红脚艾蒿的转录组解析

[目的]利用高通量测序技术解析红脚艾(Artemisia rubripes Nakai)的转录组信息特征.[方法]通过高通量测序平台Illumina HiSeq 2500对红脚艾进行转录组测序,通过Trinity软件de novo组装获得Unigene,并基于序列同源性对Unigene进行功能注释,得到红脚艾的转录组信息.[结果]测序数据经过质控后共获得24126043条高质量的reads,通过de novo组装获得173093个转录本,对组装的转录本去冗余后共获得85991个Unigene,平均长度为616.87 bp,N50为925 bp.共有47216个Unigene在NR、KEGG、COG、KOG、GO数据库获得功能注释,40802个Unigene在NR数据库注释,显示红脚艾与向日葵(Helianthus annuus)的单基因匹配率最高,16846个Unigene被KEGG数据库注释到130条代谢途径中,26171个Unigene被注释到25个KOG功能分类中,23203个Unigene被GO注释到生物过程、细胞组成和分子功能三大类51个功能分类,12810个Unigene被注释到25个COG功能分类中.[结论]利用高通量测序技术获得了红脚艾转录组信息特征,这些数据将为后期开展功能基因鉴定、解析化合物次生代谢途径及其调控机制奠定研究基础.     

养殖型暗纹东方鲀生长与发育特性

观测养殖型暗纹东方鲀，获得4个年龄组的生长数据和解剖资料。拟合出雌、雄的体长体重关系和生长方程，分析其肥满度、胸腹围特点以及性腺和肝脏发育特点。结果显示，养殖型暗纹东方鲀雌雄体长生长规律相似；但体重在2．5龄前雄鱼生长速度大于雌鱼，2．5龄后则相反。养殖暗纹东方鲀体型比野生暗纹东方纯肥胖。体重／体长和肥满度皆大于野生型。研究还发现，养殖暗纹东方鲀性腺发育不良和肝脏脂肪变性发生率高，且两者的发生存在一定关系，致使全人工育成的成鱼繁殖难以成功。     

Low-salinity tolerance of juvenile fugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

ABSTRACT:   To study the adaptability of juvenile fugu Takifugu rubripes to low-salinity environments, fish were transferred from full-strength seawater (100% SW) to freshwater (FW) and 25, 50, 75 and 100% SW, and checked for mortality over 3 days. No mortality was observed in 25–100% SW, whereas all fish died in FW. In fish transferred to 25–100% SW, blood osmolality was maintained within a physiological range. To further explore the lower limit of salinity that fugu could tolerate, fish were transferred from 100% SW to FW and 1, 5, 10, 15 and 25% SW. All fish survived in 5–25% SW, but fish died in FW and 1% SW. In fish surviving transfer to FW and 1 and 5% SW, blood osmolality was decreased to a near sublethal level of approximately 300 mOsm/kg·H₂O. Therefore, the lower limit of salinity tolerance is estimated to lie between 5 and 10% SW. Preacclimation in 25% SW for 7 days did not essentially affect the survival salinity range. Although survival rates and blood osmolality were slightly improved by preacclimation in 25% SW, blood osmolality was markedly decreased in salinities less than 10% SW, as was seen in the direct transfer. Neither chloride cell morphology nor sodium-potassium adenosinetriphosphatase activity in the gills showed a significant change following transfer to low salinities. These findings indicate that fugu can be adapted to hypoosmotic environments to some extent, exerting hyperosmoregulatory ability, although chloride cells are less likely to absorb ions in hypoosmotic environments.     

