微卫星分型方法进行中国明对虾家系系谱鉴定

微卫星(microsatellite)又称简单序列重复(simple sequence repeats,SSR),是20世纪80年代末期发展起来的DNA分子标记技术,以其数量多、分布广、多态性丰富、检测快速方便、共显性遗传等优点而被广泛应用于构建连锁图谱、个体识别、亲子鉴定、遗传多样性分析、疾病诊断、基因定位等诸多领域.微卫星研究的层次也从群体、个体到基因的表达.虽然有关微卫星起源、演化等相关的理论问题还未研究清楚,但自微卫星标记被发现始就被应用于相关研究,并显示出明显优势,是最具应用价值的标记技术之一.     

基于SAMPL的一种微卫星筛选方法在中国明对虾中的尝试

微卫星的发展主要受限于微卫星及其旁侧特异引物区的分离,在中国明对虾(Fenner-openaeus chinensis)中建立了选择性扩增多态微卫星位点(SAMPL,selective amplified mic-rosatellite polymorphic loci)技术体系,并初步尝试了基于SAMPL的一种微卫星筛选方法。通过5′端锚定引物引入微卫星序列,对传统SAMPL技术加以改进并以期富集简单重复序列。从测序胶上随机选择5条SAMPL条带(分别命名为S11,S13,S14,S22,S24)克隆测序,大小分别为161,157,147,152,298。其中S11,S13,S14,S22四个片段两端引物分别为AFLP选择性引物和SAMPL引物,S13和S22含微卫星序列重复,S24两端引物均为传统的AFLP引物,在此片断中有一(AG)39重复。     

Establishment of microsatellite-based triplex PCR for parentage analysis of Chinese shrimp Fenneropenaeus chinensis

Through exploring the microsatellite primers from the random genome sequences of Chinese shrimp (Fenneropenaeus chinensis), some microsatellite primers were obtained with rich polymorphic genetic information, and a triplex PCR was established using three primers (RS1101, RS0683 and H081 primers). By adjusting the final concentration of Mg2 , dNTP and primers, and using a touch-town PCR program, the optimum amplification parameters of PCR system were obtained, which could successfully amplify the three primers in a PCR reaction. In the denatured PAGE gel, the amplified DNA fragments of three primers RS1101, RS0683 and H081 could be easily identified each other. For the triplex PCR system, the PPE (probabilities of paternity exclusion) is 0.967 9, and the DP (discrimination power) is 0.999 327. Using the triplex PCR to test ten individuals of a parentage and their parents, an individual was excluded from the parentage in all of the three microsatellite loci, which might be mixed into the parentage for some unknown reason such as factitious misplay. The triplex PCR will be of great practical value in identifying the parentages of F. chinensis.     

中国对虾6种组织cDNA文库的构建

以中国对虾血液、眼柄、卵巢、雌虾头胸部、雄虾头胸部和三倍体对虾头胸部组织为材料,采用异硫氰酸胍-酸酚法提取总RNA;用磁珠法纯化mRNA;用Uni ZAP cDNA合成试剂盒合成双链cDNA并进行修饰,将cDNA定向连接在UniZAP载体上;经Gigapack Gold Package包装试剂盒包装成为噬菌体颗粒,转染宿主XL1 Blue MRF’菌株细胞,形成初级文库.初级文库经转染进一步扩增,形成稳定的cDNA文库.6个文库的库容在0.2×106~1.3×106之间,重组率都超过90%.从各文库随机取出6~10个清晰的噬菌斑进行PCR扩增,用琼脂糖凝胶电泳检测,其插入片段长度为500~2500bp.由初步功能基因克隆获得阳性结果.多项指标表明,所构建的对虾cDNA文库质量较高,为进一步筛选目的基因、EST测序和制作基因芯片提供了有效的工具.     

中国明对虾2个群体的杂交子一代早期分析

目前,对虾研究主要集中在基础遗传学和遗传改良的潜力研究等方面[1,2].不同的地理群体代表不同的遗传资源,尤其是对虾人工养殖迅速发展的情况下,对不同的群体生长发育性状特点进行评估是非常重要的.遗传改良的目标是提高对虾人工养殖群体的产量,这就需要育种计划不仅要建立在对虾自然群体相关性状差异的研究上,还需要进一步了解不同发育阶段主要经济性状的遗传特性.对虾幼体生长情况和存活率一直都是养殖者和科学工作者关注的问题.已经有研究证明美国大西洋牡蛎和港湾扇贝群体间在幼体生长发育和存活率方面存在遗传差异[3,4].杂种优势在生物界是一个普遍的现象.     

