性逆转石斑鱼脑垂体差异表达基因克隆的筛选

以17α甲基睾丸酮投喂赤点石斑鱼(Epinephelusakaara),成功地获得了性逆转的有功能的雄鱼。用SMARTcDNA合成和长片段PCR技术构建了性逆转前后脑垂体cDNA文库;用抑制消减杂交技术建立了性反转前后抑制性差减文库。对获得的560个雄鱼脑垂体PCR阳性克隆和350个雌鱼脑垂体PCR阳性克隆进行斑点杂交,共筛选到103个差异表达cDNA片段。对其中29个克隆进行了测序,与GenBank中的已知基因序列进行同源性比较,发现有6个片段为促性腺激素α亚基前体基因(GenBank注册号:AY207430),与同属不同种的石斑鱼促性腺激素α亚基前体基因有97%的同源性;1个片段为生长激素前体(GenBank注册号:AY207431),与同属不同种的石斑鱼生长激素前体基因有99%的同源性;其余22个cDNA片段与GenBank中的序列无明显同源性 。     

性逆转石斑鱼性腺差异表达基因的克隆和筛选

以 17α 甲基睾丸酮 (17α MT)饲喂 2～ 4龄赤点石斑鱼 (Epinephelusakaara) ,成功地促使其性转变为具有生殖功能的雄鱼 .应用抑制性差减杂交 (SSH)技术构建了石斑鱼性反转前后性腺组织的SMARTcDNA文库及其cDNA差减文库 ,从中随机挑取 12 0 0个克隆进行了PCR和斑点杂交筛选 ,得到 12 0个差异表达cDNA片段 .挑选 71个cDNA克隆测序 ,将所测序列经GenBank检索和生物信息学比较 ,发现有 5 1个cDNA片段序列无明显的同源性 ,2 0个片段与报道的基因有较高同源性 .在这 2 0个具同源性的片段中有 3个片段可能是与性别分化密切相关的重要功能基因 ,它们是钙调蛋白基因、活性蛋白激酶C受体基因和一氧化氮合酶蛋白抑制剂基因 .这 3个基因被分别命名为鱼钙调蛋白基因 (GenBankaccession :AY2 8136 3)、鱼活性蛋白激酶C受体基因 (GenBankaccession :AY2 8136 4)和鱼一氧化氮合酶蛋白抑制剂基因 (GenBankaccession :AY2 8136 5 ) .     

尼罗罗非鱼生长激素及其受体的cDNA克隆与mRNA表达的雌雄差异

尼罗罗非鱼(Oreochromis niloticus)雌雄鱼生长差异明显,为了探讨其原因,本文采用RT-PCR方法克隆了尼罗罗非鱼生长激素(Growthhormone,GH)及其受体(Growth hormone receptor,GHR)的cDNA序列,并应用半定量RT-PCR方法比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达差异。序列分析表明:GH开放阅读框为615bp,共编码204个氨基酸;GHR开放阅读框为1908bp,共编码635个氨基酸。以RT-PCR方法研究了GH、GHR在各组织的分布情况,结果表明:GH仅在垂体中检测到有表达,而GHR在所检测的18种组织中均有表达,其中以肝脏、肌肉、性腺、下丘脑、胸腺表达量较高。以半定量RT-PCR方法进一步比较了雌、雄尼罗罗非鱼垂体GHmRNA、肝脏GHRmRNA、肌肉GHRmRNA的表达量,结果表明:雄鱼垂体GHmRNA和肝脏GHRmRNA的表达量均显著高于雌鱼,肌肉GHRmRNA的表达量则无显著差异,推测垂体GHmRNA和肝脏GHRmRNA表达的雌雄差异是尼罗罗非鱼雌雄生长差异的主要原因之一。     

大口黑鲈amh基因全长cDNA克隆、表达及多克隆抗体制备

为研究大口黑鲈(Micropterus salmoides)抗缪勒氏管激素(amh)基因的表达及其在性腺发育中的潜在作用,研究利用RACE技术克隆得到了大口黑鲈amh基因,并制备Amh多克隆抗体,通过qRT-PCR、Western Blot分析Amh在大口黑鲈不同组织和不同发育阶段性腺中的表达模式,最后利用HE染色法和免疫组化观察不同发育阶段性腺的形态组织学变化及其与Amh表达的潜在关系。结果显示:大口黑鲈amh基因cDNA序列全长2050 bp,由24 bp5′非编码区、394 bp3′非编码区和1632 bp的开放阅读框组成,共编码543个氨基酸。amh基因mRNA在大口黑鲈11个组织中均有表达,其中雄鱼精巢中表达量最高,肌肉次之,雌鱼卵巢中表达量最高,肌肉次之。amh基因在雌雄鱼不同发育阶段的性腺中表达存在显著差异,精巢中表达量均显著高于卵巢(P<0.05)。同时, Western Blot结果显示Amh蛋白在精巢中表达丰度较高。amh基因在精巢中的表达量呈先上升后下降的趋势,且在孵化后65d鱼精巢中其表达量达到最高(P<0.05),免疫组化结果显示Amh表达于早期精...     

