厚壳贻贝抗菌肽mytilin和myticin的cDNA基因的克隆与序列分析

抗菌肽是贻贝免疫系统中的一个重要组成部分,也是目前海洋生物抗菌肽研究的重要内容。在贻贝抗菌肽家族中,mytilin和myticin是目前贻贝抗菌肽研究中发现成员最多,丰度最高的两个家族,在贻贝免疫系统中占有重要的地位。为了解厚壳贻贝抗菌肽mytilin和myticin的基因组成及其多样性机制,通过设计特异性引物,以厚壳贻贝血细胞cDNA文库为模板进行PCR扩增以筛选文库,共克隆到8条mytilin的同源cDNA序列和9条myticin的同源cDNA序列,并对其基因序列及推导的氨基酸序列进行了序列比对和变异位点初步分析。上述研究为将来深入研究厚壳贻贝的免疫机制及筛选具有开发价值的抗菌肽分子奠定了理论基础。     

灿烂弧菌对厚壳贻贝免疫指标和消化酶活性的影响

为探究灿烂弧菌对厚壳贻贝免疫指标和消化酶活性的影响,用1×10~6、1×10~7、1×10~8个/mL 3个浓度的灿烂弧菌刺激厚壳贻贝,探讨弧菌刺激后厚壳贻贝的一氧化氮合酶(NOS)、一氧化氮(NO)、超氧化物歧化酶(SOD)、丙二醛(MAD)等免疫指标和淀粉酶、蛋白酶等消化酶的变化情况。结果显示,灿烂弧菌刺激48 h后,厚壳贻贝足、鳃、消化腺等组织中仅消化腺的NOS活性较对照组有显著升高,且酶活性随初始细菌浓度的升高而升高,故选用消化腺来测定灿烂弧菌刺激后72 h内的免疫指标和消化酶活性的变化。灿烂弧菌刺激后,48 h内NOS活性较对照组均显著升高。NO含量较对照组均显著升高,与NOS显现相同变化趋势。SOD活性在各浓度灿烂弧菌刺激下较对照组均显著升高,而MAD含量在实验组中含量显著低于对照组。淀粉酶活性在实验组中显著低于对照组,总体呈现先下降后升高的趋势。蛋白酶活性在各实验组中均呈现先升高后下降的趋势。研究表明,灿烂弧菌对厚壳贻贝免疫指标和蛋白酶活性的升高有诱导作用,但对蛋白酶的活性有抑制作用。本研究初步探明了厚壳贻贝对灿烂弧菌的免疫应答机制,为进一步研究灿烂弧菌和厚壳贻贝相互作用机制以及厚壳贻贝免疫机制奠定了基础。     

厚壳贻贝与贻贝遗传渐渗的分子生物学鉴定

本文运用ISSR标记对厚壳贻贝与贻贝遗传渐渗进行研究。结果表明,厚壳贻贝与贻贝的特异性标记在杂交贻贝中有所反应,进一步证实了在浙江嵊泗部分厚壳贻贝与贻贝发生了杂交和基因渐渗现象。所采集的杂交群体样本中,65%与厚壳贻贝的遗传距离近,可能是杂交后与厚壳贻贝回交的后代。目前由于过度采捕、环境改变以及引进外来种等影响,应该对厚壳贻贝的种质资源进行保护。     

厚壳贻贝抗菌肽Mytilin的初步鉴定

厚壳贻贝是我国具有重大经济价值的水产养殖贝类,对其抗菌肽的研究有助于人们了解厚壳贻贝的免疫机制。为了解厚壳贻贝血清中抗菌肽Mytilin的分子组成和特性,采用多维高效液相色谱对厚壳贻贝血清进行分离纯化,从中获得3种对革兰氏阳性菌以及阴性菌均有抑制作用的抗菌肽分子,序列分析表明3种抗菌肽具有较高的序列相似性,均属于贻贝抗菌肽Mytilin家族,分别命名为Mytilin-1,Mytilin-2和Mytilin-3。其中,Mytilin-1为34个氨基酸残基构成的多肽,其分子量为3885.17u,含8个半胱氨酸,形成4对二硫键。根据所测Mytilin-1的氨基酸序列设计特异性引物,通过菌落PCR方法筛选厚壳贻贝cDNA文库,获得Mytilin-1的cDNA基因并进行了序列分析。以上研究结果表明,作为贻贝的主要抗菌肽家族,Mytilin在厚壳贻贝血清中具有较高丰度,对其蛋白质序列以及基因序列的研究为深入了解厚壳贻贝Mytilin抗菌肽的分子多样性奠定了基础。     

厚壳贻贝定向装置设计与试验

为解决厚壳贻贝前处理阶段人工劳动强度大、定向技术落后的问题,设计了一种新型厚壳贻贝振动定向装置.首先采用三点测力法测量计算出厚壳贻贝的重心位置;结合振动理论对厚壳贻贝受力分析得出运动路线;然后使用Adams软件对其进行运动学仿真;最后利用控制变量法对定向装置的主要参数进行试验.结果显示:当厚壳贻贝的初始姿态为头部朝下,...     

