太平洋牡蛎(Crassostrea gigas)类FUT2基因的克隆与组织表达

通过同源克隆的方法获得太平洋牡蛎(Crassostrea gigas)类FUT2基因的c DNA序列,分析其在牡蛎中的组织表达差异。研究结果表明,太平洋牡蛎类FUT2基因c DNA全长为1941 bp,包含180 bp的5'非翻译区、1086 bp的编码361个氨基酸的开放阅读框及675 bp的3'非翻译区。分子进化聚类分析结果显示,太平洋牡蛎类FUT2基因与家鼠(Mus musculus)等哺乳动物的FUT2基因聚为1个分支。此外,类FUT2基因m RNA在太平洋牡蛎成贝的肝胰脏、闭壳肌、外套膜、唇瓣、鳃等5个组织中均有分布,其中在唇瓣中的表达量最低,在其余4个组织中的表达量差异不显著。本研究表明,牡蛎中类A型组织血型抗原HBGA很可能存在与人A型HBGA相似的合成途径,可为进一步探索牡蛎特异性富集诺如病毒No V的分子机制奠定研究基础。     

太平洋牡蛎(Crassostrea gigas)类α-1,2-岩藻糖基转移酶的密码子优化与原核表达

牡蛎消化组织内存在的类A型血型组织抗原是其特异性富集诺如病毒的主要原因,FUT2(Fucosyltransferase 2,α-1,2-岩藻糖基转移酶)是A型血型组织抗原合成的关键酶。本研究在前期克隆了太平洋牡蛎(Crassostrea gigas)类FUT2基因cDNA全长的基础上,根据大肠杆菌密码子偏爱性优化并合成了类FUT2基因,插入原核表达载体pRSET A构建pRSET-mof,将其转化大肠杆菌BL21,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导。经SDS-PAGE分析显示,在37℃、IPTG终浓度为0.8 mmol/L的条件下,诱导4 h后出现大小约为46 k Da的特异性目的条带。利用His亲和层析柱纯化及超滤管浓缩目的蛋白,得到单一条带,说明纯化效果良好。Western blot分析显示,目的蛋白与抗6×His标签单克隆抗体、抗人FUT2单克隆抗体均能发生特异性反应,表明优化后的太平洋牡蛎类FUT2基因在大肠杆菌系统中成功表达。本研究结果为今后研究太平洋牡蛎类FUT2基因的功能,进一步探索牡蛎特异性富集诺如病毒的分子机理奠定了基础。     

4株长牡蛎(Crassostrea gigas)致病性哈维氏弧菌(Vibrio harveyi)鉴定及其毒力基因检测

哈维弧菌(Vibrio harveyi)是海水鱼养殖中一种重要的病原菌。该研究根据已知rbsB (核糖体结合蛋白)基因序列设计引物,扩增得到哈维弧菌354 (V.harveyi 354) rbsB基因的全序列(GenBank序列号MF797015),预测该序列可编码292个氨基酸,分子量为30.7 kD,理论等电点为5.05,亲水性系数为0.043,为疏水性蛋白。根据RbsB氨基酸构建的系统进化树可以发现哈维弧菌RbsB蛋白和欧文弧菌(V.owensii CAIM 1854)的关系最近。构建了pGEX-4t-1-rbsB重组质粒并转化至大肠埃希菌BL21 (DE3)中,得到的重组蛋白的相对分子量约59 kD,在37 ℃、IPTG浓度为0.6 mmol·L^–1诱导8 h时表达量最高。     

超高压处理对牡蛎(Crassostrea gigas)杀菌及贮藏品质的影响

以牡蛎(Crassostrea gigas)为研究对象,研究了不同超高压处理条件对牡蛎体内微生物的消减作用,分析了冷藏过程中挥发性盐基氮(TVB-N)和 pH 值的变化情况,对比了热加工和超高压处理对牡蛎滋味的影响。结果显示,经300 MPa 处理15 min、400 MPa 处理10、15 min 和500 MPa处理5、10和15 min 后,牡蛎体内的菌落总数均有2个对数以上的减少,表明超高压处理能有效消减牡蛎体内的微生物;牡蛎经300 MPa 处理15 min、400 MPa 处理10 min 和500 MPa 处理5 min,在4℃冷藏条件下保存,货架期延长至15 d;超高压处理能有效抑制牡蛎在4℃贮藏过程中挥发性盐基氮的产生,牡蛎经300 MPa 处理15 min,冷藏20 d 后 TVB-N 值仍小于10 mg/100 g,符合生鲜牡蛎要求;滋味方面,与加热处理相比,超高压处理后牡蛎更接近生鲜牡蛎,尤其300 MPa 处理15 min 的牡蛎同生鲜牡蛎最为接近。研究表明,选取300 MPa 处理15 min 作为牡蛎加工的最佳处理条件,即能有效消减牡蛎体内的微生物,同时能很好地保持牡蛎品质。     