氟苯尼考3种不同给药方式在中国明对虾体内的药代动力学研究

用高效液相色谱法研究静注、肌注和口服氟苯尼考在中国明对虾(Fenneropenaeuschinensis)体内的药代动力学特征,所得数据用MCP-KP程序分析。结果表明,3种给药方式的血药经时过程均符合二室开放模型,符合一级吸收二项指数方程,血窦注射、肌肉注射、口服药饵的时间对药物质量浓度的理论方程分别是:C血注=16.38e-2.58t 0.32e-0.1t,C肌注=18.7e-2.57t 0.26e-0.12t,C药饵=15.08e–1.49t 0.65e-0.09t–15.74e–10.38t。主要药物代谢动力学参数分别是:吸收半衰期(t(1/2)α)为0.269,0.270和0.465h,消除半衰期(t(1/2)β)为6.921,6.494和7.903h,表观分布容积(Vβ)为1.287,1.293和2.421L/kg,总体清除率(Cs)为0.129,0.138和0.213L/(h·kg),药时曲线下总面积(Au,c)为0.755,0.681和0.666L/(h·kg)。肌肉注射和口服给药的生物利用度(F)分别是90.20%和97.58%。     

中国对虾家系建立及不同家系生长发育的初步研究

在中国对虾(Fenneropenaeus chinensis)家系建立过程中,我们采用定向交配对子代进行选育的方法.采用4种中国对虾人工授精方法,即整个精荚移植、精荚切块移植、输精管移植和精液移植,共进行了155例雌虾的人工授精实验,有108尾雌虾成功产卵并孵化出仔虾,成功率为69.7%,建立了101个全同胞家系和29个半同胞家系.在受精卵孵化、育苗的基础上,同时进行了不同家系幼体生长情况的研究,结果表明受精率、孵化率、存活率以及初期生长在4个实验组间都没有显著性差异.另外对其他家系之间的比较也得出同样的结果,说明不同家系在早期的生长中,没有表现出显著性差异.而在养成阶段,方差分析结果表明家系间在生长上表现出差异极其显著.     

中国对虾交尾雌虾蜕皮及人工再交尾研究

为更好地利用交尾雌虾,采用温差刺激、药物刺激对养殖交尾雌虾和海捕交尾雌虾进行蜕皮实验,使交尾雌虾的精荚随蜕皮而脱落,然后对蜕皮对虾再次进行精荚移植,来建立特定杂交组合的中国对虾(Fenneropenaeus chinensis)家系。实验结果表明,养殖交尾雌虾蜕皮实验中,在只有温差刺激的条件下,对虾蜕皮率为30%,且对虾成活率为100%,说明温差刺激在保持对虾较高存活率的情况下,可以促使中国对虾蜕皮;在温差刺激的基础上进行药物刺激,不同茶籽饼质量浓度梯度下对虾的蜕皮率存在显著差异,且随着药物浓度的增加,对虾蜕皮率也随之增加,3个茶籽饼质量浓度(30,60,90 g/m3)下的对虾蜕皮率分别为40%,60%,70%,茶籽饼质量浓度为0的对虾蜕皮率与60,90 g/m3质量浓度下的对虾蜕皮率存在显著差异(P0.05);从对虾的死亡情况看,当药物质量浓度达到90 g/m3时,对虾出现死亡,经x2检验表明,90 g/m3质量浓度与0～60 g/m3质量浓度下的对虾死亡率存在显著差异(P0.05)。在海捕交尾雌虾蜕皮实验中,对虾蜕皮率不高,只有在药物质量浓度为30,90 g/m3条件下才有对虾蜕皮,蜕皮率仅为20%,10%;每个药物浓度下均有对虾死亡,死亡率随着茶籽饼质量浓度升高而升高,不同药物浓度下的对虾死亡率存在显著差异。对蜕皮后雌虾进行精荚移植,共获得18个中国对虾家系,平均受精率和平均孵化率分别为51.2%,88.6%。本研究通过对交尾雌虾进行蜕皮后再交尾,获得了不同杂交组合的中国对虾家系,为进一步的中国对虾家系选育工作打下了基础。     

