荆州地区草鱼生长状况及血清生化指标参考值的初步探讨

选取湖北省荆州地区池塘养殖草鱼,针对其生长、生物学特性及12项血清生化指标进行了检测,这将对草鱼血清生化指标参考值的建立以及健康养殖和调控,具有一定的参考意义。由于影响动物血清生化指标的因素较多,该试验中草鱼样本数相对较少,在今后的研究中尚需加大样本量进行更深入的探讨。     

草鱼羧肽酶A1基因(CPA1)部分片段的单核苷酸多态性(SNP)多态性及其与生长性状的关联分析

羧肽酶A (EC 3.4.16)是一类水解蛋白和多肽底物C端芳香族氨基酸或脂肪族氨基酸残基的消化酶.在草鱼生长相关的功能基因上研究单核苷酸多态性(SNP)与生长的相关性可为草鱼的分子辅助育种提供依据.本研究根据草鱼(Ctenopharyngodon idella)EST-SNP库的羧肽酶A1基因(CPA1)重叠群的2个Contig扩增该基因的序列片段,采用直接测序法,经过序列比对,共筛到2个颠换SNP位点:C+412A和A36C,分别位于CPA 1基因外显子5的34bp和内含子3的36 bp处,前者为错义突变.然后用一个群体的296尾草鱼对这2个位点用SnaPshot的方法进行检测和分型,统计基因型频率:A36C位点的AA基因型占26.7％,AC基因型占52.0％,CC基因型占21.3％.C+412A位点的AA基因型占15.5％,AC基因型占40.5％,CC基因型占43.9％.利用一般线性模型分析2个SNP位点与草鱼体质量、体长等重要生长性状的关系.关联分析结果显示,C+36A位点不同基因型在体质量、眼间距均值上存在显著差异(P＜0.05).并且AA基因型和CC基因型在体质量、体长、体宽和眼间距上存在显著差异(P＜0.05).AA基因型各项指标均值显著高于CC基因型.C+412A位点在体质量等生长性状上差异不显著(P＞0.05),该位点和生长不相关,但是CC基因型和AC基因型在体重和肛前距上差异显著(P＜0.05),该位点CC基因型的6个生长性状均值都高于AA基因型.由两个位点组成的5种双倍型在体质量上存在显著差异(P＜0.05).双倍型D3和D5在体质量上存在差异显著(P＜0.05).双倍型D3和D8在体质量、体宽、体长、体长/头长等生长性状上都存在显著差异(P＜0.05).D3的各项指标均值最高,D8的各项指标均值最低.本研究结果显示,可以考虑将草鱼CPA1基因作为候选基因,用于草鱼的分子辅助育种.     

Microsatellite marker isolation and cultured strain identification in <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.     

锦鲤保护性组织RNA提取及引物扩增研究

从养殖锦鲤各种组织中提取完整的RNA, 是分子生物学研究锦鲤应激表达、体色变异、生长发育等功能基因表达的关键实验。在不伤害养殖锦鲤个体健康的情况下, 取用鱼体的鳍条、鳞片、鳃丝、血液4种保护性组织,可以最大程度地减少对珍贵锦鲤个体的损伤, 并达到研究相关基因表达的目的。本研究利用TR izo l法提取RNA, 获得的各样品RNA条带完整、纯度高。结果表明, 4 种保护性组织提取的RNA 经过逆转录反应, 得到了高质量的CDNA。内标基因( B- actin)、热激蛋白基因( hsp70)、金属硫蛋白基因(MT)扩增得到清晰的条带, 通过NCB I数据上的BLAST比对证明, 各序列的同源性在95%以上。利用本研究的方法, 可以充分保护养殖锦鲤个体的完好性, 用于进一步的分子生物学研究。     

鳙鳃凝集素的分离纯化及理化性质的研究

利用DEAE-Sepharose FF离子交换和Sephacryl S-200 HR及Superdex 200 10/300 GL分子筛层析技术,从鳙鳃组织中分离纯化到一种岩藻糖专一的凝集素,命名为GANL.在还原SDS-PAGE电泳上显示单一蛋白染色带,其亚基相对分子质量为37 kD.经Superdex 200凝胶过滤层析测得其天然相对分子质量为220 kD.GANL的中性糖含量为13.4%.因此,GANL作为一种由相同亚基组成多亚基糖蛋白.GANL对兔红细胞有专一的凝集活性,其凝集活性不依赖Ca~(2+).在被测的单糖、双糖及糖蛋白中,仅岩藻糖能抑制其凝集活性.氨基酸分析表明GANL的Asp、Glu、Leu、Val、Lys的含量较高,Cys-S的含量为0.81%.GANL的最适pH为8～9;具有很高的热稳定性,最适温度为50 ℃.     