厚壳贻贝组蛋白H2的免疫响应及其衍生肽抑菌活性

为深入了解组蛋白及其衍生肽在贻贝免疫过程中的作用,实验采用荧光定量PCR(qPCR)技术对厚壳贻贝组蛋白H2A和H2B进行微生物胁迫后的表达量变化进行分析。在此基础上,对厚壳贻贝组蛋白H2A和H2B基于其N端序列设计的衍生肽段(分别为Coruscusin-Ⅰ和Coruscusin-Ⅱ)进行固相化学合成、圆二色性光谱分析以及抑菌活性验证。结果显示,厚壳贻贝组蛋白H2A和H2B在不同组织中,对不同微生物胁迫均产生明显的表达量上调,且在血细胞中表现出对真菌和革兰氏阴性菌的敏感性,在鳃组织中表现为对革兰氏阳性菌的敏感性;而在消化腺组织中,H2A对革兰氏阳性菌较为敏感,但H2B对真菌和革兰氏阴性菌较为敏感。厚壳贻贝组蛋白衍生肽段Coruscusin-Ⅰ和Coruscusin-Ⅱ在非极性溶液中,其螺旋含量明显上升,且二者对革兰氏阳性菌和革兰氏阴性菌均具有明显的抑菌活性,但Coruscusin-Ⅱ对真菌的抑制活性较Coruscusin-Ⅰ强。进一步的螺旋结构预测结果表明,2种衍生肽序列中碱性氨基酸的种类及分布特征可能是其抑菌活性差异的内在原因。本研究为了解组蛋白及其衍生肽在贻贝免疫过程中的作用及其机...     

厚壳贻贝筏式养殖技术

正厚壳贻贝隶属于瓣鳃纲、翼形亚纲、贻贝目、贻贝科;自然分布于我国东海、黄海、渤海,是我国重要的海水养殖贝类。厚壳贻贝市场价格高于紫贻贝,养殖效益好,深受养殖业主的推崇。厚壳贻贝自产业化人工育苗技术突破后,在浙江嵊泗海域的养殖规模快速增加。目前,浙江嵊泗县已成为我国厚壳贻贝养殖的重要产区,     

GABA受体拮抗剂荷包牡丹碱和CGP52432对厚壳贻贝幼虫附着变态的影响

为研究GABA及其受体拮抗剂荷包牡丹碱（GABA_A受体）和CGP52432 （GABA_B受体）在厚壳贻贝幼虫附着变态中的调控作用,实验通过分析厚壳贻贝不同发育阶段幼虫转录组数据获得GABA受体相关蛋白基因（GABARAP）和GABA_B2受体基因(GABA_B2R),发现GABA_B2R在变态前期的眼点幼虫阶段相较于其他阶段幼体显著高表达。通过实时荧光定量PCR验证也得到相似的表达模式,显示GABA_B2R可能参与调控厚壳贻贝幼虫变态发育。药理学实验结果显示,10^-4 mol/L GABA对厚壳贻贝幼虫的变态具有诱导活性（27.2%±3.0%）。在拮抗剂实验中,10^-6～10^-4 mol/L荷包牡丹碱和CGP52432均显著抑制了厚壳贻贝幼虫变态,且抑制作用随浓度升高而增强。实验还发现GABA及其受体拮抗剂都抑制了厚壳贻贝幼虫的游泳行为。研究表明,GABA_A或GABA_...     

生态因子对厚壳贻贝眼点幼虫生长和存活的影响

为了实现厚壳贻贝人工育苗技术突破和推广,研究了海水盐度、温度、pH等生态因子对厚壳贻贝眼点幼虫的生长和存活的影响,筛选出最佳的繁育条件.结果表明:厚壳贻贝眼点幼虫对盐度的适应范围为23.5～39.1,最适为27.4～35.3;对温度的适应范围为18～27℃,最适为21～24℃;对pH值的适宜范围为7.5～9.0,最适为7.8～8.5.掌握了厚壳贻贝眼点幼虫对主要生态因子的适应范围,对厚壳贻贝人工育苗技术的突破具有重要的指导意义.     