不同盐度波动幅度对中国明对虾稚虾蜕皮和生长的影响

研究了盐度波动幅度对中国明对虾稚虾(Fenneropenaeus chinensis)蜕皮和生长的影响.实验在水族箱内进行,实验对虾的初始体重为(0.553 2±0.000 1)g,投喂人工配合饲料.实验结果表明:(1)不同盐度波动幅度对中国明对虾稚虾的蜕皮周期有显著的影响(P<0.05),其中S10能明显抑制对虾的蜕皮,S2则能促进对虾的蜕皮,前者较后者蜕皮周期延长4.97 d;(2)不同盐度波动幅度对中国明对虾的特定生长率有显著影响(P<0.05),其中,S4和S7两组对虾的特定生长率最大,分别大于S0组28.11%和22.6%,并且与S0和其他处理组相比差异均达到显著水平(P<0.05);(3)不同盐度波动幅度对中国明对虾的摄食量有显著影响(P<0.05),S4和S7两组分别高于S0组15.96%和11.46%;(4)不同盐度波动幅度对中国明对虾的食物转化效率未产生显著的影响P>0.05,但S4和S7两组对虾的食物转化效率较高.     

Expression profiles of penaeidin from Fenneropenaeus chinensis in response to WSSV and vibrio infection by real-time PCR

Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemoeyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a “U” like expression profile resulted. Expression levels of ponaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimierobial activities. In WSSV infection experiments, the expression profile appeared as an inverse ““““U““““ with the expression ofpenaeidin gradually decreasing to below baseline level after 24 h.The expression of antimierobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms ofvirns to haemoeytes and microbial to haemoeytes are completely different. Decline of ponaeidins expression levels may be due to haemoeytes being destroyed by WSSV or that the virus can inhibit the expression ofponaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.     

中国明对虾囊胚和原肠胚细胞的分离和培养

尝试了多种方法分离中国明对虾(Fenneropenaeus chinensis)囊胚细胞和原肠胚细胞并进行了细胞培养.结果表明,解剖法适用于囊胚细胞,自行设计的压片法适用于原肠胚细胞.在培养起始阶段,通过降低血清浓度和缩小培养体系可使中国明对虾囊胚细胞和原肠胚细胞迅速贴壁并铺展,囊胚细胞铺展后有明显的生长晕,中后期原肠胚细胞较易达到半汇聚状态.值得注意的是,中国明对虾囊胚细胞在不同培养液中培养可发生形态上的分化.本实验方法有望用于对虾细胞分化机理和对虾细胞建系的研究.     

盐度和Ca2+浓度对中国明对虾稚虾耗氧率的影响

设计双因子实验研究了水温25.0℃±0.5℃下,盐度(5,15,30)和Ca2 质量浓度(175,350,700,1 400,2 800 mg/L)对中国明对虾(Fenneropenaeus chinensis)稚虾耗氧率的影响,实验对虾的湿体质量为0.301 g±0.041 g。实验结果表明:(1)不同盐度下,中国明对虾稚虾耗氧率的大小顺序为R5>R30>R15。其中,盐度15下对虾的耗氧率显著低于盐度5和30下的耗氧率(P<0.05);(2)不同Ca2 质量浓度下,中国明对虾稚虾耗氧率的大小顺序为R2 800>R175>R700>R1 400>R350。其中,Ca2 质量浓度为350 mg/L组对虾的耗氧率显著低于其它处理组,Ca2 质量浓度为2 800 mg/L组对虾的耗氧率显著高于其它处理组(P<0.05),而Ca2 质量浓度为175,700和1 400 mg/L组间对虾的耗氧率差异不显著(P>0.05);(3)盐度和Ca2 质量浓度的交互作用显著影响中国明对虾稚虾的耗氧率(P<0.05)。     