北方地区鲤鱼出血病流行病学分析

文章通过对北方地区鲤鱼出血病病原、症状、流行情况以及影响疾病流行的主要因素进行分析，提出了鲤鱼出血病防治的主要措施。     

异育银鲫耗氧率和窒息点的研究

为了解昼夜变化、体重及水温对异育银鲫耗氧量、耗氧率和窒息点的影响，应用流水呼吸室法和静水呼吸室法分别测定异育银鲫在不同条件下的耗氧量，结果表明：异育银鲫日间平均耗氧高于夜间平均耗氧，耗氧量随体重增加而增加，呈显著正相关关系；而耗氧率和窒息点随体重增加而减少，呈显著负相关关系，耗氧量、耗氧率、窒息点随水温增加而增加，呈正相关关系。     

松浦镜鲤与德国镜鲤选育系（F4）的可量性状、鳞片数比较

对松浦镜鲤（SP）和德国镜鲤选育系（F4）（DJ）的可量性状及体表鳞片数进行比较，SP与DJ的可量性状与体表鳞被存在差异。SP的体长／体高（2．44±0．15）、头长／眼间距（2．24±0．08）明显小于DJ，体长／头长（3．86±0.20）、头长／吻长（2．99±0．23）明显大于DJ，SP体表的鳞片数明显少于DJ。结果表明，从DJ到SP遗传选育过程中，体型和鳞片的变化已达到了选育目的。     

Effects of dietary medium-chain triacylglycerols (tricaprylin and tricaproin) and phospholipid supply on survival, growth and lipid metabolism in common carp (Cyprinus carpio L.) larvae

The present study investigated the interaction of dietary medium-chain fatty acids (MCFA) and phospholipids (PL) on survival, growth and lipid metabolism in common carp larvae. Nine diets based on casein and dextrin and with a variable lipid part were tested in triplicate for 22 days post first feeding. The 3×3 design consisted of three triacylglycerols (3% of diet) combined with three different lipid supplements. Tested triacylglycerols were triolein (TOL), tricaprylin (TC8) and tricaproin (TC6), and lipid supplements were 2% soybean oil (low-fat diets without PL), 2% soybean lecithin (low-fat diets with 2% PL) or both 2% soybean lecithin and 6% TOL (high-fat diets with 2% PL).

In the first step, both TC6 and TC8 resulted in improved survival and growth rates compared to TOL, irrespective of the PL supply. In the second step, TC8 decreased survival and growth rates, whereas the difference between TC6 and TOL became less. Histological signs of impaired intestinal absorption of neutral lipids were evidenced in larvae fed TOL without PL and also in high-fat diets with 2% PL. The latter diets also resulted in poorer growth rates compared to low-fat diets with 2% PL. These results suggest that the quantitative PL requirement of larvae increases as the dietary level of long-chain triacylglycerols increases. Larvae fed TC6 or TC8 showed enlarged liver and hepatocyte volume and a decreased level of body neutral lipids. Based on β-hydroxybutyrate (β-HBA) measurements in whole larvae, TC8 was found to be more ketogenic than TC6. TC6 and TC8 affected differently the fatty acid profile of larval body neutral lipids. TC6 did not induce the appearance of MCFA, whereas TC8 feeding resulted in a low level of 8:0 and relatively high levels of 10:0 (3.8% of total fatty acids). Neither 8:0 nor 10:0 were found in larval polar lipids.

This study confirmed the essentiality of PL in common carp larval diets and underlines differences in the utilization of TC6 and TC8, which both initially stimulate growth during the first week, but only temporarily in the case of TC8.     

Purification and characterization of two anionic trypsins from the hepatopancreas of carp

SUMMARY: Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH₂-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively.