱼肌肉的蛋白质含量（P〈0．05）；鳜鱼、斑鳜和杂交鳜的18种氨基酸含量及其组成、18种氨基酸总量（TAA）、人体所需8种必须氨基酸总量（HEAA）及4种呈味氨基酸（天门冬氨孽、谷氨酸、甘氨酸和丙氨酸）的总量（FAA）均无显著差异（（P〉0．05）。     

Determination of primary structure of amberjack myosin heavy chain and its relationship with structural stability of various fish myosin rods

The structural stability of fish myosin depends upon species and temperatures of water in which fish live. Primary, secondary, and quaternary structures of myosin heavy chain (MyHC) from three species of fish living at different temperature ranges have been compared with those of rabbit MyHC in order to investigate the differences in stability. Primary structure of MyHC, although being accessible for warm-water and cold-water fish (carp and walleye pollack), was not available in previous for tropical-water fish literature; so in this study primary structure of MyHC of the tropical-water fish amberjack has been determined by cloning and sequencing its cDNA. The MyHC has 1938 amino acid residues (AA), which are almost as much as as those of carp and walleye pollack. The amberjack MyHC is 91–95% homologous with other fish and rabbit MyHCs. There is a discernible difference between animal species with stable myosin rod (amberjack, carp, and rabbit) and walleye pollack with unstable rod. Stable rod species have a high probability of forming coiled-coil around the COOH-terminal end of the rod, while the pollack has a low coiled-coil formation probability. In addition, the average scores of the coiled-coil for myosin rod were rabbit (1.738) > amberjack (1.691) > carp (1.680) > walleye pollack (1.674) which correlated exactly with the observed stability. The results suggest that coiled-coil forming ability, particularly around the COOH-terminal end, directs structural stability of fish myosin rod.     

Seasonal changes in atrophy-associated proteins of the sonic muscle in the big-snout croaker, Johnius macrorhynus (Pisces, Sciaenidae), identified by using a proteomic approach

In most sciaenids, males possess sonic muscles and produce sound through the contraction of these muscles and amplification of the swim bladder. The sonic muscles in some fishes exhibit seasonal changes in size. For example, they are hypertrophic in the spawning season, and atrophic in the non-spawning months. The protein profiles of the sonic muscle, red muscle, and white muscle in the Johnius macrorhynus were shown by two-dimensional electrophoresis (2-DE) and were compared to reveal differential protein expressions. About 80 up-regulated protein spots in the sonic muscle, and 30 spots related to six contractile proteins (fast muscle myosin heavy chain, skeletal alpha actin, alpha actin cardiac, tropomyosin, myosin light chain 2, and myosin light chain 3), four energy metabolic enzymes (enolase, acyl-CoA synthetase, creatine kinase, and cytochrome P450 monooxygenase), and two miscellaneous proteins (DEAD box protein and cyclin H) were identified. Seasonal hypertrophy and atrophy of the sonic muscles related to the reproductive cycle were verified in male big-snout croaker. The contents of some proteins were significantly different in the muscles under these conditions. The levels of cytochrome P450 monooxygenase, fast muscle myosin heavy chain, DEAD box proteins, isocitrate dehydrogenase, and creatine kinase were up-regulated in the hypertrophic muscle, but the levels of alpha actin cardiac, myosin light 2, and myosin light 3 were lower than in the atrophic muscle. Potential reasons for these differences in protein expression related to physiological adaptation are discussed.