Leptin in fish: possible role in sexual maturation in male Atlantic salmon

Leptin plays an important role in energy homeostasis and reproductive function in mammals, while its function in fish is still poorly understood, especially its role in reproduction. In the present study, leptin gene expression and circulating leptin plasma levels were measured during sexual maturation in male Atlantic salmon parr. In maturing male salmon, higher hepatic leptin (lepa1) gene expression levels were observed during mid-spermatogenesis compared to immature fish. An upregulation of leptin during sexual maturation has also been observed in a few other teleost species. The physiological significance of elevated leptin levels during the maturational process in teleosts remains to be explored.     

草鱼NK-lysin基因的克隆、原核表达与活性分析

为研究草鱼抗菌肽NK-lysin基因的组成结构、在组织中的表达差异及其抑菌活性,实验根据已知鱼类NK-lysin基因保守区序列设计上下游引物,采用RT-PCR和RACEPCR技术克隆草鱼NK-lysin基因(Cinkl),RT-PCR进行各组织间的表达分析,并构建Cinkl成熟肽的原核表达载体,采用琼脂糖孔穴扩散法检测重组蛋白的抑菌活性。结果显示,Cinkl c DNA全长768 bp,编码121个氨基酸;基因组DNA全长3 361 bp,包含4个外显子和3个内含子。经多序列比对分析和结构域预测表明,Ci Nkl氨基酸序列具有SAPLIP家族的特征:含有6个保守的半胱氨酸和saposin B蛋白结构域,与斑马鱼Nklc、Nkld的相似性分别为57.98%和63.03%。构建NK-lysin氨基酸序列系统进化树,发现Ci Nkl与斑马鱼Nklc、Nkld聚为一支。RT-PCR检测结果显示,Cinkl m RNA在所有检测的草鱼组织中均有表达,在脾脏中表达量最高,心脏、鳃、中肾和头肾的表达量次之,皮肤、脑、肝脏和肠有微量表达。p ET-28b-MBP与Ci Nkl成熟肽序列构建的原核表达质粒在宿主菌Rosetta(DE3)表达出约57 ku的可溶性融合蛋白,利用Ni-NTA His·Bind树脂进行纯化,Ci Nkl重组蛋白对G–细菌如大肠杆菌M15株、嗜水气单胞菌,以及G+细菌如金黄色葡萄球菌均具有抑菌活性。基于草鱼NK-lysin的组织表达特征和重组蛋白的抑菌活性,推测NK-lysin在草鱼的先天性免疫防御中发挥调节作用。     

The effects of Coriandrum sativum L. as feed additive on mucosal immune parameters,antioxidant defence and,immune‐related genes expression in zebrafish (Danio rerio)

This study investigates the effects of Coriander (C. sativum) on the growth, antioxidant, and immune‐associated genes and serum and mucosal immune parameters in zebrafish (D. rerio). The experimental fish were treated with 0 [control], 5, 10 and 20 g/kg coriander supplemented diets for 8 weeks. The results revealed that coriander remarkably increased mucosal immune parameters (the total Immunoglobulin, protease and lysozyme activity). The highest levels of the measured parameters were noticed in 20 g/kg CP treatment. Also, the zebrafish fed 20 g/kg coriander powder showed significantly higher expression gene for lysozyme and TNF‐alpha. The same results were noticed in case of IL‐1B gene expression. In case of sod gene expression there was no significant difference between treatments. However, regarding cat gene expression, significant difference was noticed between 20 g/kg CP treatment and other groups. In addition, no significant changes were observed between coriander fed zebrafish and control treatments regarding GH gene expression level, although IGF‐I remarkable up‐regulate in 20 g/kg coriander powder treatment compared other groups. In conclusion, it can be proposed that dietary Coriander powder can improve mucosal immune parameters and immune and antioxidant genes expression.     

一种免疫诱导型鲤启动子的克隆与功能分析

为发掘适用于基因工程抗病育种的鱼类启动子,通过实时荧光定量PCR实验对鲤Rab GTP酶(Ras-associated binding-GTPases 1a3,Rab1a3)基因的表达模式进行了分析,证实该基因在鳃、头肾等与机体免疫防御功能密切相关的组织内转录水平较高,且免疫激活后转录显著增强,符合基因工程抗病育种所需的外源免疫基因转录模式。从鲤细菌人工染色体文库中,使用Rab1a3基因特异引物筛选获得包含该基因区域的文库克隆,测序获得该基因完整序列,以及上下游调控序列。通过生物信息学手段,预测到长度为1014 bp的鲤Rab1a3基因启动子序列,该启动子不具有典型的TATA盒或CpG岛特征,存在多个免疫相关转录因子结合位点。在草鱼肾组织细胞系内验证该启动子活性,结果显示,绿色荧光蛋白基因和萤火虫荧光素酶基因都能够在该启动子驱动下表达,证实该片段具有启动子活性,且启动子活性在受到免疫诱导后增强,双荧光素酶报告基因检测结果显示,该启动子活性在免疫刺激后增强至免疫刺激前的8.67倍。研究表明,鲤Rab1a3基因启动子有望被开发成为免疫诱导型的基因工程元件,驱动外源免疫基因在鱼体内适时表达,抵御外界病原感染,同时避免非必要条件下的过度表达形成生长负担。     

大口黑鲈leptin A基因的组织表达及其对急性低氧胁迫的响应

瘦素(leptin)是一种重要的激素,参与调节动物的摄食、繁殖和能量消耗,其可以通过抑制食欲和促进能量代谢的方式维持机体能量稳态。大多数鱼类具有双重瘦素基因。在本研究中,利用PCR技术克隆了大口黑鲈leptin A基因的编码区,其开放阅读框(ORF)为486 bp,编码161个氨基酸蛋白。通过与其他物种进行同源性比对,发现鲈形目的 leptin基因的保守性较高,一致性高达91.46%,在大口黑鲈leptin A中几乎不存在基因特异性突变位点,而且大口黑鲈leptin A氨基酸序列与鳜同源性最高(92.59%),与花鲈(89.51%)、布氏鲳鲹(87.04%)、斜带石斑鱼(83.87%)的同源性次之。组织分布结果显示,leptin A基因在肝脏中的表达量最高,在心脏、头肾、脑、中肾、肠、脾脏、鳃、肌肉和性腺中的表达量均较低。此外,对大口黑鲈进行不同程度急性低氧胁迫[重度低氧(1.2±0.2)mg/L和中度低氧(3.5±0.2)mg/L],发现低氧胁迫初期时,严重低氧组和中度低氧组的leptin A的表达量都升高,显著高于对照组Leptin A的表达。这表明急性低氧能够诱导大口黑鲈leptin A在肝脏中的表达。综上所述,大口黑鲈leptin A基因与鳜leptin A基因的同源性最高,leptin A主要在大口黑鲈肝脏中显著表达,急性低氧胁迫能显著诱导leptin A的表达。     

Cortisol induces masculinization of XX medaka through gonadal soma-derived growth factor (GSDF) and anti-Müllerian hormone receptor type 2 (AMHR2)

The medaka Oryzias latipes is a teleost fish with an XX/XY sex determination system similar to that of mammals. However, under high-temperature conditions, XX medaka are masculinized by an elevation of the key teleost glucocorticoid, cortisol. Cortisol inhibits female-type proliferation of germ cells and induces masculinization of XX medaka during gonadal sex differentiation. To identify masculinization mechanisms downstream of cortisol, we analysed the functions of gonadal soma-derived growth factor (gsdf) and anti-Müllerian hormone receptor type 2 (amhr2); these genes are known to play important roles in the inhibition of germ cell proliferation and male differentiation. We investigated the impact of gsdf and amhr2 on the proliferation of germ cells using gsdf knockout (KO) and amhr2 KO medaka. At hatching stage, loss of gsdf or amhr2 function recovered female-type proliferation in germ cells under cortisol treatment. Moreover, cortisol treatment of gsdf KO or amhr2 KO medaka did not induce masculinization of XX medaka. These results suggest that cortisol inhibits female-type proliferation of germ cells and induces masculinization of XX medaka through GSDF and AMHR2. This study thereby provides the first evidence that GSDF and AMHR2 are involved in cortisol-induced masculinization.

     

Promoter analysis of two aromatase genes in the serial-sex changing gobiid fish, Trimma okinawae

Despite numerous endocrine studies on sex change in teleost, no general mechanism that mediates sex change has emerged. The gobiid fish, Trimma okinawae, can change sex in both directions repeatedly. This phenomenon of sex change in goby assigns it as an excellent animal model to elucidate the understanding mechanisms of sex change. In hermaphrodite fishes, estrogen plays a particularly important role in natural and experimentally induced sex change. To investigate the role of estrogen in the serial-sex changing fish T. okinawae, we cloned and analyzed the 5′-flanking regions of P450arom genes from goby genome DNA. Both regions have consensus sequences of TATA, CRE and ERE. Ad4 binding site was restricted in the region of P450aromA. These findings indicate that different regulators control the expression of the two P450arom genes.     

Establishing a zebrafish transgenic line expressing tilapia lysozyme with enhanced antibacterial activity

In recent years, infectious disease caused by Streptococcus agalactiae has increased in aquaculture, threatening the healthy development of tilapia and resulting in substantial economic losses. Cultivating disease‐resistant tilapia by transgenic technology is one strategy that has been developed to address this issue. Here, we used a transposon system to investigate gene expression and tissue lytic activity in transgenic zebrafish expressing the tilapia lysozyme. The transpose vector contained the Tgf2 transposon skeleton, tilapia heat shock protein 70 promoter (Hsp70) and two target genes (tilapia C‐type lysozyme 3 [lysozyme‐C3] and green fluorescent protein [GFP]). The transgenic zebrafish F₀ generation was obtained by microinjecting the donor vector and transposase mRNA into fertilized zebrafish eggs. There was 45% green fluorescence in the zebrafish F₀ generation, which was spread over the entire body based on microscope observation. The F₀ generation was reared to sexual maturity, at which point individuals were mated with wild‐type zebrafish to produce the F₁ generation. Tilapia lysozyme‐C3 expression was detected in the liver of F₁ generation by RT‐PCR and western blotting, but was not detected in the skin, intestine, or muscle. Significantly higher liver bacteriolysis activity was detected in transgenic zebrafish compared with wild‐type zebrafish. Therefore, transgenic zebrafish may have an increased potential for bacterial resistance.     

食蚊鱼CYP19a基因的克隆与序列分析

硬骨鱼类CYP19基因与生物的性别分化和激素调节相关,因此可开发用来探究环境激素污染与基因表达的关系。本研究首次克隆和分析了食蚊鱼CYP19a cDNA的全系列,为将CYP19基因作为监测环境激素生物标志物的研究提供了全面的实验数据。根据CYP19a基因c DNA保守区域设计引物,扩增保守区域并测序。采用RACE法扩增食蚊鱼CYP19a基因c DNA序列全长,对其蛋白序列进行同源性分析,并将序列应用于CYP19a mRNA转录水平的RT-PCR法检测中。成功克隆食蚊鱼CYP19a基因全长,获得CYP19a基因总长为2020 bp,ORF为238~1791 bp,共编码518个氨基酸,对其编码的蛋白质进行有关信号肽、跨膜螺旋、亲水性/疏水性、一级结构、二级结构和三级结构分析,与其他硬骨鱼类底鰆、青鰆、平鲷、鲫、鲤和斑马鱼的性腺CYP19a基因作同源性比较,其基因相似度分别为93%、84%、84%、71%、71%和66%。用MEGA6.0软件对19个物种的CYP19a基因进行聚类分析,食蚊鱼CYP19a基因与底鰆、青鰆同源性最高,说明芳香化酶在进化上相对保守。确定从食蚊鱼性腺所克隆的CYP19a基因是芳香化酶基因,证明食蚊鱼的芳香化酶是由CYP19a和CYP19b两种基因编码的。食蚊鱼卵巢芳香化酶具有3个高度保守的片段,并具有催化活性。     

鲤神经球蛋白基因序列与低氧表达分析

为深入了解鲤珠蛋白家族的基因组成和表达模式,及其与低氧适应能力的相关性,本实验通过鲤基因组框架图比对和全长cDNA文库筛查,获得鲤神经球蛋白(neuroglobin,Ngb)基因完整序列,证实鲤不仅具有独特的脑组织特异表达的II型肌红蛋白(myoglobin-2,Mb-2)基因,也具有Ngb珠蛋白基因,实时荧光定量PCR实验显示,该基因在脑组织特异性表达,并呈现出低氧应答特征。基因结构和系统发生分析表明,鱼类Ngb基因高度保守,鲤Ngb蛋白可能与斑马鱼直系同源蛋白具有相似的结构及功能,而与鲤Mb-2存在明显差异。鲤Ngb基因表达量在两个品系间存在显著差异,耐低氧能力强的散鳞镜鲤Ngb基因表达量高于荷包红鲤抗寒品系。研究表明,鲤Ngb基因可能以与Mb-2基因分工协作的方式共同实现脑组织供氧,执行应对低氧胁迫的神经保护功能,在鲤低氧适应中具有重要作用。     

白斑狗鱼新基因lincRNA-Abcc10的克隆、表达及敲除模型构建

长非编码RNA(lncRNAs)作为一种新的表观遗传因子,在研究鱼类性别决定和性别分化过程中具有重要的作用。本研究利用RACE技术,对白斑狗鱼(EsoxLucius)性别特异性lncRNAs基因Novel01784进行克隆,获得了全长共1328bp的序列。在线预测工具CPC2和CPAT的分析结果显示,该基因不具有编码蛋白质的能力。通过Blast比对分析将其定位在9号染色体,且位于sp2和Abcc10基因之间,根据其位置关系将其命名为lincRNAAbcc10。从共定位分析结果显示, lincRNA-Abcc10可能参与调控离子运输、卵泡细胞发育和线粒体活性,其表达模式与部分邻近基因相似。其次,对白斑狗鱼lincRNA-Abcc103个时期在8个组织中的qRT-PCR结果显示,该基因在卵巢中特异性表达,随着时间增长表达量逐渐下降,说明其可能在卵巢早期分化过程中起着重要的作用。此外,通过对白斑狗鱼126 d卵巢核质RNA分离, qRT-PCR结果显示, lincRNA-Abcc10分布于细胞质内,可能以顺式或反式方式调控邻近基因的翻译水平。最后,利用CRISPR/Cas9技术建立了linc...