东方鲀生长激素基因3'-UTR多态性分析

以暗纹东方鲀（Takifugu. Obscurus）、红鳍东方鲀（Takifugu. Rubripes）、星点东方鲀（Takifugu. niphobles）共82个个体为对象,运用单链构向多态性（SSCP）技术和测序技术分析生长激素（Growth Hormone,GH）基因3＇非翻译区的多态性.结果表明3个群体中3＇非翻译区存在两种长度多态性,分别为320bp和317bp,与GenBank（登录号为：FRU63807）的序列（316bp）有差异;共检测到6种基因型,分别命名为aa、bb、ab、bc、cd、dd,变异频率达4.36%,其中在暗纹东方鲀中检测到四种aa、bb、ab、bc,cd和dd基因型分别只在于星点东方鲀和红鳍东方鲀检测到;7个突变位点中有1处颠换即位点212（T→G）,6处转换即位点120、180、227、265、287（C→T）和位点199（A→G）.     

基于轻量级卷积神经网络的红鳍东方鲀个体身份无损识别方法

文章充分利用红鳍东方鲀(Takifugu rubripes)体侧纹理特征提出了一种基于轻量级卷积神经网络的鱼类个体身份识别方法,可在无损前提下实现红鳍东方鲀个体身份的高精度识别。首先,采用SOLOv2模型进行前景分割,并结合红鳍东方鲀体型特点,通过质心和哈希值计算方法完成数据集生成和筛选;随后,从多维度分别测试主流深度学习图像分类骨干网络和不同损失函数在红鳍东方鲀身份识别中的效果;继而,在MobileNet v2骨干网络基础上,耦合Softmax Loss函数,建立了一种适用于红鳍东方鲀的个体身份无损识别的最优组合方法。研究结果表明,文章方法准确率可达90.2%,优于其他相关主流方法(准确率73.6%—89.3%),相关研究成果将为循环水养殖鱼类个体身份无损识别和精准生物量估算提供技术支撑。     

红鳍东方鲀干扰素γ基因的原核表达北大核心CSCD

旨在探究一种简单高效的获取重组红鳍东方鲀IFNγ蛋白的方法。提取红鳍东方鲀(Takifugu rubripes)肾脏组织的总RNA,并以其为模板进行RT-PCR扩增干扰素γ(IFNγ)基因序列。将IFNγ成熟肽序列与表达载体p ET-32a(+)相连,成功构建了重组表达载体PET-32a-IFNγ。将其转化入E.coli BL21(DE3)感受态细胞后获得了重组表达IFNγ的基因工程菌。经IPTG诱导,p ET-32a-IFNγ在BL21中得到了表达。通过对表达产物的纯化和Western blotting检测,结果显示红鳍东方鲀IFNγ基因在大肠杆菌中的表达准确,且目的蛋白表达量较高。     

暗纹东方鲀线粒体COI及其侧翼tRNA基因的克隆与序列分析

以暗纹东方鲀(Takifugu fasciatus)肝脏的线粒体DNA为模板,按照红鳍东方鲀线粒体DNA序列设计合成特异引物进行PCR扩增,克隆并测定了线粒体细胞色素氧化酶I亚基（COI）及其侧翼tRNA基因的全序列,结果显示,克隆了暗纹东方鲀COI基因1546bp及其5′端上游的tRNATyr基因和3′端下游的tRNASer基因序列共1766bp。用DNA分析软件对暗纹东方鲀与GenBank中10个目13种鱼类的COI序列进行比较分析,显示暗纹东方鲀与这些鱼类的COI基因具有较高的同源性,与同属红鳍东方鲀的同源性最高为97.6%,与同目不同科的矛尾翻车鲀和翻车鲀的同源性为76.5%和75.4%。根据暗纹东方鲀与其他13种鱼的COI基因序列同源性所建立的进化树,与传统的分类地位基本吻合。推定的这二种tRNA的二级结构都具有典型的三叶草型结构。     

