大亚湾核电站邻近水域马氏珠母贝的种群生态

１９９３年７月－１９９４年１２月,在大亚湾东山珍珠养殖场进行马氏珠母贝的生长和死亡率研究,结果表明：中、小贝种群个体大小组成呈正态分布,进入中贝以后,正态分布不明显。小贝生长快,壳高增幅大,死亡率较低,中贝以后,生长较慢,死亡率较高。     

能自体受精的雌雄同体黄边胡鲇的性腺结构研究

能自体受精的雌雄同体黄边胡鲇的性腺结构研究刘少军,姚占州,刘筠（湖南省生物研究所,长沙４１００８１）关键词黄边胡鲇,性腺,雌雄同体,自体受精ＳＴＵＤＩＥＳＯＮＴＨＥＧＯＮＡＤＳＴＲＵＣＴＵＲＥＳＯＦＨＥＲＭＡＰＨＲＯＤＩＴＩＳＭＳＰＥＲＦＯＲＭＩＮＧ...     

马氏珠母贝Caspase-3基因克隆和组织表达分析

半胱氨酸蛋白酶3 (Caspase-3)作为细胞凋亡通路中重要的效应蛋白,在细胞凋亡过程中发挥着重要作用.为初步探究马氏珠母贝Caspase-3(PmCaspase-3)的生物学功能,本研究利用cDNA末端快速扩增(RACE)技术克隆获得PmCaspase-3基因cDNA的全长序列并对其序列特征进行分析;同时利用实时荧光定量PCR (RT-qPCR)方法分析了PmCaspase-3基因mRNA在马氏珠母贝不同组织和不同发育时期的表达差异.结果 显示,PmCaspase-3基因cDNA全长为2233 bp,其中5'端非编码区长度为80 bp,3'端非编码长度为31 bp,开放阅读框长度为2088 bp,共编码695个氨基酸;生物信息学分析显示,PmCaspase-3含有Caspase家族特有的CASc结构域和半胱氨酸蛋白酶家族p20、p10活性位点以及多种磷酸化位点,经进化分析以及多序列比对可知与其他物种Caspase-3蛋白同源性较高.RT-qPCR结果表明,PmCaspase-3在肝胰腺中的表达量最高,在闭壳肌中的表达量最低;在发育过程中D型幼虫期和眼点期的表达量较高.     

温度、盐度和pH对马氏珠母贝稚贝清滤率的联合效应

清滤率(Clearance rate, CR)与滤食性贝类生长发育密切相关,采用Box-Behnken设计(BBD)和响应曲面法,在实验室条件下研究了温度(18-34℃)、盐度(20-40)和pH(6.5-9.5)对马氏珠母贝(Pinctada martensii)稚贝清滤率(CR)的联合效应,旨在建立温度、盐度和pH对马氏珠母贝稚贝清滤率的定量关系模型,并通过统计优化方法得出温度、盐度和pH的最佳组合。结果表明:温度的一次效应、温度和pH的互作效应、盐度和pH的互作效应以及温度、盐度和pH的二次效应对马氏珠母贝稚贝清滤率的影响均极显著(P<0.01);盐度的一次效应、pH的一次效应以及温度和盐度的互作效应对清滤率无显著影响 (P>0.05)。实验得出的清滤率模型决定系数为0.9950,预测决定系数为0.9284,表明该模型建立有效并可用于预测马氏珠母贝稚贝的清滤率。通过采用优化方法得出,在温度26.95℃,盐度29.69,pH8.09时,稚贝清滤率达到最大,最大值为1.4894×10^-3L/h,满意度为0.9886。研究结果可为马氏珠母贝滤食生理研究及稚贝培育提供理论依据。     