中国明对虾caspase基因的克隆与表达分析

利用细胞凋亡来清除多细胞器官受损伤或有害的细胞,为动物发育过程中保持机体平衡起到关键作用。为了研究细胞凋亡在甲壳类动物应对病毒感染后所起到的作用,作者利用同源克隆技术获得了中国明对虾(Fenneropenaeus chinensis)caspase基因(FcCasp)的cDNA序列,并分析了该基因在对虾受到白斑综合症病毒(white spot syndrome virus,简称WSSV)刺激后的表达变化规律。结果表明,该基因的cDNA含有954个核苷酸,编码317个氨基酸,具有caspase家族典型的QACRG五肽蛋白酶活性位点(190-194氨基酸)。对注射了WSSV的中国明对虾FcCasp的表达进行分析,在5个时间点取其肝胰脏样本,经real-time PCR检测发现,WSSV刺激3 h后,对虾肝胰脏中FcCasp的表达呈现显著性上调,说明FcCasp的表达与WSSV感染密切相关,提示FcCasp基因与细胞凋亡相关。     

中国对虾选育群体“黄海1号”和野生群体不同组织基因组DNA甲基化的MSAP分析

DNA甲基化在调节动物生长发育和组织分化中发挥了重要作用。本研究主要从酶切、预扩和选扩等方面优化了中国对虾DNA甲基化分析的MSAP技术,给出了适合中国对虾MSAP分析的最近反应程序和体系,并采用该技术分别对中国对虾选育群体"黄海1号"和野生群体的肌肉、鳃和血细胞三种组织基因组DNA的CCGG甲基化水平进行分析。研究结果表明,中国对虾野生群体肌肉、鳃和血细胞的DNA甲基化比例分别为23.1%、22.3%和19.7%,选育群体"黄海1号"肌肉、鳃和血液的甲基化比例分别为21.4%、19.6%和18.9%。鳃组织的DNA甲基化水平在两群体中差异极显著(P <0.01),肌肉间的甲基化水平差异显著(P <0.05),而血细胞中甲基化水平差异不显著(P >0.05)。中国对虾野生群体和"黄海1号"同一组织间的甲基化水平不同(P ≤ 0.05),而不同组织间的甲基化水平也各不相同(P ≤ 0.05)。DNA甲基化多态性分析表明,野生群体和选育群体"黄海1号"的鳃和血细胞组织的甲基化多态性比例变化明显,而肌肉组织甲基化水平较稳定,这些变化趋势与CCGG位点的甲基化和去甲基化有关。     

中国对虾细胞色素P450基因CYP4原核表达条件优化

根据克隆得到的中国对虾（Fenneropenaeus chinensis）CYP4基因开放阅读框设计引物,构建了中国对虾CYP4基因原核表达载体p28a-CYP4,并对重组菌株Rosetta／28a-CYP4的原核表达条件进行了优化。结果表明：p28a—CYP4转化Rosetta后可实现CYP4基因的原核表达,SDS—PAGE分析显示其在56．0kDa处有显著诱导条带;通过对诱导温度、IPTG浓度、诱导时机（OD600）及诱导时间的优化表明,重组菌株Rosetta／p28a-CYP4的最佳诱导温度为37℃,最佳IPTG浓度为1．2mmol／L,最佳诱导时机及诱导时间分别为0．59和6h。     

黄芩苷在中国对虾体内对诺氟沙星消除及细胞色素P450 酶的影响

对照组中国对虾(Fenneropenaeus chinensis)连续投喂不含药物饵料,诺氟沙星组和联合用药组以50 mg/kg体质量剂量连续投喂含诺氟沙星药饵5 d后,诺氟沙星组投喂不含药物饵料10 d,联合用药组以100 mg/kg体质量剂量投喂含黄芩苷饵料10 d。从投喂诺氟沙星药饵时开始,在不同时间点采集中国对虾血液、肝胰腺、鳃和肌肉组织进行药物残留和肝药酶活性测定。结果表明:与诺氟沙星组相比,联合用药组各组织中诺氟沙星消除较快,血淋巴、肝胰腺、鳃和肌肉的理论休药期比诺氟沙星组分别缩短了23.92%、22.73%、25.92%和21.92%;与对照组相比,诺氟沙星对中国对虾CYP1A(ECOD)和CYP2(APND)酶活性有一定的抑制作用,但随着诺氟沙星的消除抑制作用逐渐减弱,最后恢复至对照水平;黄芩苷在加速诺氟沙星在中国对虾体内消除的同时,对中国对虾CYP450酶也有较强的诱导作用。因此,药物使用应注意药物之间的相互作用,以免造成药物中毒或疗效下降现象。     