红鳍东方鲀干扰素γ基因的原核表达

旨在探究一种简单高效的获取重组红鳍东方鲀IFNγ蛋白的方法。提取红鳍东方鲀(Takifugu rubripes)肾脏组织的总RNA,并以其为模板进行RT-PCR扩增干扰素γ(IFNγ)基因序列。将IFNγ成熟肽序列与表达载体p ET-32a(+)相连,成功构建了重组表达载体PET-32a-IFNγ。将其转化入E.coli BL21(DE3)感受态细胞后获得了重组表达IFNγ的基因工程菌。经IPTG诱导,p ET-32a-IFNγ在BL21中得到了表达。通过对表达产物的纯化和Western blotting检测,结果显示红鳍东方鲀IFNγ基因在大肠杆菌中的表达准确,且目的蛋白表达量较高。     

红鳍东方鲀精原干细胞鉴定、分离及纯化

为获得高比例精原干细胞, 开展了不同月龄红鳍东方鲀精原干细胞分离纯化研究。采用组合酶消化法制备不同月龄的精巢单细胞悬液, 通过形态学观察和生殖细胞特异基因vasa免疫荧光鉴别精原干细胞, Percoll不连续梯度(10%、30%和50%)纯化精原干细胞。结果显示: 14月龄雄鱼精巢生殖细胞主要为A型精原细胞, 精原干细胞占比极显著性地高于22月龄(P<0.05)。纯化后精原干细胞主要分布在10%—30% Percoll梯度带中, 14月龄雄鱼生殖细胞主要分布在此层, 且纯化后精原干细胞占比远高于纯化前以及22月龄纯化前后。结果表明, 14月龄更适用组合酶消化分离, 并通过Percoll梯度离心法获得高比例的红鳍东方鲀精原干细胞。     

基于MiSeq测序技术分析红鳍东方鲀养殖环境菌群多样性

养殖环境中的微生物在物质循环及能量流动中发挥着重要的作用。对养殖环境中微生物多样性研究,不仅可以为养殖过程中病害防治提供基础,还可以发现新的微生物资源。本文利用Mi Seq测序技术分析了大连某养殖公司红鳍东方鲀养殖环境中室内养殖环境水、室外入水口水和底泥中菌群结构特征,从室内养殖环境水、室外入水口水和室外入水口底泥样品中分别获得27164、41146和30474条有效序列。结果表明:红鳍东方鲀养殖环境中具有较高的菌群多样性,其中室外入水口底泥中菌群多样性较其相应养殖水环境中菌群多样性高;3个样品中的细菌可归为33个门类,除常见报道的绿弯菌门、拟杆菌门、浮霉菌门、蓝藻门、酸杆菌门、变形菌门、梭杆菌门、疣微菌门、厚壁菌门和放线菌门外,还有23个鲜见报道的门类的菌群被检测到;各样品中菌群组成虽有差异,但变形菌门和拟杆菌门为优势菌,约占68.33%~93.37%。本文揭示了大连某养殖公司红鳍东方鲀池塘养殖环境中的菌群多样性和丰度,比较了其菌群结构及差异,为红鳍东方鲀健康养殖提供参考。     

暗纹东方鲀线粒体基因组核苷酸全序列测定与分析

以暗纹东方鲀(Takifugu fasciatus)肝的线粒体DNA为模板,参照红鳍东方鲀(T.rubripes)等近源鱼类的线粒体基因组DNA序列,设计合成14对特异引物,进行PCR扩增并测序,首次获得了暗纹东方鲀线粒体基因组全序列。结果表明,暗纹东方鲀线粒体基因组序列全长16 444 bp(GenBank登录号为GQ409967),A+T含量为55.8%,其mtDNA结构与其他脊椎动物相似,由22个tRNA基因、2个rRNA基因、13个蛋白质编码基因和1段819 bp非编码的控制区(D-loop)所组成。蛋白质基因除COⅠ和ND6的起始密码子为GTG、CCT以外,均为典型的起始密码子ATG。ND1、ATPase8、COⅢ、ND4L、ND5、Cyt b使用典型的终止密码子TAA,其他的使用不完全终止密码子。除ND6和tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer、tRNAGlu、tRNAPro在L-链上编码之外,其余基因均在H-链编码。基因排列顺序与已测定的鲀类一致,这显示了鲀类线粒体基因排列顺序上的保守性。tRNA基因核苷酸长度为64～73nt,预测了22个tRNA基因的二级结构,均呈较为典型的三叶草状。基于19种鲀类mtDNA全序列构建的进化树表明,暗纹东方鲀与红鳍东方鲀、中华东方鲀(T.chinensis)聚成一个姊妹群。结果还支持东方鲀属鱼类为一单系类群。     

鲀形目3种鱼的染色体组型分析

采用胸腔注射植物血球凝集素(phytohemagglutinin,PHA)及秋水仙素溶液,取活体头肾细胞经低渗、固定、空气干燥法,分析比较了中华单角鲀(Monacanthus chinensis)、黄鳍东方鲀(Takifuguxanthopterus)、红鳍东方鲀(T.rubripes)的核型。结果表明,3种海水鱼中期染色体均为二倍体,未发现异型性染色体、随体和次缢痕。其核型如下:中华单角鲀的核型为2n=34(34t),臂数:NF=34;黄鳍东方鲀的核型为2n=44(12m+8sm+24t),臂数:NF=64;红鳍东方鲀的核型为2n=44(14m+6sm+24t),臂数:NF=64。中华单角鲀的核型与后两者存在较大差异。同时,将此3种鱼的核型与前人报道的其他鲀形目鱼类核型作了比较。     

重组白介素2对红鳍东方鲀的血液生化指标及免疫指标的影响

为研究重组白介素2(Recombinant interleukin-2,r IL-2)对红鳍东方鲀(Takifugu rubripes)血液生化指标及免疫指标的影响,选用体重为(205.9±30.5)g的红鳍东方鲀进行了重组白介素2的免疫应答实验。设计r IL-2免疫剂量为2.5、5、7.5、10μg,并在免疫后的4 h、8 h和12 h进行采血。结果表明:经r IL-2免疫后红鳍东方鲀血清中尿素氮含量在各个浓度组的8 h时间点显著高于同组内其它时间点(P<0.05),4 h时间点甘油三酯含量有随着免疫浓度增加而增加的趋势,谷草转氨酶活性在8 h时间点要显著高于其他时间点(P<0.05),2.5μg、5μg浓度组过氧化氢酶的活性在4 h时间点显著高于其他各组(P<0.05),对免疫球蛋白的含量也有显著影响。本研究发现,r IL-2的免疫可引起血液生化指标和免疫指标的变化,尤其是超氧化物歧化酶和谷草转氨酶的变化较为显著(P<0.05),可在一定程度上促进红鳍东方鲀的免疫反应。     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

Scurvy in capybaras bred in captivity in Argentine

In order to determine if the absence of vitamin C in the diet of capybaras (Hydrochoerus hydrochaeris) causes scurvy, a group of seven young individuals were fed food pellets without ascorbic acid, while another group of eight individuals received the same food with 1 g of ascorbic acid per animal per day. Animals in the first group developed signs of scurvy-like gingivitis, breaking of the incisors and death of one animal. Clinical signs appeared between 25 and 104 days from the beginning of the trial in all individuals. Growth rates of individuals deprived of vitamin C was considerably less than those observed in the control group. Deficiency of ascorbic acid had a severe effect on reproduction of another population of captive capybaras. We found that the decrease in ascorbic acid content in the diet affected pregnancy, especially during the first stages. The results obtained suggest that it is necessary to supply a suitable quantity of vitamin C in the diet of this species in captivity.     

Changes in lactate dehydrogenase activity in chicken tissues in hyperthyreosis in ontogenesis

The lactate dehydrogenase activity in reactions of lactate oxidation and synthesis was studied in subfractions of the chicken brain, heart and liver at the embryonal, early postembryonal and adult stages of development after thyroxine administration. It has been shown that during embryogenesis thyroxine predominantly enhanced the rate of lactate oxidation in the mitochondrial tissues. A marked increase in the lactate synthesis was found in cytoplasm of the adult chicken tissues. Specificity of enzyme activity alterations was detected in the chicken brain during ontogenesis after thyroxine administration.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.