野生和养殖裂腹鱼血液学指标的比较研究

对齐口裂腹鱼和重口裂腹鱼的野生和养殖个体血液学指标进行了比较研究.结果显示:重口裂腹鱼野生组的红细胞长径和短径、红细胞核长径和短径、嗜中性粒细胞长径和短径、淋巴细胞长径和短径、总蛋白和甘油三酯与齐口裂腹鱼野生组相应指标差异显著;齐口裂腹鱼野生组的嗜中性粒细胞长径、嗜中性粒细胞短径、淋巴细胞长径、淋巴细胞短径和淋巴细胞核长径显著大于养殖组;重口裂腹鱼野生组的红细胞短径明显小于养殖组重口裂腹鱼;齐口裂腹鱼野生组血液中TP、GLU和TG显著低于齐口裂腹鱼养殖组;重口裂腹鱼野生组TG、T-CHO与养殖组重口裂腹鱼差异显著.这些差异说明齐口裂腹鱼和重口裂腹鱼在生理适应方面存在差异,养殖活动对它们的生理状况有一定的影响.     

齐口裂腹鱼PGC-1a基因编码区的克隆及其在肌肉组织中的表达

目的：获得齐口裂腹鱼过氧化物酶体增殖物激活受体Y辅助活化因子1a（PGC-1a）cDNA序列,探讨其在齐口裂腹鱼肌肉组织中的表达规律。方法：根据斑马鱼基因PGC-1a序列设计引物,提取齐口裂腹鱼肌肉组织总RNA,经RT-PCR扩增PGC-1a仪基因序列;利用半定量RT-PCR分析齐口裂腹鱼PGC-1a基因在红肌和白肌中的mRNA表达特性。结果：获得齐口裂腹鱼PGC-1a基因序列2633bp,GenBank登陆号为JNl95738,其中ORF为2631bp,编码876个氨基酸残基,与同鲤科的斑马鱼、草鱼和金鱼的同源性较高,为88％～93％,但与哺乳动物和禽类如人、小鼠、大鼠、猪、牛和鸡的同源性较低,为49％～50％。在基础状态下,PGC-1a在齐口裂腹鱼红肌中的表达显著高于白肌,禁食可显著诱导PGC-1a在红肌和白肌中的表达水平。结论-首次克隆得到齐口裂腹鱼PGC-1a基因序列,其在红肌中的表达显著高于白肌中,禁食可显著诱导其在红肌和白肌中的表达,为研究PGC-1a在齐口裂腹鱼肌肉中的作用提供了理论依据。     

基于mtDNA Cyt b序列分析齐口裂腹鱼群体遗传多样性

研究测定了长江上游4个齐口裂腹鱼(Schizothorax prenanti)野生群体(重庆巫溪、重庆城口、四川雅安、四川阿坝)共104个个体的线粒体Cyt b基因部分序列, 以探讨齐口裂腹鱼野生群体的遗传多样性和遗传结构。结果表明: 在104个个体Cyt b序列中共检测到43个多态性位点, 25个单倍型。4个齐口裂腹鱼群体的单倍型多样性介于0.704—0.884, 核苷酸多样性介于0.007—0.012。群体间Kimura双参数遗传距离介于0.008—0.017, 其中四川雅安群体与四川阿坝群体间遗传距离最近, 基因交流频繁。重庆城口群体与四川雅安群体间遗传距离最远, 基因交流受阻。AMOVA分析表明, 齐口裂腹鱼的遗传分化主要来自群体内部, 且组群间、组群内群体间和群体内存在显著的遗传分化。中性检验得到Tajima’s D和 Fu’s F_s的值不显著, 且歧点分布图呈多峰, 表明长江上游4个齐口裂腹鱼野生群体未经历过种群扩张。研究旨为齐口裂腹鱼野生资源保护提供必要参考意见, 同时为齐口裂腹鱼种质资源合理开发和利用提供理论依据。     