马氏珠母贝Sox11基因的克隆及时序表达模式分析

为探究Sox(SRY-related HMG-box genes)基因家族在马氏珠母贝个体发育及性别分化中的作用, 研究首先利用兼并引物从马氏珠母贝基因组中克隆到一个HMG框(high mobility group box), 利用RACE-PCR技术从SMART cDNA文库中克隆到一个Sox基因的cDNA全长, 通过Clustal X和MEGA 4软件对该序列进行比对分析并构建系统进化树; 通过荧光定量PCR技术对该基因在不同组织及发育不同时期性腺中的表达情况进行分析。结果显示, 马氏珠母贝该Sox基因的cDNA全长为1579 bp, 其中开放阅读框(ORF)为1008 bp, 编码336个氨基酸, 5'非编码区为126 bp, 3'非编码区为445 bp。同源性分析表明, 马氏珠母贝Sox基因与太平洋牡蛎Sox11基因的同源性(Identity)最高, 为80%, 故命名为pmSox11; 系统进化树分析也显示pmSox11与太平洋牡蛎Sox11基因的亲缘关系最近。荧光定量PCR分析组织表达特异性显示, pmSox11在马氏珠母贝神经节分布较多的组织如外套膜、鳃、足、消化盲囊等大量表达, 在神经节相对较少的闭壳肌和卵巢中表达量较少; 时序表达图谱显示, pmSox11在3月龄幼贝性腺和1年龄发育早期精巢中表达量最高, 在2年龄成熟精巢和2年龄性转换性腺中表达量降低, 而在2年龄卵巢中表达量最低。研究表明, pmSox11基因可能在马氏珠母贝早期神经系统发育和性别发育的调控方面起到重要作用。     

马氏珠母贝galectin-4基因cDNA的克隆及表达分析

半乳糖凝集素(Galectins)属于一种多功能凝集素家族,在机体抵抗微生物的感染中起重要作用。本研究利用cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术克隆获得马氏珠母贝galectin-4(PmGal-4)基因cDNA的全长,并对其序列进行分析。PmGal-4基因cDNA全长1 071 bp,5′非翻译区(5′UTR)长75 bp,3′非翻译区(3′UTR)长72 bp;其开放阅读框(open reading frame,ORF)长度为924 bp,编码307个氨基酸组成的前体肽,理论分子量约为34.6 kD,理论等电点为8.80。多序列比对结果显示各物种间galectin-4具有高保守性。序列分析结果显示PmGal-4的氨基酸序列具有典型富含半胱氨酸(cysteine-rich domain,CRD)的结构域。实时荧光定量PCR分析表明,PmGal-4基因在马氏珠母贝所检测的组织中呈组成型表达特征,但在闭壳肌和外套膜中表达量最高。上述结果表明PmGal-4基因可能参与了马氏珠母贝多种生理功能,特别是在免疫防御反应中具有重要的作用。     

马氏珠母贝(Pinctada fucata martensii)Pm-CTSL基因的克隆及表达分析

组织蛋白酶L (cathepsin L, CTSL)是无脊椎动物体内非特异性免疫的重要组成部分。为了研究马氏珠母贝CTSL (Pm-CTSL)的功能,本实验利用RACE技术克隆获得了Pm-CTSL基因,并对其结构和组织表达模式进行了分析。结果表明,Pm-CTSL基因c DNA序列全长为1 237 bp,其中开放式阅读框957 bp,5'UTR69 bp,3'UTR 211 bp,共编码氨基酸318个。结构域分析发现Pm-CTSL具有CTSL两个典型的结构域Inhibitor_I29和Pept_C1。Pm-CTSL也具有信号肽、保守序列ERFNVN和GNFD、两个潜在的N-糖基化位点和催化活性位点三联体残基(Cys, His和Asn)。多序列比对结果表明Pm-CTSL与太平洋牡蛎的同源性最高,为42%;系统进化分析发现,Pm-CTSL与虾夷扇贝等贝类聚为一支。实时荧光定量分析发现,Pm-CTSL在肝胰腺中显著高表达(p<0.05),表明Pm-CTSL可能参与马氏珠母贝的免疫应答。本研究为进一步探讨CTSL在马氏珠母贝非特异性免疫应答中的作用提供了基础资料。     

Characterization of 31 EST-derived microsatellite markers for the pearl oyster Pinctada martensii (Dunker)

We developed and characterized 31 microsatellite markers from expressed sequence tags of Pinctada martensii (Dunker). The number of alleles per locus ranged from 4 to 18 as determined in 44 individuals from a wild population. The expected heterozygosity ranged from 0.4121 to 0.9436, while the observed heterozygosity ranged from 0.4054 to 0.7273. Most of the loci are in Hardy-Weinberg equilibrium. These markers should be useful for population genetics studies, parentage and genome mapping in this species.     