牡蛎诺如病毒受体类Lewis抗原合成相关基因CgFUT5的克隆与表达鉴定

Lewis抗原被认为是诺如病毒特异性结合受体,作为诺如病毒传播载体,牡蛎中也存在着类Lewis抗原,但牡蛎合成这种碳水化合物的途径尚未阐明。为解析牡蛎中诺如病毒受体类Lewis抗原的合成路径,利用cDNA末端快速扩增(Rapid amplification of cDNA ends, RACE)技术克隆得到太平洋牡蛎(Crassostrea gigas)的CgFUT5基因全序列并进行生物信息学分析,通过实时荧光定量聚合酶链式反应(RT-qPCR)分析其在5种组织中的表达情况。构建原核表达质粒转化大肠杆菌(Escherichia coli)实现异源表达,并通过免疫印迹法(Western blot)鉴定免疫原性。克隆得到了具有1 173 bp开放阅读区的CgFUT5基因cDNA序列,系统发育树显示CgFUT5基因与多个物种具有合成Lewis抗原功能的岩藻糖基转移酶基因遗传学关系较近。重组CgFUT5蛋白可在大肠杆菌中过量表达,且表达的重组CgFUT5蛋白与抗人FUT5抗体及抗6×His标签抗体均能特异性结合。研究发现CgFUT5基因在牡蛎鳃组织中大量表达,CgFUT5蛋白与人FUT5蛋白具...     

长牡蛎(Crassostrea gigas)壳宽快速生长选育群体遗传多样性及遗传结构的微卫星标记分析

为了监测长牡蛎(Crassostrea gigas)在选育过程中的遗传变异、分析选育对其遗传结构的影响,本研究以选育目标为壳宽快速生长的长牡蛎为实验材料,利用微卫星(Simple Sequence Repeats)标记技术,对长牡蛎基础群体(P0)和连续两代选育群体(F1和F2)进行遗传多样性评估。结果发现,所有微卫星位点在3个群体中都表现出了较高的多态性,P0、F1和F2代群体的平均等位基因数分别为16.5、12.2和12.8;P0、F1和F2代群体多态性信息含量(Pic)的平均数值分别为0.9068、0.8982和0.8836。所有群体10个位点的观测杂合度值(Ho)均小于期望杂合度值(He),观测杂合度平均值的大小范围为0.5775–0.6484,期望杂合度范围为0.8594–0.9279。哈迪-温伯格平衡(HWE)结果显示,3个群体在10个位点上有24个群体的位点组合显著偏离HWE(P<0.05),说明人工选育对选育群体的遗传结构有一定的影响。3个群体在10个位点上的Fis值均为正值,平均范围为0.1541–0.2341,表明群体内各位点上的杂合子比例有所下降;各群体间Fst值范围为0.0093–0.0245,遗传分化程度较弱。此研究表明,以壳宽快速生长为选育目的,长牡蛎连续选育群体仍具有很高遗传多样性,人工选育过程中保持一定选择压力,仍然会使长牡蛎的优良生长性状得到不断提高。     

牡蛎疱疹病毒囊膜蛋白(ORF111)的基因克隆及表达

本研究对编码牡蛎疱疹病毒(Ostreid herpesvirus 1, OsHV-1)囊膜蛋白的orf111基因进行了生物信息学分析、克隆和表达。首先,通过特异性PCR扩增得到orf111基因全长序列,并对其编码囊膜蛋白的理化性质、高级结构、跨膜区和抗原决定簇等进行生物信息学分析。结果显示,orf111编码一种稳定的疏水性蛋白,具有5个跨膜结构域和9个抗原决定簇,同时,氨基酸序列中还包含1个高度保守的精氨酰–甘氨酰–天冬氨酸(Arg-Gly-Asp, RGD)结构域。随后,构建了pET28a-orf111重组质粒,并将其转化到大肠杆菌DH5α感受态细胞中。最后,通过使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,表达产物分子量约为32 kDa。本研究应用原核表达得到了含RGD结构域的OsHV-1囊膜蛋白,为进一步制备ORF111蛋白单克隆抗体及开展牡蛎疱疹病毒侵染机制的研究奠定了重要基础,为将来OsHV-1的防控工作提供新的思路。     