齐口裂腹鱼在低照度下的趋光行为

以野生亚成体齐口裂腹鱼为实验对象,进行了光色选择实验和趋光性实验,旨在找到异齿裂腹鱼的偏好光色和光强。实验变量设置5种光色(红、黄、蓝、绿和黑暗)及3种流速工况(0、0.15和0.30 m·s-1)。在光色选择实验中,研究了齐口裂腹鱼在静水、光强照度为10 lx下不同光环境下的偏好行为。结果表明:齐口裂腹鱼对5种光色的喜好顺序为绿色蓝色黑色红色黄色,且在绿光和蓝光区域中的分布率和选择指数显著大于红光和黄光区域(P0.05)。在趋光性实验中,研究了光源光照强度均为20 lx,不同水流流速(0、0.15和0.30 m·s-1)时齐口裂腹鱼的趋光性,发现:无论在静水还是动水工况下,齐口裂腹鱼均对蓝光和绿光表现为正趋光性,对红光和黄光表现为负趋光性;齐口裂腹鱼属弱趋光性鱼类,且其趋光性不随水流的增大而增大,趋光阈值为1.40~2.65 lx。依据实验结果,我们建议在工程中采用光强照度为20 lx的绿光进行诱集齐口裂腹鱼。     

齐口裂腹鱼和鲤鱼H-FABP基因的克隆及其表达谱

心型脂肪酸结合蛋白（heart fatty acid binding protein, H-FABP）的水平与影响肉质性状的肌内脂肪含量有关,鱼类H-FABP的表达水平对其肌内脂肪含量是否相关仍未见报道.本研究获得齐口裂腹鱼和鲤鱼心脏型脂肪酸结合蛋白基因序列,利用半定量RT-PCR分析其表达特性并测定肌内脂肪含量,比较H-FABP基因在不同生活环境的2种鲤科鱼肌内脂肪沉积中的作用.结果显示,齐口裂腹鱼和鲤鱼H-FABP基因的ORF为402 bp,编码133个氨基酸,它们的氨基酸序列相同,与人、猪、小鼠、斑马鱼、大西洋鲑、虹鳟等的同源性为71.3%~ 90%;H-FABP基因在2种鲤科鱼的心、肌肉、脂肪、肝、脑、脾、肾和鳃等组织中均有表达,肝中的表达量显著高于其它组织（P<0.05）,H-FABP基因的肌肉表达谱在齐口裂腹鱼和鲤鱼中存在明显差异：齐口裂腹鱼中的表达随生长发育呈上升趋势,在大体重鱼（500 g）中的表达显著高于小体重鱼（P<0.05),其表达与肌内脂肪含量呈显著正相关（R=0.370,P<0.05）;H-FABP基因在鲤鱼生长发育中呈下降趋势,而小体重鱼（50~60 g）中的表达显著高于其它大体重鱼(P<0.05),其表达与肌内脂肪含量呈显著负相关（R=-7.083,P<0.01）.据此推测,齐口裂腹鱼和鲤鱼肌肉组织H-FABP基因表达与肌内脂肪关联性的差异可能与2种鱼的生活环境不同有关.     

黄芪多糖对齐口裂腹鱼生长、体组成和免疫指标的影响

试验研究黄芪多糖对齐口裂腹鱼生长性能、体组成及免疫指标的影响。以450尾健康的齐口裂腹鱼[体重(6.98±0.43)g;体长(9.11±0.25)cm]为试验对象,随机分为5组(C1、C2、C3、C4、C5),每组3个重复,每重复30尾试验鱼。C1、C2、C3、C4、C5组分别投喂在等氮等能(蛋白质含量38.29%,能量15.73 mJ/kg)的基础料中分别添加0、0.02、0.04、0.06、0.08%的黄芪多糖制成5种试验饲料,养殖齐口裂腹鱼50d。结果表明:饲料中未添加黄芪多糖组的增重率(WGR)、特定生长率(SGR)、饲料蛋白效率(PER)均显著低于黄芪多糖添加组(P<0.05),而饵料系数(FCR)则显著高于黄芪多糖添加组(P<0.05)。当黄芪多糖添加水平为0.04%时,试验鱼的WGR、SGR、PER均达到最大(分别为110.31%、1.86%/d和182.07%),FCR最低(1.44),与其他各组差异显著(P<0.05);以WGR、SGR、PER、FCR为指标,利用直线和抛物线回归分析表明,齐口裂腹鱼生长性能最佳时黄芪多糖添加水平为0.045%—0.074%;对齐口裂腹鱼机体组成分析表明,黄芪多糖对鱼体粗灰分和水分影响不显著(P>0.05),黄芪多糖添加水平为0.06%时机体粗蛋白最高,但与黄芪多糖添加水平为0.04%时无明显差异(P>0.05);粗脂肪含量在黄芪多糖添加水平为0.04%时最高,与其他各组差异显著(P<0.05);未添加黄芪多糖组血清免疫酶活性显著低于黄芪多糖添加组,齐口裂腹鱼血清免疫酶活性在一定范围内随黄芪多糖的增加而增强。黄芪多糖添加水平为0.04%时,碱性磷酸酶(ALP)活性最高;酸性磷酸酶(ACP)活性在黄芪多糖添加水平0.06%时最大;黄芪多糖添加水平应在0.06%—0.08%时,溶菌酶(LSZ)、超氧化歧化酶(SOD活性趋于稳定。这说明黄芪多对齐口裂腹鱼的生长和免疫力有明显的促进作用。综合考虑齐口裂腹鱼生长性能和免疫能力最佳时黄芪多糖添加水平为0.04%—0.074%。     