Identification of EGFR in pearl oyster (Pinctada fucata martensii) and correlation analysis of its expression and growth traits

Marine pearl production is directly influenced by the growth speed of Pinctada fucata martensii. However, the slow growth rate of this organism remains the main challenge in aquaculture production. Epidermal growth factor receptor (EGFR), an important receptor of tyrosine kinases in animals, plays versatile functions in development, growth and tissue regeneration. In this study, we described the characteristic and function of an EGFR gene identified from P. f. martensii (PmEGFR). PmEGFR possesses a typical EGFR structure and is expressed in all studied tissues, with the highest expression level in adductor muscle. PmEGFR expression level is significantly higher in the fast-growing group than that in the slow-growing one. Correlation analysis represents that shell height and shell weight show positive correlation with PmEGFR expression (p < 0.05), and total weight and tissue weight exhibit positive correlation with it (p < 0.01). This study indicates that PmEGFR is a valuable functional gene associated with growth traits.     

马氏珠母贝3个野生种群及种群间杂交后代遗传多样性的ISSR分析

运用ISSR分子标记技术分析了马氏珠母贝(Pinctada martensii)广西北海(BW)、广东大亚湾(DW)和海南三亚(SW)野生种群及其自繁子一代(BB1、SS1、DD1)和杂交子一代(BS1、BD1、DS1)9个群体各50个个体的遗传多样性.结果表明:3个野生群体遗传多样性分别是0.2585、0.2607和0.2571;3个自繁群体子一代遗传多样性分别是0.2504、0.2545和0.2527,3个杂交子一代遗传多样性分别是0.2747、0.2659和0.2784.种群间杂交增加了子代的遗传多样性,同时增加了杂交子一代与杂交亲本间的遗传距离,而自繁群体的遗传多样性与其来源的野生种群比较,遗传多样性降低.本文指出利用野生群体进行杂交育种应该纯化亲本才能获得杂种优势,人工育苗或选择性育种需要保持足够数量的繁殖亲本以避免遗传多样性降低.     

Realized heritability and genetic gains of three generation for superior growth in the pearl oyster Pinctada martensii

The pearl oyster Pinctada martensii is an important economic shellfish species and mainly cultured in the southern provinces of China. The species is cultured for marine pearl production. However, pearl quality has recently decreased because the cultured stocks had slow growth and mass mortalities caused by inbreeding depression. We have initiated a selective breeding program to improve the cultivated stocks since 2002. In 2004, a base population was developed by collecting the breeders from Beibuwan and Liushagang stocks. During the period of 2005–2007, a two successive generation selection for shell length in the base population was carried out to produce the second generation selected and unselected lines. In May of 2008, three types of lines were produced by selecting the breeders from the second generation selected and unselected lines. The three types of lines were designated as SS (Selected for three generations), SC (selected for the two generations) and CC (unselected for three generations). Realized heritability for the third generation, cumulative (over three generation) and current (for the third generation) genetic gains were evaluated by comparing the growth performance of three types of lines at days 8, 16, 45, 75, 180 and 360. It was found that the SS lines had significant larger mean shell length and shell height than the SC and CC lines at all sampling ages (P < 0.05). The realized heritability estimate for adult shell length for the third generation was 0.41, similar with those detected for the first and second generation. The cumulative and current genetic gains for adult shell length were 20.94% and 13.27%, respectively. The present results indicate that there exists a high genetic variation in the population and mass selection is potential to improve pearl oyster stocks.     

The influence of cooling rate,developmental stage,and the addition of sugar on the cryopreservation of larvae of the pearl oyster Pinctada fucata martensii

This paper examines the effects of cooling rate, developmental stage, and the addition of sugar on the cryopreservation of the larvae of the pearl oyster, Pinctada fucata martensii. The survival rates of frozen-thawed trochophores was 43.1% at a cooling rate of 1.0 degrees C/min. The survival rate of frozen-thawed larvae increased with developmental stage, except for umbo stage larvae, and the late D-shaped larvae showed a survival rate as high as 91%. The addition of sugar (0.2M glucose or sucrose) improved the survival rate of larvae. These results indicate that the preferred cooling rate, developmental stage, and sugar for the cryopreservation of pearl oyster larvae are 1 degrees C/min, late D-shaped larvae and 0.2M glucose or sucrose.     