牙鲆(Paralichthys olivaceus) CD59基因的原核表达与功能探究

本研究分析了CD59基因在牙鲆(Paralichthys olivaceus)感染鳗弧菌(Vibrio anguillarum)前后的表达模式.结果显示,感染前,CD59基因在检测的4种免疫器官中均不表达;感染后,该基因均有不同程度的表达,其中,肾脏的表达量最高.对牙鲆CD59基因进行原核表达,构建原核表达载体pET-32a-CD59,并将其转化入大肠杆菌(Escherichia coli) BL21,使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导,获得约为29 kDa的重组蛋白pET-32a-CD59.该蛋白以包涵体形式存在,通过His亲和层析柱纯化和超滤管浓缩目的蛋白后,经SDS-PAGE检测得到单一条带,纯化效果较好.Western blotting分析结果显示,重组蛋白pET-32a-CD59可以与抗His单克隆抗体发生特异性反应,说明牙鲆CD59基因在大肠杆菌系统中成功表达.对重组蛋白进行透析复性,复性结束后,蛋白无析出或絮状沉淀,初步表明复性成功.选取嗜水气单胞菌(Aeromonas hydrophila)、鳗弧菌、金黄色葡萄球菌(Staphylococcus aureus)和大肠杆菌4种致病指示菌,测定复性重组蛋白pET-32a-CD59的抑菌活性.研究表明,重组蛋白pET-32a-CD59对嗜水气单胞菌具有一定的抑菌活性.本研究旨在探究牙鲆免疫调节机制,并为提高牙鲆养殖效率提供一定的分子理论基础.     

香港牡蛎淋巴因子基因tal的克隆与功能

为了研究淋巴因子基因(T-cell acute lymphocytic leukemia,tal)在香港牡蛎中的免疫功能,实验采用c DNA末端快速扩增技术(rapid-amplification of cDNA ends,RACE)克隆了香港牡蛎tal的c DNA全长序列,命名为Chtal,该基因全长812 bp,其中5?非编码区17 bp,3?非编码区135 bp,开放阅读框(open reading frame,ORF)660 bp,编码219个氨基酸,相对分子质量25.11 ku,理论等电点5.80。多序列比对和系统进化树结果显示,Chtal与长牡蛎、美洲牡蛎和海蜗牛tal同源性较高,证明了该基因是软体动物tal家族的一个成员。荧光定量PCR结果显示,Chtal在香港牡蛎血淋巴细胞中表达量最高,其次是心脏、鳃和消化腺。在溶藻弧菌和溶血性葡萄球菌刺激后,Chtal表达量显著上调,分别在24和12 h达到最高水平,随着时间的增加该基因的表达量逐渐下降。亚细胞定位结果显示,Chtal在细胞核中表达。活体注射重组TAL蛋白能够显著提高淋巴细胞的数量,通过Edu增殖实验进一步证实了tal具有促进香港牡蛎血淋巴细胞再生的功能。本研究初步揭示了香港牡蛎tal参与了淋巴细胞再生,为深入研究该基因在牡蛎抗病中的功能提供了依据。     

溶藻弧菌HY9901鞭毛蛋白flaB基因的克隆及原核表达

参照GenBank上登录的弧菌鞭毛蛋白flaB基因序列设计引物,PCR扩增溶藻弧菌HY9901株的flaB全长基因,序列分析结果显示该基因为1134bp,编码377个氨基酸。与GenBank中其它弧菌的同源基因序列比对显示,溶藻弧菌flaB基因与副溶血弧菌flaB基因的同源性最高(92%)。将该基因定向克隆到原核表达载体pET-32a(+)中,在大肠杆菌BL21(DE3)中成功表达出带His-tag的融合蛋白,分子量大小与预期一致。优化的表达条件为28℃,0.4mmol/LIPTG浓度诱导10h。用纯化后的融合蛋白免疫SPF级小鼠,制备了多克隆抗体。Western-blotting结果表明鼠抗FlaB血清不仅能与诱导后的重组蛋白发生反应,而且能与天然的溶藻弧菌全菌蛋白发生反应,提示鞭毛蛋白FlaB可能是溶藻弧菌的重要保护性抗原之一,为下一步进行FlaB蛋白免疫原性的研究以及疫苗的制备奠定了基础。     

