藏鸡主要组织相容性复合体B-LBⅡ基因第二外显子序列遗传变异分析

根据鸡主要组织相容性复合体B-LBⅡ基因序列设计特异性引物,在藏鸡基因组中扩增了一个包括其第二外显子和第二内含子在内长度为374 bp的片段,并通过克隆和PCR直接测序获得了该片段的核苷酸序列。发现了15个B-LBⅡ新等位基因。对18个B-LBⅡ等位基因核苷酸序列和其所编码的MHCB-LBⅡ分子β1结构域的氨基酸序列分析显示,第二外显子核苷酸序列遗传多态性异常丰富,存在着62个多态变异位点(共包括80个突变),其中41个为简约性多态位点;衡量该序列遗传多样性的π值为0.0718;反映其群体内遗传变异度的平均遗传距离为0.056±0.008,低于在5个外来品种所估算的平均遗传距离。该编码区核苷酸相对异义替换率(15.61±2.69%)显著高于其同义替换率(3.25±0.94%),进一步分析表明,基因重组和平衡选择机制可能是引起B-LBⅡ基因序列变异的主要因素。在β1结构域氨基酸序列中,存在11个同义替换和27个异义替换;在24个肽结合位点中有12个变异位点;与其他6个中国地方鸡品种和一个外来品种比较发现,有11个异义氨基酸替换仅出现在藏鸡群体中,并被认为与藏鸡的免疫特异性有关,可为鸡的抗病力研究提供分子依据。     

中国地方鸡种MHC B-LBⅡ新等位基因的遗传多态性研究

为研究鸡MHC B-LBⅡ基因的遗传多态性,首先在8个中国地方鸡种(藏鸡、仙居鸡、北京油鸡、固始鸡、斗鸡、丝羽乌骨鸡、白耳鸡和狼山鸡)B-LBⅡ基因第二外显子扩增了一长度为 175 bp 的 DNA 片段并进行 SSCP 基因型分析;在8 个地方鸡种共 467 个个体中检测到 37 个 PCR-SSCP 基因型;从被检样品中筛选出不同基因型的个体,并在其 B-LBⅡ基因组中扩增了一个包括其第二外显子和第二内含子在内长度为374 bp的片段,通过克隆和测序获得了该片段的核苷酸序列。经序列分析,在前述地方鸡种被筛选出的 30 个无血缘关系的个体中发现了 31 个 B-LBⅡ新等位基因,并参照哺乳动物 MHC II 类 B 等位基因命名规则进行了命名。对这 31 个 B-LBⅡ新等位基因长度为 374 bp 的 DNA 片段进行比对表明,在其第二外显子序列上共有 68 个多态性变异位点,其中简约性信息位点 51 个,单变异位点 17 个,具有丰富的遗传多态性。在这些多态性变异位点中,出现在遗传密码子第一和第二位上的碱基替换率分别为 36.76% 和 35.29%。等位基因序列间的相似性估测为 90.6%-99.5%;B-LBⅡ基因第二外显子的错义替换率和同义替换率分别为 14.64±2.67%和 2.92±0.94%。结果表明,B-LBⅡ基因的丰富遗传多态性主要是由基因重组和平衡选择效应所引起的。对 B-LBⅡ等位基因第二外显子所编码的 B-LBⅡ分子β1 结构域氨基酸序列比对发现,31 个 B-LBⅡ新等位基因属于 26 个等位基因主型;在β1结构域氨基酸序列的 33个变异位点上,存在 6 个同义替换和 27 个错义替换。分析认为,那些发生在多肽结合位点上的氨基酸错义替换与鸡 MHC B-LBⅡ分子的免疫特异性有关。该结果可为鸡的抗病育种研究提供分子生物学依据。     

日本牙鲆主要组织相容性复合体DAB等位基因的多态性

根据牙鲆(Paralichthys olivaceus)主要组织相容性复合体(MHC)DAB基因序列设计特异性引物,在日本牙鲆基因组中扩增了包括DAB基因完整外显子2和内含子1在内的长度为408 bp的DNA片段.对该片段克隆测序后,发现了37个MHC-DAB等位基因,各等位基因的频次以及各主型中亚型数目分布都不平衡.在第二外显子273 bp核苷酸序列中有50个位点发生变异,核苷酸多样性PI值为0.0618,在编码的氨基酸序列中存在多态变异位点30个,其中简约信息位点29个,单变异位点1个.非同义替代与同义替代的比率在抗原结合区(PBR)和非PBR编码区分别为10.0和1.62,分析表明正向选择机制可能是产生牙鲆MHC-DAB多态性的主要因素.估计的核苷酸的转换和颠换数随着遗传距离的增加而增加.各等位基因间的系统发生关系表明,19个主型分为两个群.对氨基酸组分和密码子的偏倚性以及分布频率的分析表明各同义密码子的使用频率不均衡.本研究为牙鲆MHC-DAB基因的遗传进化和分子标记辅助育种的研究提供了理论基础.     