马氏珠母贝(Pinctada fucata martensii)细胞粘附分子3(Pm-CAM3)基因的克隆与表达分析

细胞粘附分子3 (cell adhesion molecule 3, CAM3)是免疫球蛋白家族(immunoglobulin family, IgSF)的一员,在细胞黏连、外源病原体的识别等方面具有重要作用。本实验利用末端快速克隆(RACE)技术,克隆获得马氏珠母贝(Pinctada facata martensii)细胞粘附分子3的全长序列(Pm-CAM3),并使用荧光定量PCR(Real-time PCR, q RT-PCR)技术检测了Pm-CAM3在马氏珠母贝不同组织中的表达模式。结果显示,Pm-CAM3基因全长2 245 bp。其中5'UTR 335 bp,3'UTR 166 bp,开放阅读框(ORF)长度为1 744 bp,共编码581个氨基酸;预测其相对分子量为63.21 kD,等电点为5.07,脂溶指数(aliphatic index)为67.21;总平均亲水性(grand averageofhydropathy, GRAVY)为-0.549,属于亲水性蛋白;Pm-CAM3具有跨膜结构域,其胞外区域具有一个Ig SF家族典型的Ig结构域以及一个Ig-like结构域。多序列比对结果显示,Pm-CAM3在物种间的保守性较低,其中Pm-CAM3与太平洋牡蛎的CAM3 (Crassostrea gigas, Cg-CAM3)的氨基酸序列相似性最高,但仅为37%。qRT-PCR分析表明Pm-CAM3在马氏珠母贝的9个组织中均有表达,其中在鳃中的表达量最高(p<0.05)。     

马氏珠母贝(Pinctada fucata martensii)Pm-HSP70基因的克隆及其对温度胁迫的响应

本实验利用RACE技术克隆获得了马氏珠母贝HSP70 (Pm-HSP70)基因,并对其基因和氨基酸序列结构特征进行了生物信息学分析,同时采用荧光定量PCR技术分析了该基因在不同组织的表达模式及不同温度下的时序表达模式。序列分析表明,Pm-HSP70 cDNA序列全长为2 215 bp,其中开放式阅读框1 899 bp,5'UTR 107 bp,3'UTR 209 bp,编码632个氨基酸,理论蛋白分子量为69.44 kD,理论等电点为5.61。该蛋白具有HSP70家族典型的结构域HSP70,以及ATP结合位点、细胞质特征性保守序列和3个HSP70家族标签。多序列比对结果表明Pm-HSP70与菲律宾蛤仔HSP70同源性最高,为80%;系统进化分析发现,Pm-HSP70与菲律宾蛤仔等贝类HSP70聚为一支。组织表达定量分析结果显示,Pm-HSP70在马氏珠母贝多个组织中均有表达,在肝胰腺中表达量最高,其次是性腺;对不同温度下鳃组织中Pm-HSP70的时序表达分析发现,在处理后各时间点高温组(32℃)基因表达水平最高,且均显著高于对照组(22℃)和低温组(17℃)。以上结果表明Pm-HSP70可能参与马氏珠母贝的高温胁迫响应。该研究为进一步探索Pm-HSP70在马氏珠母贝温度适应性中的作用提供了基础资料。     

Realized heritability and genetic gains of three generation for superior growth in the pearl oyster Pinctada martensii

The pearl oyster Pinctada martensii is an important economic shellfish species and mainly cultured in the southern provinces of China. The species is cultured for marine pearl production. However, pearl quality has recently decreased because the cultured stocks had slow growth and mass mortalities caused by inbreeding depression. We have initiated a selective breeding program to improve the cultivated stocks since 2002. In 2004, a base population was developed by collecting the breeders from Beibuwan and Liushagang stocks. During the period of 2005–2007, a two successive generation selection for shell length in the base population was carried out to produce the second generation selected and unselected lines. In May of 2008, three types of lines were produced by selecting the breeders from the second generation selected and unselected lines. The three types of lines were designated as SS (Selected for three generations), SC (selected for the two generations) and CC (unselected for three generations). Realized heritability for the third generation, cumulative (over three generation) and current (for the third generation) genetic gains were evaluated by comparing the growth performance of three types of lines at days 8, 16, 45, 75, 180 and 360. It was found that the SS lines had significant larger mean shell length and shell height than the SC and CC lines at all sampling ages (P < 0.05). The realized heritability estimate for adult shell length for the third generation was 0.41, similar with those detected for the first and second generation. The cumulative and current genetic gains for adult shell length were 20.94% and 13.27%, respectively. The present results indicate that there exists a high genetic variation in the population and mass selection is potential to improve pearl oyster stocks.     

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.