马氏珠母贝精子低温保存主要影响因素的研究

采用二甲基亚砜(DMSO)和甘油作为抗冻剂,以海水或体液等为基础液,配制体积分数为6％、8％、10％和12％的抗冻保护液．将马氏珠母贝精液和抗冻保护液以1：2、1：5、1：10和1：20(体积比)混合后,置4℃中平衡30和60min,再于-18℃中冷冻4、24和72h,解冻后观察精子的活动状态和受精率．结果表明,抗冻剂种类、体积分数以及精液与保护液的比例对精子的活动状态有很大的影响;4℃的平衡时间对精子的存活率影响不显著,但对受精率却有明显的影响．在-18℃时,精液和抗冻保护液体积比1：20,DMSO体积分数为10％和12％的两组保存效果较好．精子存活率随着保存时间的增长而降低,但基础液的类型对精子受精率没有明显影响．     

马氏珠母贝TLR2基因cDNA的分子特征及组织表达分析

TLR2（toll like receptor 2）是TLR模式识别受体家族的重要成员之一,参与有机体的免疫识别。本研究采用cDNA末端快速扩增（RACE）技术克隆获得了马氏珠母贝（Pinctada martensii）TLR2基因（PmTLR2）cDNA的全长序列并对其序列特征进行分析;同时检测了PmTLR2基因的mRNA在马氏珠母贝六个组织中的表达。结果表明：PmTLR2基因的cDNA全长2614 bp,包含41 bp的5'UTR,299 bp的3'UTR,27 bp的polyA,开放阅读框为2274 bp,编码758个氨基酸残基。多序列比对显示PmTLR2与牡蛎TLR2的序列相似性最高,为51%;Smart软件分析显示PmTLR2的蛋白序列中含有8个亮氨酸重复序列（Leucine rich repeats,LRRs）, 1个C端亮氨酸富集区（Leucine-rich repeat C-terminal,LRR-CT）,1个N端亮氨酸富集区（Leucine-rich repeat N-terminal,LRR-NT）,1个TIR（Toll/IL-1R）同源结构域（TIR）;荧光定量PCR分析表明PmTLR2在马氏珠母贝肝胰脏、性腺、血淋巴、鳃、外套膜和闭壳肌六个组织中均有表达,鳃中表达量最高。本研究为进一步阐述PmTLR2在马氏珠母贝免疫应答中的作用机制提供理论基础。     

日本珍珠贝人工繁殖苗在热带海区的养殖生长观察

日本珍珠贝引进海南陵水热带海区养殖,人工繁殖幼贝苗1·5~2·0mm下海养殖。经1年的养殖生长观察,结果为:平均壳长59·2mm、壳高59·7mm、壳宽20·7mm,平均个体重34·8g;养殖90d,生长速度较快,壳长平均增长值为11·9mm,相对增长率为55·4%;壳高平均增长值11·2mm,相对增长率61·2%;壳宽平均增长值5·9mm,相对增长率为190·3%。壳长、壳高和体重增长之间具有非常显著的相关关系。研究表明,该贝在海南热带海区养殖的生长速度快,是适宜引进的养殖对象。     

Estimating the heritability for growth-related traits in the pearl oyster,Pinctada fucata martensii (Dunker)

Twelve paternal half-sib families (or 36 full-sib families) of the pearl oyster, Pinctada fucata martensii (Dunker), were produced according to the requirements of hierarchical genetic mating design. A total of 4320 individuals, aged 15 months, were measured for seven growth-related traits. Predicated upon the additive-dominance genetic analysis model, varying genetic variance components and then heritabilities of the growth-related traits of interest were estimated using analysis of variance. Results showed that seven growth-related traits had larger additive genetic variances (P<0.05); the dominance genetic variance of shell weight (SW) was smaller (P>0.05), the dominance genetic variances of other six traits were all larger (P<0.05). Narrow- and broad-sense heritabilities for the seven traits were, respectively, 0.64 ± 0.10 and 0.78 ± 0.12 for shell length, 0.49 ± 0.06 and 0.63 ± 0.09 for shell height, 0.38 ± 0.14 and 0.54 ± 0.16 for shell breadth, 0.41 ± 0.17 and 0.56 ± 0.11 for hinge length, 0.53 ± 0.11 and 0.68 ± 0.08 for body weight, 0.35 ± 0.07 and 0.55 ± 0.08 for tissue weight and 0.67 ± 0.10 and 0.75 ± 0.16 for SW. All heritability estimates were statistically significant (P<0.05). According to these results, the mass selection procedure is suggested for the breeding of P. martensii.     