厚壳贻贝亲贝及苗种培育技术要点

<正>厚壳贻贝俗称海红,系暖温性底栖贝类,主要以浮游硅藻类和有机碎屑为食,栖息于低潮线至水深20米的海底,足部退化,以足丝附着在岩礁或物体上生活。其贝壳呈楔形,壳大而厚,壳长大于壳高,两壳大小相等而对称。其壳面被黑褐色厚壳皮,壳内面紫褐色或灰白色,有珍珠光泽。厚壳贻贝雌雄异体,体外受精,2龄可达性成熟。研究组对厚壳贻贝进行了人工育苗试验并对其人工育苗技术进行了研究,取得了成功,现将厚壳贻贝亲贝及苗种培育技术     

Chemical Compositions and Functional Properties of Protein Isolated from By-Product of Triangle Shell Pearl Mussel Hyriopsis cumingii

Hyriopsis cumingii is a freshwater mussel widely cultured in China to produce cultured pearls. However, after the pearls are harvested, the mussel is discarded. To make effective utilization of pearl production wastes, proteins were recovered from the pearl mussel meat using pH shift technology. The protein recovery conditions, chemical compositions, and functional properties of the recovered protein were investigated. Results show that the proteins could be well extracted from the meat by 5 volumes of alkali water (pH 11, 20°C, 1 h) followed by acidic precipitation (pH 5.2). The recovered product contained 94.7% of protein with high levels of essential and semiessential amino acids (48.9%). The most abundant essential amino acid was sulfur-containing amino acids (12.82%), followed by lysine (8.89%), phenylalanine (4.69%), and threonine (4.37%). Compared with soybean protein isolate and egg protein, the recovered protein had better water/oil absorption capability, better foaming ability, and similar emulsion capability. These results suggest that the protein isolated from the pearl mussel meat might be utilized as ingredients for the food industries.     

Biochemical,physiological, and immunological changes during starvation in juveniles of Litopenaeus vannamei

In an attempt to know how the protein level modulates catabolism and its effects on the immune response, we studied juvenile L. vannamei that had been starved for varying period after being conditioned on diet containing either maintenance or optimal dietary protein levels (DPL). The effect of dietary protein level on nutritional reserves management of shrimp and its relation with immune condition was also addressed. Juvenile shrimp were fed for 21 days on diets containing 5% and 40% dietary protein. Hemolymph metabolites (glucose, cholesterol, protein, acylglycerols, and lactate), hemocyanin, osmoregulatory capacity, digestive gland glycogen and lipids, and immune conditions (hemocytes characterization, phenoloxidase activity, respiratory burst: basal and activated) were evaluated and considered as initial condition. After that time, shrimp were starved for 21 days. During starvation time every 7 days nutritional, physiological and immunological condition were evaluated. A reduction in all physiological and immunological indicators was observed with starvation. The protein level of the conditioning diet had a significant effect on this response; generally, the effect was smaller with shrimp previously fed 40% dietary protein. In this sense the present results demonstrate that shrimp are well adapted to tolerate food deprivation for some time but that this tolerance is closely related to its previous nutritional condition. In the case of shrimp fed 40% DPL, wet weight, nutritional and immune condition was significantly affected after 14 days of starvation. In shrimp previously fed 5% DP, tolerance to starving condition was limited to only a few days (7 days) as a result of low reserves of circulatory and mussel proteins. All these results demonstrate that dietary protein levels can governor the immune condition of shrimp through the management reserves metabolism, indicating that a shrimp with a good nutritional condition can tolerate until 14 days without modifying the evaluated immune responses. In this sense it can be concluded that protein metabolism have a central role for shrimp.     