温度对黄鳍东方鲀胚胎发育和仔鱼存活的影响

观察了不同温度条件下(20、23、26、29、32℃)黄鳍东方鲀受精卵的培育周期、孵化周期、孵化率和畸形率;并对黄鳍东方纯初孵仔鱼进行耐饥饿试验,测定其不投饵存活系数。试验结果表明,黄鳍东方纯受精卵孵化的适宜温度为20～26℃(最适温度为23℃);仔鱼生存的适宜温度为20～26℃(最适温度为23℃)。黄鳍东方鲀受精卵和仔鱼对温度这种环境因子的耐受范围基本相同。     

红鳍东方鲀幼鱼对饲料中蛋氨酸需求的研究

在半精制饲料中分别添加0、0.30%、0.60%、0.90%、1.20%、1.50%蛋氨酸制成6组等氮等脂饲料,各组蛋氨酸的实际含量分别为0.61%、0.85%、1.10%、1.39%、1.60%、1.84%,对初始体重为(13.83±0.63) g的红鳍东方鲀(Takifugu rubripes)幼鱼在室内流水系统中进行了76 d的喂养实验,探讨其蛋氨酸的需求量。研究表明,当饲料中蛋氨酸水平由0.61%升高至1.60%时,红鳍东方鲀的特定生长率(Specific growth rate,SGR)、饲料效率(Feed efficiency,FE)、蛋白效率比(Protein efficiency ratio, PER)均显著升高(P0.05),且都在1.60%组取得最大值。其中,特定生长率在1.39%、1.60%实验组无显著差异(P0.05),但随着蛋氨酸的进一步升高,特定生长率、饲料效率、蛋白效率比均呈现出下降的趋势;蛋氨酸水平对于蛋白沉积率没有显著影响(P0.05);饲料中蛋氨酸水平对全鱼粗脂肪含量呈现出先升高再降低的趋势,但各组间粗蛋白、水分和粗灰分无显著差异(P0.05);随着蛋氨酸水平升高,肝体比(Hepatosomatic index, HSI)和脏体比(Viscerosomatic index, VSI)呈现先升高再下降,在蛋氨酸含量为1.60%时达到最高,显著高于蛋氨酸0.61%组(P0.05),与其他组无显著差异;血清中甘油三酯、胆汁酸的含量,随着蛋氨酸水平的升高呈现出先升高再降低的趋势,饲料蛋氨酸水平对血清中总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、总蛋白水平及谷草转氨酶、谷丙转氨酶的活性无显著影响(P0.05)。以特定生长率为评价指标进行一元二次回归曲线分析,得出体重为(13.83～82.47) g的红鳍东方鲀幼鱼的蛋氨酸需求量为1.38%,占饲料蛋白质的2.71%。     

Enrofloxacin pharmacokinetics in Takifugu flavidus after oral administration at three salinity levels

The present study was designed to explore the pharmacokinetics of enrofloxacin in Takifugu flavidus at different salinity levels (10‰, 20‰ and 30‰). The concentrations of enrofloxacin in plasma and tissues (kidney, liver and muscle) of T. flavidus after a single oral dose of 10 mg kg^?1 were simultaneously determined using HPLC. The parameters of pharmacokinetics were calculated using non‐compartmental model based on statistic moment theory. The peak concentrations of enrofloxacin were higher and T_max values in plasma, liver and muscle tissues of T. flavidus were lower at the salinity of 20‰ and 30‰ than that of 10‰, except the kidney tissue. These demonstrated the absorption of enrofloxacin was more quickly and more quantity at the high salinity. The elimination half‐life (t_1/2z) of enrofloxacin in the plasma (45.22, 69.91 and 95.45 h) was increasing with the increase in salinity. And the t_1/2z values of enrofloxacin in liver (76.44, 44.21 and 33.48 h) and kidney (212.16, 157.43 and 35.61 h) both decline with the increase in salinity, indicating the elimination of enrofloxacin was faster in liver and kidney but slower in plasma at the high salinity. The content of enrofloxacin in liver was increasing with the increase in salinity, suggesting that salinity affects tissue distribution and metabolism in T. flavidus. In addition, the AUC_0–∞ data of enrofloxacin (kidney > plasma > liver > muscle) at different salinity levels were essentially consistent, and AUC_0–∞ data of kidney were declined with the increase in salinity, supporting that the kidney was the main organ and the role of extent of kidney on excretion was different in different salinity levels.     