黄颡鱼HSC70基因及其组织表达分析

热休克蛋白70（HSP70）与生物体的抗胁迫能力密切相关。本文采用RACE (Rapid amplification of cDNA ends) 技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白（HSC70）基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF) 1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子（873bp）位于5′端非编码区,其余内含子（长度在80－251bp之间不等）均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。     

抚仙金线鲃促性腺激素亚基基因克隆及组织表达分析

抚仙金线鲃Sinocyclocheilus tingi是云南抚仙湖特有种,虽然实现了人工繁殖,但在塘养环境下仍无法自然繁衍。鱼类的生殖活动受下丘脑-垂体-性腺轴的调控,其中促性腺激素在该过程中起重要作用。本实验利用实时荧光定量PCR和c DNA末端快速扩增技术从抚仙金线鲃垂体中克隆了GTHα、FSHβ和LHβ3种基因的c DNA序列,其中GTHα的开放阅读框(ORF)长度为357 bp,编码118个氨基酸,有10个半胱氨酸残基和2个N-糖基化位点;FSHβc DNA全长为856 bp,ORF长度为393 bp,编码130个氨基酸,有11个半胱氨酸残基和1个N-糖基化位点;LHβc DNA全长为930 bp,ORF长度为441 bp,编码146个氨基酸,有12个半胱氨酸残基和1个N-糖基化位点。实时荧光定量PCR结果显示,GTHα和LHβ都只在雄鱼和雌鱼的垂体中表达。FSHβ在垂体中表达量最高,在雌鱼和雄鱼的脂肪、肌肉和雄鱼的精巢和肝脏中有少量表达。本研究为抚仙金线鲃的人工繁育提供了重要的基础数据。     

西伯利亚蓼钙调蛋白基因的克隆及其在盐胁迫下的表达

已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增（RACE）技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列（GenBank登录号GQ988382）,命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5＇非翻译区为63bp,3＇非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。     

中华鳖Dmrt1基因的克隆、表达及在性逆转中的变化分析

在大部分脊椎动物中,Dmrt1基因在雄性性别决定和性腺分化中起重要的调控作用.本文从m RNA和蛋白水平分析Dmrt1基因的组织差异性表达、在不同发育阶段性腺中的细胞定位及在性逆转中的表达变化,研究Dmrt1基因在中华鳖性别分化中的调控作用.Rapid-amplification of c DNA ends(RACE)结果显示,Dmrt1基因c DNA序列全长2409 bp,其中5′非编码区为230 bp,3′非编码区为1072 bp,开放阅读框为1107 bp,编码368个氨基酸,具有一个高度保守的DM结构域.荧光定量PCR和免疫组化结果显示,Dmrt1在性腺分化之前的第16期雄性性腺中开始表达,先于Amh和Sox9基因表达.随着性腺的发育,Dmrt1蛋白主要定位于性腺Sertoli细胞的细胞核上,在雌性性腺发育过程中并未见其表达.此外,在雌二醇诱导的雄性转雌性性逆转胚胎性腺中,Dmrt1表达显著下调;在芳香化酶抑制剂诱导的雌性转雄性性腺中,Dmrt1表达则显著上升.上述研究表明,Dmrt1基因是中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别决定中起重要的调控作用.     

棉花咖啡酰辅酶A-O-甲基转移酶基因的克隆及特征分析

根据棉花纤维特异表达cDNA文库得到的咖啡酰辅酶 A-O-甲基转移酶基因EST序列设计引物,采用RT-PCR技术从棉花中克隆了一个CCoAOMT基因,命名为GhCCoAOMT2.GhCCoAOMT2基因cDNA(GenBank登录号为FJ376606)具有一个747 bp的开放阅读框,5′非编码区为12 bp,3′非编码区为243 bp,编码248个氨基酸,预测分子量约为28.023 kD,等电点为5.39.GhCCoAOMT2基因组序列长度为1 442 bp,包含4个外显子和3个内含子.氨基酸同源分析发现,GhCCoAOMT2与来自毛白杨、烟草和苎麻的CCoAOMT同源性较高.半定量RT-PCR检测表明,GhCCoAOMT2基因在棉花各个组织中都有表达,其中茎部的表达量最高.原核表达分析表明,最佳诱导表达条件为0.2 mmol/L IPTG在37℃下诱导6 h.     

金钱鱼Sox9 cDNA克隆及其表达分析

为了解Sox9基因在金钱鱼(Scatophagus argus)性腺分化中的作用,本研究利用c DNA末端快速扩增法(RACE)克隆了金钱鱼Sox9基因c DNA全长序列,同时研究了投喂芳香化酶抑制剂来曲唑(LE)(50 mg/kg)后该基因的表达及其性腺的组织学变化。金钱鱼Sox9 c DNA全长2 759 bp,包括5′非翻译区(5′-UTR)31 bp、3′非翻译区(3′-UTR)1 288 bp和开放阅读框(ORF)1 440 bp,编码479个氨基酸。该氨基酸序列104~172位为HMG保守盒,在该盒内存在一个特征性基序,两个核定位信号NLS及一个富含亮氨酸的核输出信号NES。该序列与赤点石斑鱼(Epinephelus akaara)的相似性最高,为96.0%,与非洲爪蟾(Xenopus laevis)、人(Homo sapiens)、原鸡(Gallus gallus)、小家鼠(Mus musculus)等物种的相似性为61.5%~76.1%。Real-time PCR显示,金钱鱼Sox9基因在脑、鳍、精巢中表达量较高。组织学研究表明,芳香化酶抑制剂来曲唑能有效诱导金钱鱼发生不同程度的性逆转,性腺中卵母细胞退化,精原细胞增殖。Real-time PCR结果显示,金钱鱼Sox9基因在来曲唑处理20 d后开始不断上升,于处理后第40天时达到最高值,在第60天时表达量迅速下降。上述结果表明,金钱鱼Sox9基因高度保守,可能在金钱鱼性腺雄性化过程中起重要作用。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.