齐口裂腹鱼CDH5基因特性及其对温和气单胞菌感染的应答

探讨齐口裂腹鱼(Schizothorax prenanti)血管内皮黏附分子(Cadherin 5,CDH5)的基因特性。用生物信息学软件分析齐口裂腹鱼CDH5基因序列,用Real-time PCR检测CDH5基因在齐口裂腹鱼感染温和气单胞菌后0 h、24 h和48 h的表达变化。获得CDH5基因全长c DNA序列,Gen Bank登录号为KT329441。该序列长4 825 bp,开放阅读框长2 313 bp,编码770个氨基酸。蛋白预测结果显示,该蛋白相对分子量为85.19 k D,等电点为5.01。存在信号肽序列,二级结构以随机卷曲、延伸链、α-螺旋为主。齐口裂腹鱼CDH5氨基酸序列与斑马鱼同源性达74%,与人的相似性为43%。CDH5在感染温和气单胞菌后在各组织中均有表达。脾脏中CDH5基因在24 h表达量显著高于0 h和48 h(P0.05)。肝脏、肾脏和肌肉中CDH5在48 h的表达量均显著高于24 h(P0.05)。心脏和肠道中CDH5基因在48 h的表达量显著高于0 h(P0.05)。CDH5基因在可能参与了齐口裂腹鱼抗温和气单胞菌感染的免疫应答,为深入研究CDH5在齐口裂腹鱼中的功能奠定基础。     

齐口裂腹鱼白介素-1β基因的克隆及表达谱

探讨齐口裂腹鱼白介素-1β(Interleukin-1β,IL-1β)基因的基本特点。用RT-PCR方法以齐口裂腹鱼脾脏c DNA作为模板扩增IL-1β基因,用生物信息学软件分析其序列,用RT-PCR方法检测IL-1β基因在齐口裂腹鱼6种组织的表达情况。克隆获得的IL-1β序列长1 252 bp,共编码276个氨基酸。Gen Bank序列号为KU886235。IL-1β蛋白相对分子量为31.25 k D,等电点5.45,预测为亲水性蛋白。二级结构主要结构元件为随机卷曲和延伸链,有4个N-糖基化位点和16个磷酸化位点。NJ法系统进化树显示,齐口裂腹鱼与鲤鱼、鲫鱼的亲缘关系最近。组织表达分析显示,IL-1β基因主要在脾脏和肾脏中表达。这为深入研究鱼IL-1β的生物学功能提供理论依据。     

雅鲁藏布江中游裂腹鱼类的分布及栖息地特征

分别于2015年4月和10月对雅鲁藏布江中游裂腹鱼类的分布与栖息地环境特征进行了调查。结果表明:雅鲁藏布江中游现分布有裂腹鱼类6种,即异齿裂腹鱼(Schizothorax oconnori)、双须叶须鱼(Ptychobarbus dipogon)、拉萨裸裂尻鱼(Schizothorax younghusbandi)、拉萨裂腹鱼(Schizothorax waltoni)、巨须裂腹鱼(Schizothorax macropogon)和尖裸鲤(Oxygymnocypris stewarti);尖裸鲤仅在雅鲁藏布江仁布以上江段出现,异齿裂腹鱼和拉萨裸裂尻鱼在所有调查断面均有分布。RDA分析显示,河宽、流速和pH是影响裂腹鱼类栖息地选择的主要因子。针对当前雅鲁藏布江中游存在的过度捕捞、外来物种入侵和梯级水电开发等问题,建议管理部门采取划定保护区、禁捕、宣传放生土著鱼类等措施来维持裂腹鱼类栖息地的有效性。     

基于雅砻江两种裂腹鱼游泳能力的鱼道设计

为探究雅砻江两种裂腹鱼的游泳能力,给过鱼设施设计和鱼类游泳行为学研究提供基础参数,本研究采用递增流速法对长丝裂腹鱼、齐口裂腹鱼的感应流速、临界游泳速度、突进游泳速度进行测试,采用固定流速法对长丝裂腹鱼的耐久游泳速度进行测试。结果表明: 长丝裂腹鱼与齐口裂腹鱼的感应流速随着体长的增加均出现了先增加后平稳的趋势,但最大感应流速均小于0.2 m·s^-1;长丝裂腹鱼的临界游泳速度与突进游泳速度分别为(0.81±0.20)和(1.49±0.26) m·s^-1,相对临界游泳速度为(4.90±1.73) BL·s^-1,相对突进游泳速度为(9.77±1.72) BL·s^-1(BL为体长);齐口裂腹鱼的临界游泳速度与突进游泳速度分别为(0.73±0.24)和(1.17±0.39) m·s^-1,相对临界游泳速度为(6.88±2.82) BL·s^-1,相对突进游泳速度为(11.75±2.77) BL·s^-1。耐久测试发现,随着流速增加(0.7～1.5) m·s^-1,长丝裂腹鱼持续游泳时间与水流速度呈负相关,疲劳时间(T)与水流速度(V)的关系可以拟合为lgT=-2.52V+5.59,预测鱼道长度(d)与鱼道内可通过的最大平均水流速度(V_fmax)的关系式为V_{f max}=-0.17lnd+1.74。根据试验结果,当以长丝裂腹鱼和齐口裂腹鱼为主要过鱼对象时,建议鱼道内最小水流速度应大于0.2 m·s^-1,进口及竖缝处水流速度为0.73～1.67 m·s^-1,休息池主流水流速度为0.2～0.7 m·s^-1。     

Physical principles of protein structure and protein folding

Physical principles determining the protein structure and protein folding are reviewed: (i) the molecular theory of protein secondary structure and the method of its prediction based on this theory; (ii) the existence of a limited set of thermodynamically favourable folding patterns of α- and β-regions in a compact globule which does not depend on the details of the amino acid sequence; (iii) the moderns approaches to the prediction of the folding patterns of α- and β-regions in concrete proteins; (iv) experimental approaches to the mechanism of protein folding. The review reflects theoretical and experimental works of the author and his collaborators as well as those of other groups.     

Patterns of protein protein interactions in salt solutions and implications for protein crystallization

The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, alpha-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions. Comparison of the interaction trends in terms of the dimensionless second virial coefficient b(2) shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b(2) with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b(2). When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b(2) measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b(2) trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization.     

In-cell protein NMR and protein leakage

In-cell nuclear magnetic resonance spectroscopy is a tool for studying proteins under physiologically relevant conditions. In some instances, however, protein signals from leaked protein are observed in the liquid surrounding the cells. Here, we examine the expression of four proteins in Escherichia coli. We describe the controls that should be used for in-cell NMR experiments and show that leakage is likely when the protein being studied exceeds ～20% of the total cellular protein.     

Prediction of protein function from protein sequence and structure

The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a number of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologues often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein-protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as indicators of functional properties. Even if it is possible to ascribe a particular function to a gene product, the protein may have multiple functions. A fundamental problem is that function is in many cases an ill-defined concept. In this article we review the state of the art in function prediction and describe some of the underlying difficulties and successes.     

Bridging protein local structures and protein functions

One of the major goals of molecular and evolutionary biology is to understand the functions of proteins by extracting functional information from protein sequences, structures and interactions. In this review, we summarize the repertoire of methods currently being applied and report recent progress in the field of in silico annotation of protein function based on the accumulation of vast amounts of sequence and structure data. In particular, we emphasize the newly developed structure-based methods, which are able to identify locally structural motifs and reveal their relationship with protein functions. These methods include computational tools to identify the structural motifs and reveal the strong relationship between these pre-computed local structures and protein functions. We also discuss remaining problems and possible directions for this exciting and challenging area.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.