Inhibitory modes of Kunitz and Bowman-Birk soybean trypsin inhibitors to tryptic and chymotryptic proteinases of tiger puffer and yellowtail

SUMMARY: An inhibitory effect of Kunitz and Bowman-Birk soybean trypsin inhibitors (K- and BB-SBTI) was investigated on tryptic and chymotryptic proteinases of a brood of tiger puffer Takifugu rubripes and yellowtail Seriola quinqueradiata . These assist to comprehend the differences in dietary soybean meal (SBM) utility by fish species and to develop low cost SBM diets with low contents of fish meal. The K-SBTI strongly inhibited tryptic proteinase and weakly inhibited chymotryptic proteinase from the hepatopancreas and intestine including pyloric ceca of the fishes. The BB-SBTI strongly inhibited both proteinases of the fishes. The K-SBTI inhibition for tryptic protease and the BB-SBTI inhibition for tryptic and chymotryptic proteases were relatively constant in tiger puffer with growth of 12–199 g bodyweight. In yellowtail with growth of 7–672 g bodyweight, the K-SBTI inhibition for tryptic protease and the BB-SBTI inhibition for chymotryptic protease were also relatively constant. The BB-SBTI inhibition for yellowtail tryptic protease typically fell in the stage from 7 g to 57 g bodyweight, and then decreased slightly or was maintained constantly. These results indicate that there are different inhibitory modes of K- and BB-SBTI and there is inter- and intraspecific diversity of tryptic protease conformation between the two species.     

红鳍东方鲀防御素db-1基因的原核表达分析

采用原核表达方法高效表达红鳍东方鲀Takifugu rubripes防御素db-1基因,并获得该基因的重组表达产物。通过RT-PCR扩增获得红鳍东方鲀防御素db-1基因成熟肽编码序列,将其连入pET32a+载体,构建原核表达载体pET32a-m DB-1,然后将其转入至大肠杆菌表达菌株Rosetta(DE3)感受态细胞中,经IPTG诱导目的基因表达,通过SDS-PAGE和Western Blotting检测该蛋白,并利用Ni-NTA亲和层析纯化该蛋白,最后以Anti-Mus defensin Beta2抗体为探针的Elisa反应鉴定该蛋白。结果表明:红鳍东方鲀db-1基因在大肠杆菌中高效表达,并纯化得到了这种重组防御素融合蛋白。本研究中提供了一种简单、高效获取重组防御素蛋白的方法,可为其应用和生产提供支持。     

暗纹东方鲀亲鱼产后护理的初步研究

采用降温、防感染处理和加强营养等方法对野生暗纹东方亲鱼进行产后护理,使野生亲鱼产后的成活率达到75%,人工养殖亲鱼产后成活率达到100%。暗纹东方亲鱼产后死亡的一般原因多为鱼体受伤、催产不当、忽视产后亲鱼护理所致。对产后亲鱼采取适当的护理措施,可提高亲鱼利用率。     

红鳍东方鲀SREBP-1基因的电子克隆及生物信息学分析

利用斑马鱼的SREBP-1 的氨基酸序列NP_001098599.1 为探针,对红鳍东方鲀基因组数据库进行BLAST 分析,获得红鳍东方鲀（Takifugu rubripes）的固醇调节元件结合蛋白-1 （Sterol regulatory element bindingprotein-1,SREBP-1）的cDNA 序列及相应的氨基酸序列。采用生物信息学方法对其基本理化性质、蛋白质结构及功能结构域进行分析。结果发现红鳍东方鲀SREBP-1 基因的开放阅读框全长3 330 bp,共编码1 109 个氨基酸;红鳍东方鲀SREBP-1 的氨基酸序列与黄斑蓝子鱼、大西洋鲑、斑马鱼、人、牛及原鸡的同源性分别为81%、73%、72%、55%、55%、54%和54%;系统进化分析结果表明,红鳍东方鲀SREBP-1 与黄斑蓝子鱼的进化关系最为密切,亲缘关系最近;红鳍东方鲀SREBP-1 蛋白中α-螺旋较多,该蛋白质氨基端包含1 个bHLH-zip 的结构。