Contribution of donor and host oysters to the cultured pearl colour in Pinctada martensii

Cultured free round pearls are produced by implanting a spherical nucleus with a small piece of nacre‐secreting mantle graft from a donor oyster into the gonad of a recipient oyster (host). To examine the possible contribution of host and donor oysters to the colouration of the harvested pearls, the CSE‐1 Imaging and Color‐Measuring System were used to quantitatively measure the L*a*b* values of donor and host shells and the produced pearls in Pinctada martensii. Results showed that the colour of pearls had significant positive correlation (r = 0.1–0.22, P = 0.00) with that of donors, but had no correlation with that of host oysters, thus convincingly confirmed the contribution of nacre colour of donor to the realization of pearl quality of colour. To further clarify the relationship between the donor and the pearl colour, the donors from pearls of good and poor colour quality were further compared and the results demonstrated the significant difference in L* values (P < 0.05) and insignificant difference in a* and b* values, suggesting the necessity of selecting donors with bright and lustrous nacre in pearl production.     

4种防治马氏珠母贝多毛类寄生病方法的效果比较

多毛类寄生病是我国养殖马氏珠母贝普遍存在的病害,同时也是危害最大的病害。本研究比较了4种防治多毛类寄生病方法(淡水浸泡法、饱和盐水浸泡法、SBS涂料涂覆法和水泥涂覆法)的效果,通过分析处理后10d和处理后60d试验贝中的活虫数、有虫率和贝死亡率,用￡检验同对照组比较。结果表明,水泥涂覆法在处理后前期的杀虫效果明显,在长时间养殖中对提高试验贝的成活率方面有显著的优势,该法成本低、易操作。     

Fertilization,Hatching, Survival,and Growth of Third-Generation Colored Pearl Oyster (Pinctada martensii) Stocks

In April of 2009, third-generation black (BS₃), red (RS₃), yellow (YS₃), and white (WS₃) colored stocks of pearl oyster Pinctada martensii were compared in terms of fertilization, hatching, survival, and growth. Among stocks, there were no significant differences in fertilization rate (P > 0.05; values ranging from 94.8%–98.9%). Hatching rates of WS₃ were significantly lower (P < 0.05) at 61.3 ± 6.1% than other stocks (74.3 ± 5.4%, 75.9 ± 5.2%, 73.9 ± 4.9%, for BS₃, RS_3, and YS_3, respectively). Larval survival rates of the BS₃ stock (65.8 ± 9.4%), RS₃ (61.5 ± 8.7%), and YS₃ (58.2 ± 9.2 %) were significantly (P < 0.05) higher than that of the WS₃ stock (44.8 ± 11.2%). On day 8, there were no significant differences in mean shell length among the four colored stocks (P > 0.05). At days 15, 23, 90, 150, 210, and 325, however, significant differences in mean shell length among the four colored stocks were observed (P < 0.05). The BS₃ stock had consistently larger mean shell length than the other colored stocks.     