培育密度对长牡蛎面盘幼虫生长影响的对比试验

长牡蛎育苗期间经常出现面盘幼虫生长参差不齐现象,影响单位水体出苗量。笔者在正常培养密度范围内6～12个 ml作对比试验:低密度6～8个 ml培养的幼虫生长速度快,个体差异小;重视亲贝促熟,加强优质新鲜饵料的供给,有良好的水环境,能够减少幼虫差异显著的现象。     

Consumer Preference and Sensory Properties of the Pacific Cupped Oyster (Crassostrea gigas) and the European Flat Oyster (Ostrea edulis)

ABSTRACT

Experts in the oyster supply chain (farmers, retailers, and gourmet chefs) in The Netherlands have suggested that the European flat oyster (Ostrea edulis) has superior sensory qualities compared to the Pacific cupped oyster (Crassostrea gigas). However, scientific evidence is lacking. The aim of this study was to evaluate consumer preferences for these two oyster species and to determine the sensory properties of both species by consumers. Two-alternative forced choice tests were performed with the oysters involving a panel of 74 naïve consumers. No significant differences in preferences between the oyster species were observed by the consumers. Nine sensory attributes of both oyster species were evaluated. Consumers did not observe significant differences between species in the sensory attributes: overall odor intensity, sea odor, mud odor, sweetness, pungency, and firmness. The Pacific cupped oyster was perceived as significantly saltier and more intense in greenness than the European flat oyster. We concluded that only a few sensory properties of the European flat oyster and the Pacific cupped oyster are perceived as being different by untrained consumers.     

Standardization of photometric measurement of sperm concentration from diploid and tetraploid Pacific oysters, Crassostrea gigas (Thunberg)

To provide necessary standardization of procedures for cryopreservation of sperm, a spectrophotomeric method was developed to determine the sperm concentration of diploid and tetraploid Pacific oysters, Crassostrea gigas. Wavelengths of 380, 550, 581 and 780 nm were compared, and 550 and 581 nm were found to be the most sensitive and reliable. A linear relationship between sperm concentration and photometric absorbance was observed for sperm concentrations between 2 × 10⁷ and 2 × 10⁹ cells mL^?1. The regression equation for the standard curve at 550 nm for sperm of diploid oysters was Y=?8.528+1.165 log X. The equation for sperm of tetraploid oysters was Y=?8.844+1.236 log X. The equation at 581 nm for sperm of diploid oysters was Y=?8.07+1.104 log X. The equation at 581 nm for sperm of tetraploid oysters was Y=?8.331+1.167 log X. Comparisons derived from the standard curves at 581 nm between observed values and the predicted values indicated good agreement for sperm from diploid (coefficient of determination, r²=0.983) and tetraploid (r²=0.980) oysters.     

长牡蛎高糖原含量新品系正反交家系的生理代谢

为了探究糖原含量差异显著的长牡蛎(Crassostrea gigas)自交家系和正反交家系在生理代谢水平上的差异,本研究开展了在一定温度(20℃)和盐度(32)下自交组和正反交组的滤水率、耗氧率和排氨率实验。结果显示,滤水率方面,自交组AA最大,BB最小,二者差异显著(P0.05);正交组AB和反交组BA二者差异不显著且大小介于2个自交组之间,正反交组的杂种优势值为8.44%。耗氧率方面,自交组BB最大,正交组AB最小;自交组AA与BB差异显著(P0.05),正反交组杂种优势值为-43.87%;耗氧率与糖原含量呈负相关,相关系数为-0.884,达到极显著性水平(P0.01)。排氨率方面,各组之间的排氨率无显著差异,大小为0.04 mg NH_3-N/(g·h)。研究表明,糖原含量品系正反交家系的差异在生理代谢上主要表现为滤水率和耗氧率的差异。     

Sex-specific growth and condition of the Pacific oyster (Crassostrea gigas Thunberg)