Isolation and characterization of a novel 45 kDa calponin-like protein from anterior byssus retractor muscle of the mussel Mytilus galloprovincialis

SUMMARY: A calponin-like protein of 45 kDa was isolated from mussel anterior byssus retractor muscle (ABRM) and its inhibitory effects on actomyosin Mg²⁺-ATPase was demonstrated. The 2-D electrophoresis for ABRM myofibrils gave a spot of 45 kDa protein in addition to myofibrillar proteins such as myosin and actin. The 45 kDa protein, which was more basic and showed a slightly higher molecular weight than actin, was isolated by ion-exchange chromatography and subjected to chymotryptic digestion. N-terminal amino acid sequencing of polypeptide fragments produced gave two sequences, ASQKGMTSFGAVRHH and GMDRALISKMGSKYDSGL, both of which showed a high homology to those of vertebrate calponins and invertebrate calponin-related proteins. Furthermore, the 45 kDa protein strongly reacted with commercially available antibody raised against chicken smooth muscle calponin, demonstrating that the mussel ABRM 45 kDa protein is a new member of the calponin family. Then, actomyosin Mg²⁺-ATPase activity of ABRM was measured in the presence and absence of the 45 kDa protein. The 45 kDa protein clearly inhibited actomyosin Mg²⁺-ATPase activity in a dose-dependent manner as in the case of other vertebrate calponins. These results indicate that the 45 kDa calponin-like protein is involved in the thin filament-associated regulation of molluscan smooth muscle contraction, possibly of a unique contraction called catch.     

Blue mussel meal as feed attractant in rapeseed protein‐based diets for turbot (Psetta maxima L.)

Antinutritional factors in rapeseed products have been identified to reduce feed palatability and growth performance of turbot. Therefore, we evaluated the potential of blue mussel (Mytilus edulis L.) meal as feed attractant in rapeseed protein‐based diets for turbot. Triplicate fish groups received isonitrogenous and isocaloric diets with fish meal (FM) protein replacements of 50% or 75% by rapeseed protein concentrate (RPC 50, RPC 75). These diets were supplemented with 0%, 2%, 4% or 8% of blue mussel meal. In contrast with RPC 50 diets, fish fed with RPC 75/0 showed significantly reduced daily feed intake (DFI) and specific growth rate (SGR). With increasing mussel meal inclusion, RPC 75 diets resulted in increased DFI and SGR, suggesting mussel meal as attractant in rapeseed protein‐based diets for turbot. Feed conversion was unaffected by any treatment. Protein productive value and apparent digestibility coefficients were unaffected by either RPC or mussel meal inclusion. With regard to the whole body composition, no differences in crude protein, crude lipid and ash content could be determined. Haematological characteristics were unaffected among the treatments indicating good nourished and unstressed fish. In conclusion, we demonstrated that the utilization of blue mussel meal improved the palatability of rapeseed protein‐based diets for turbot.     

Expression of HSP70 in response to heat-shock and its cDNA cloning from Mediterranean blue mussel

Expression of HSP70 in response to heat-shock was investigated at the protein and mRNA levels in Mediterranean blue mussel. Western and Northern blot analyses revealed that HSP70 was expressed following heat-shock in the mantle at both protein and mRNA levels, suggesting that gene expression of HSP70 is implicated in the cellular response to heat-shock stress in mussel. It was then attempted to clone HSP70 cDNA in order to determine the primary structure of mussel HSP70. As a result, two full-length cDNA encoding HSP70 were isolated from a cDNA library prepared from the heat-shocked mantle. The isolated cDNA consist of single open reading frames of 2067 bp and 1911 bp which encode proteins of 689 amino acids and 637 amino acids, respectively. Both HSP70 cDNA encode an ATPase do main, and a substrate-binding do main in addition to a Glu-Glu-Val-Asp (EEVD) peptide motif that is specific for cytosolic HSP70. These findings suggest that the cDNA clones obtained in the present study encode cytosolic HSP70.     

草鱼干扰素3蛋白多克隆抗体制备及其应用

为探讨干扰素3(Interferon 3,IFN3)在抗病毒免疫应答中的作用,以草鱼(Ctenopharyngodon idella)巨噬细胞cDNA为模板,PCR扩增IFN3成熟肽基因序列,制备草鱼IFN3蛋白的多克隆抗体,同时研究了IFN3在草鱼不同免疫组织中的蛋白表达,以及草鱼呼肠孤病毒(grass carp reovirus,GCRV)感染草鱼肾细胞系(Ctenopharyngodon idella kidney,CIK)后不同时间点的表达。结果显示,细菌表达的重组IFN3大小约为45 kD,主要以包涵体形式存在;抗体效价约为1∶3 200,制备的多克隆抗体既能识别原核表达的重组蛋白,也能识别个体水平上和细胞水平上的内源蛋白。草鱼主要免疫组织中,肝胰腺和皮肤检测到相应条带。CIK细胞感染病毒后12 h开始检测到IFN3蛋白,随感染时间的延长,IFN3蛋白表达量有所增加。蛋白水平上检测IFN3的表达,为深入研究草鱼的抗病毒免疫机制奠定了基础。     