马氏珠母贝TNFR27基因克隆与功能初探

肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)是一类重要的细胞因子受体,主要参与细胞凋亡、宿主免疫防御、炎症等生物过程。本研究采用cDNA末端快速扩增(RACE)技术克隆了马氏珠母贝TNFR27(PmTNFR27)的cDNA全长并进行生物信息学分析,运用实时荧光定量PCR(qPCR)技术检测了PmTNFR27 mRNA在马氏珠母贝不同组织、脂多糖(LPS)、聚肌胞苷酸(Poly(I:C))及镉胁迫后的表达模式。结果显示：PmTNFR27 cDNA全长为1 524 bp,5′UTR长为186 bp,3′UTR长为248 bp,包含28 bp的poly(A)尾巴,开放阅读框(ORF)为1 062 bp,编码353个氨基酸;结构域预测表明PmTNFR27具有一个典型的CRD结构域和一个跨膜蛋白结构域,符合肿瘤坏死因子受体超家族特征;多序列比对结果表明贝类种间的相似性不高,但功能结构域位置较保守。系统进化树结果显示马氏珠母贝与其他贝类聚为一簇。qPCR结果显示,PmTNFR27 mRNA在马氏珠母贝各组织中均有表达,在鳃中表达量最高。LPS刺激后,PmTNFR27基因在鰓中的表达量于3 h达到最高,于72 h降到最低;Poly(I:C)刺激后,PmTNFR27基因在鰓中的表达量在6 h、12 h显著上升并达到最高至96 h时降到最低。镉胁迫后,3 h表达量达到最高,24 h、48 h表达量显著下降。研究结果显示PmTNFR27可能参与了马氏珠母贝的免疫应答反应。     

马氏珠母贝正常贝肉与黑壳病贝肉成分对比

为了研究黑壳病造成马氏珠母贝肉成份的变化,首先采用水蒸气蒸馏法分别提取正常马氏珠母贝和黑壳病马氏珠母贝肉的挥发性成份进行分析,正常马氏珠母贝肉检测出58种,黑壳病贝肉检测出59种。但黑壳病贝肉中仅有7种化合物与正常贝肉的挥发性成分相同。在黑壳病贝中,含量较高的成分有二十一烷和十八醛,分别为18.52%和17.95%,但在正常贝肉未检出。其次,采用茚三酮作柱后衍生试剂测定氨基酸的方法检测正常马氏珠母贝和患黑壳病的马氏珠母贝肉中牛磺酸的含量,结果分别为72.47mg/g、72.75mg/g,牛磺酸含量基本一致。通过分析黑壳病引起马氏珠母贝体内挥发性成分和牛黄酸成分变化,为研究马氏珠母贝患黑壳病的病变机理提供一定的科学依据。     

利用家蚕细胞表达重组蝎毒素蛋白及其杀虫效果分析

用天然毒素蛋白杀灭害虫是农林病虫害防治的一个重要发展方向。利用家蚕核型多角体病毒(BmNPV)多角体蛋白基因的部分序列与蝎毒素蛋白基因(BmK ITa1)融合表达,SDS-PAGE分析通过融合表达策略构建的含有蝎毒素蛋白基因的重组病毒具有较高的蝎毒素蛋白表达水平,重组病毒vBmBac(polh-)PH5B/35B-BmK ITa1.6His,在家蚕BmN细胞内的BmKITa1蛋白表达水平分别为0.4、0.2 mg/mL。以家蚕和棉铃虫为供试昆虫,探讨融合蝎毒素蛋白的杀虫效果,结果表明:经口添食含融合蝎毒素蛋白的BmN细胞液对不同龄期家蚕幼虫均具有毒性,尤其对低龄幼虫的毒性较强,蚁蚕添食后的死亡率达80%;给1龄期的家蚕和棉铃虫幼虫经口添食含有不同剂量融合蝎毒素蛋白的BmN细胞破碎液,当添食剂量在2.8μg/头时,家蚕的死亡率即达100%,棉铃虫的死亡率达74%。研究结果提示:BmNPV多角体蛋白编码基因的部分序列与蝎毒素蛋白基因融合表达,可提高蝎毒素蛋白的表达水平,且重组病毒表达的蝎毒素蛋白具有较好的杀虫活性,在较低剂量下即可对低龄昆虫产生良好的杀灭效果。