In order to study the possibility of gaining commercial benefit from culturing an excess of one sex of the Pacific oyster (Crassostrea gigas), comparative data on the growth rate and condition of male and female oysters are reported. Historically, measurement of sex‐specific growth rate in oysters has been overlooked or confounded by protandric sex. The recent conclusion that the sex ratio of Pacific oysters is predominantly under genetic rather than environmental control introduces the possibility of manipulating sex ratio for commercial gain if they exhibit asynchronous sex‐specific growth rates. Pacific oysters were cultured intertidally in Smoky Bay, south Australia. The observations, made over the 7‐month gametogenic cycle from August to February to ensure no sex reversal, were of growth rates of male and female oysters and ambient chlorophyll a concentrations. Mean shell growth of female oysters was significantly faster than that of males (4.5 ± 3.3 compared with 3.8 ± 3.2 μm day^?1 mm^?1 total length). Sex‐specific asymmetries in length and weight were generally significant and increased in magnitude during the 7‐month study period, suggesting potential commercial benefits from increasing the proportion of cultured female oysters. The fastest increase in the sex‐specific disparity in growth and condition came after the October chlorophyll a peak, suggesting that females utilize blooms more efficiently than males. Our results compare favourably with methods currently used to increase oyster growth (e.g. triploidy can provide growth gains of 13–51%).     

基于微卫星标记整合长牡蛎遗传图谱

为了提高长牡蛎遗传图谱上的微卫星标记密度,实验采用6个家系图谱间的共有微卫星标记作为锚定标记,构建了长牡蛎的整合图谱.该整合图谱共有161个微卫星标记,覆盖10个连锁群,图谱长度和平均间距分别为615.4和3.8 cM.各连锁群的标记数介于10 ～ 24个之间,连锁群长度为47.3～73.3 cM,是目前密度最高的长牡蛎微卫星图谱.不同作图家系连锁群上的标记分组保持一致,但标记顺序出现差异,可能与长牡蛎自然群体中存在大量的染色体重排现象有关.结果表明,该图谱可以为今后长牡蛎的遗传育种研究提供新的遗传工具.     

低渗诱导太平洋牡蛎三倍体以及与其他诱导方法的比较

通过低渗方法抑制受精卵第二极体(PB2)的释放,诱导太平洋牡蛎(Crassostrea gigas)三倍体.在水温为23～25℃的条件下,分别采用不同低渗海水(盐度分别为4、6、8、10、12、14、16),在不同处理时机,即第1个第一极体(PB1)出现、30%的PB1出现、40%的PB1出现、50%的PB1出现以及第1个第二极体(PB2)出现时,对太平洋牡蛎受精卵进行10 min、15 min、20 min、25 min持续处理.处理后,于正常盐度海水中孵化,收集D形幼虫,通过流式细胞仪进行倍性测定,确定最佳诱导条件.结果表明,当太平洋牡蛎受精卵PB1出现40%时,在盐度为8的低渗海水中,持续处理15 min,得到最高三倍体诱导率为(89.16±1.39)%.与对照组相比,低渗诱导获得的三倍体幼虫表现出明显的生长优势.将低渗方法同高温(32℃)、低温(2℃)和6-DMAP(450μmo1/L)处理等多倍体诱导方法进行比较,结果显示,低渗组的卵裂率和孵化率显著高于高温、低温和6-DMAP诱导的太平洋牡蛎三倍体(P＜0.05).6-DMAP诱导的三倍体率虽略高于低渗诱导,但差异不显著(P＞0.05).通过综合评价指数(Ie)比较,认为低渗诱导多倍体的方法比高温、低温和6-DMAP诱导具有明显的优势.     

Reproduction, condition and mortality of the Pacific oyster Crassostrea gigas (Thunberg) in Sonora, México

Starting in 1997, mortality outbreaks in cultured oysters Crassostrea gigas have been reported in northwestern México. Previous studies have shown that massive die‐offs result from multi‐factor processes related to reproduction of the species. We studied the reproductive cycle and the condition index of cultured oysters in the coastal lagoon of El Soldado, Sonora, as well as the relationship of the life cycle with environmental parameters. We used oocyte diameter to determine reproductive stages and the condition index to describe the physiological state of oysters. Additionally, the temperature, salinity, seston and chlorophyll a were recorded at the study site. The results showed that C. gigas began accelerated reproductive activity in March under the influence of high temperature and increased concentrations of food. No spawning events were recorded and gametes were reabsorbed within the gonad in September and October. The results showed a period of nutrient storage during autumn–winter and another period of gamete production in spring–summer. A mortality event occurred at the end of winter, tied to significant increases in temperature and availability of food and in accelerated reproductive activity and high condition index. These conditions were very similar to those reported in other countries during summer die‐offs of C. gigas.