弧菌生物被膜的动态演替对厚壳贻贝附着的影响

为探讨生物被膜动态演替过程中如何影响海洋无脊椎动物附着,实验选取了对厚壳贻贝附着具有不同诱导活性的弧菌Vibrio cyclitrophicus、V. chagasii和Vibrio sp. 22形成单一生物被膜,观察弧菌动态演替中生物被膜细菌密度、膜厚度和胞外产物等生物学特性变化,探究其对厚壳贻贝稚贝附着的影响。结果显示,弧菌生物被膜动态演替过程中,被膜细菌随着时间推移出现聚集现象,细菌密度和膜厚度也随着时间变化呈先增多后减少。除了Vibrio sp. 22,V. cyclitrophicus和V. chagasii生物被膜细菌密度和膜厚度与稚贝附着均有不同程度的相关性。所测弧菌生物被膜胞外产物的显微激光共聚焦结果分析发现,胞外多糖随着时间先增多,然后开始下降。相对比而言,胞外蛋白和胞外脂质无显著性变化。因而,胞外多糖变化规律与稚贝在被膜上附着变化相一致,表明胞外多糖是生物被膜动态演替过程中调控厚壳贻贝附着的重要因素。本实验初步探讨了生物被膜动态演替特征及其对厚壳贻贝稚贝附着的影响,对于后续进一步在海区开展生物被膜的动态演替与海洋底栖动物附着相互关系研究具有重要的学术价值,同时对于人工鱼礁礁体生物附着机理的研究具有重要的实践价值。     

Effect of Washing on the Quality of Surimi-Like Preparation Obtained from Soft Tissue of Freshwater Mussel Sinanodonta woodiana (Lea, 1834)

ABSTRACT

The research analyses the influence of the number of washings and different washing solutions, on the recovery of dry matter and protein in a preparation made from freshwater mussel Sinanodonta woodiana, (Lea, 1834). The study involved qualitative and quantitative characterisation of mussel protein preparation (MPP) obtained by electrophoretic separation and differential scanning calorimetry (DSC). The type of washing agent had the greatest influence on dry matter and protein content. Washing the homogenate with NaCl solutions and water was more effective on protein extraction than using only water. Two bands of proteins with molecular weights of 270–273 kDa and 42–43 kDa were characterized by the highest band intensity in electropherograms. The DSC analysis showed that on all denaturation curves of the samples there was one main peak of transformation ranging 56.69–62.94°C. The research revealed that washing homogenates with NaCl solutions caused the appearance of additional peaks at lower transformation temperatures, i.e. from 19.68°C to 36.45°C. The highest enthalpy value (4.53 Jg^?1) was observed for the sample washed once NaCl solution and then water. The number of washings (1 or 2) and the use of a 0.5% NaCl solution or water were found to be the most favorable parameters for MPP production.     

Effect of Dietary Inclusion of Blue Mussel Extract on Growth and Body Composition of Japanese Flounder Paralichthys olivaceus

Dietary inclusion of a water-soluble fraction of blue mussel Mytilus galloprovincialis was examined as a feeding stimulant for juvenile Japanese flounder Paralichthys olivaceus . The control diet mainly consisted of fish meal, potato starch, and pollack liver oil. Five, 10, and 20% (weight/weight) of the control diet was exchanged with aqueous extracts of blue mussel meat in experimental groups. Fish of about 10 g in initial body weight were fed each diet to satiation, twice daily, 6 d per wk for 6 wk at 20 C. The final body weight, weight gain, and feed efficiency of fish fed the diets containing blue mussel extracts were significantly higher than those of fish fed the control diet. However, these parameters were not different among experimental groups containing blue mussel extract independent of the inclusion level of extract. A similar trend was shown in protein efficiency ratio as fish fed the control diet had a significantly lower protein efficiency ratio than the other dietary groups. Compared to the control diet, higher plasma protein and lower triglyceride were found in fish fed the diets with the extract, while other blood constituents were relatively similar for the dietary groups tested. On the other hand, whole-body crude lipid content and lipid retention of fish fed the diets with the extract were generally significantly higher than those of fish fed the control diet. Whole body crude protein was identical regardless of the dietary composition; however, protein retention of fish showed a similar trend to lipid retention.