Estimation of genetic parameters and breeding values in shrimp Fenneropenaeus chinensis using the REML/BLUP procedure

An analysis of a selection experiment was used to assess the impact of various animal model structures on REML estimates of variance components.The analyses were carried out based on 162 d body mass (BM) of 1 287 animals from 21 paternal half-sib groups of Fenneropenaeus chinensis.Estimated breeding values (EBV) of BM of all individuals were estimated using eight statistical models (A,AB,ABC,ABDC,ABMFC,ABMDC,ABFDC and ABMFDC) and BLUP (best linear unbiased prediction).These models were designed involving factors such as sex,spawn date as fixed effects,maternal genetic effects,full-sib family effects as random effects,mean BM of families at tagging and age at recording (covariate).The results demonstrate the importance of correct interpretation of effects in the data set,particularly those that can influence resemblance between relatives.The data structure and the particular model that was applied markedly influenced the magnitude of variance component estimates.Models based on few effects obtained upward biased estimates of additive genetic variance.The accuracy of genetic parameters and breeding value estimated by ABFDC model was higher than other models.The results imply that additive genetic direct value,full-sib family effects,and covariance effects besides sex and spawn date as fixed effects were very important for estimating genetic parameters and breeding value of body mass.This model had a heritability estimate of 162 d BM of 0.44.The comparison of the efficiency of selection based on breeding values or phenotypic value revealed great difference:average breeding value of the best 24 families selected by the 162 d BM breeding value and phenotype were 0.577 g and 0.366 g,respectively,representing a 36.57% higher efficiency in the former.In conclusion,selection based on breeding value was more effective than selection based on phenotypic value.Our results indicate that effects influencing the magnitude of estimates should be taken into account when estimating heritability and breeding values for BM.     

Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis

A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus （ Litopenaeus ） setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen.     

中国对虾(Fenneropenaeus chinensis)耐低温和生长性状的遗传参数估计

The inability of Fenneropenaeus chinensis to tolerate low temperatures is of major economic concern in temperate climates,as it reduces their growing season and leads to over-winter mortality.In this study,the heritability of body weight under low grow-out temperature and cold tolerance in F.chinensis were first investigated and estimated using 88 ful-sib families,which might provide crucial information in Chinese fleshy prawn breeding programs.The heritability for body weight under suitable and low temperature of F.chinensis were both moderate(0.158 0±0.307 5 and 0.132 0±0.026 9 respectively);the large coefficient of variation(approximately 21%) and moderate estimate of heritability for body weight indicated substantial potential for selective breeding.The heritability estimate for cold tolerance was low(0.019 2±0.023 5),and showed no significant differences from zero(P0.05).A weak genetic correlation between cold tolerance and body weight was also estimated in the present study,also showing no significant differences from zero(P0.05).Thus,more research needs to be conducted on the more accurate heritability estimate of cold tolerance and genetic correlations between traits in F.chinensis to further improve the achievement of breeding goals.     

中国明对虾血蓝蛋白C末端片段在毕赤酵母中的表达及其抗菌活性

为研究中国明对虾(Fenneropenaeus chinensis)血蓝蛋白C末端(FcHC-C)的抗菌功能,将血蓝蛋白基因FcHC的2个C末端基因片段连接到毕赤酵母表达载体pPIC9K中,构建酵母表达载体pPIC9K/FcHC-C。该载体经SalI酶切后,采用PEG法转化毕赤酵母(Pichia pastoris GS115)。转化子经过PCR鉴定后,阳性克隆通过含有G418的YPD平板筛选,获得高拷贝重组子。重组毕赤酵母利用甲醇诱导表达目的基因。经Tricine-SDS-PAGE和Western blot分析结果表明,利用酵母工程菌成功表达了血蓝蛋白C末端片段(rFcHC-C1和rFcHC-C2)。抑菌活性鉴定实验结果显示,重组蛋白rFcHC-C1和rFcHC-C2作为阴离子抗菌肽具有抗真菌和抗细菌的活性。