基于Cyt b基因序列分析的松毛虫种群遗传结构研究

为了揭示松毛虫种群的遗传结构,采用DNA序列测定的方法测定了松毛虫不同种群的线粒体细胞色素b (Cyt b)基因的部分序列,并利用分子生物学软件分析其核苷酸组成、转换和颠换、氨基酸组成、遗传距离及亲缘关系。结果显示:在获得的Cyt b 基因387bp的序列中碱基A,T,C,G平均含量分别为40.1%、33.5%、9.5%、16.9%,A+T含量明显高于G+C含量表现出强烈的A、T偏向性,密码子第3位点的A+T含量高达86.5%,这种偏向性在种群间无明显差异。碱基替换主要发生在密码子第三位,转换大于颠换,且种群内替换高于种群间。该序列片段中共有39个核甘酸位点发生变异,遗传距离为0.000-0.100,显示出较小的遗传变异。蛋白质氨基酸由除谷氨酸以外的19种氨基酸组成。聚类分析结果表明马尾松毛虫和油松毛虫亚种遗传距离较近,种群间的遗传分化与生态环境有关。     

牙鲆MHC-DAA结构及其等位基因多态性

为了探讨牙鲆MHC-DAA等位基因的多态性, 根据牙鲆 (Paralichthys olivaceus) MHC-DAA的cDNA序列设计特异引物, 扩增内含子序列。牙鲆DAA基因由4个外显子和3个内含子组成, 与大西洋鲑 (Salmo salar) DAA基因结构相同, 在内含子2中存在一个高度多态的微卫星位点(GT)n。根据获得的MHC-DAA基因组序列设计特异性引物, 在45尾牙鲆中扩增了包括完整外显子2和内含子2的长度约670 bp的DNA片段, 克隆测序后共发现30个MHC-DAA等位基因, 各等位基因的频次及其各主型中亚型的数目都不均衡。在249 bp的外显子2序列中共有55个位点发生变异, 核苷酸多样性指数为0.0887, 编码的氨基酸序列中多态变异位点31个, 其中简约信息位点30个, 单变异位点1个。非同义替代与同义替代的比率在PBR(Peptide binding region)和非PBR结合区分别为3.30和2.43, 分析证明平衡选择是牙鲆存在众多DAA等位基因的发生机制。     

基于线粒体 CO玉及 Cyt b 基因的7种龙虾类分子系统关系

运用PCR法扩增湛江沿海海域7种龙虾的线粒体COⅠ和Cytb基因并对其序列进行分析,以分析7种龙虾的分子系统关系。从7种龙虾中扩增到的COⅠ基因片段长度均为650bp,共存在224个核苷酸位点变异,变异率为35.22%,简约信息位点161个;扩增到的Cytb基因片段长度均为536bp,共存在148个核苷酸位点变异,变异率为29.48%,简约信息位点66个。所有的扩增序列中,没有发现碱基的缺失以及插入,序列中的转换大于颠换,且碱基替换多发生于密码子的第3位。以帝加洛真龙虾(Palinurus delagoae)、普通真龙虾(Palinurus elephas)、吉氏真龙虾(Palinurus gilchristi)为外群,对COⅠ和Cytb两个基因序列采用最大简约法(maximum parsimony,MP)和贝叶斯推论法(bayesian inference,BI)构建龙虾类分子系统树。结果显示:日本龙虾和密毛龙虾亲缘关系比较近,而其它5种龙虾与真龙虾属的3种关系较近,与传统的分类存在一定分歧。     

基于线粒体 CO玉及 Cyt b 基因的7种龙虾类分子系统关系

运用 PCR 法扩增湛江沿海海域7种龙虾的线粒体 CO玉和 Cyt b 基因并对其序列进行分析,以分析7种龙虾的分子系统关系.从 7种龙虾中扩增到的 CO玉基因片段长度均为650 bp,共存在224个核苷酸位点变异,变异率为35.22%,简约信息位点161个;扩增到的 Cyt b 基因片段长度均为536 bp,共存在148个核苷酸位点变异,变异率为29.48%,简约信息位点66个.所有的扩增序列中,没有发现碱基的缺失以及插入,序列中的转换大于颠换,且碱基替换多发生于密码子的第3位.以帝加洛真龙虾(Palinurus delagoae)、普通真龙虾(Palinurus elephas)、吉氏真龙虾(Palinurus gilchristi)为外群,对 CO玉和 Cyt b 两个基因序列采用最大简约法(maximum parsimony, MP)和贝叶斯推论法(bayesian inference, BI)构建龙虾类分子系统树.结果显示:日本龙虾和密毛龙虾亲缘关系比较近,而其它5种龙虾与真龙虾属的3种关系较近,与传统的分类存在一定分歧.     

合作猪SLA-DQA基因外显子2多态性分析

旨在研究合作猪DQA基因外显子2多态性,确定其等位基因数、核苷酸多态位点、氨基酸多态位点及各个等位基因之间的遗传关系,分析其进化意义。选用PCR-SSCP对439只合作猪SLA-DQA基因外显子2的多态性进行检测;测序群体内因变异而产生的各等位基因序列,并分析序列数据。结果显示,在合作猪SLA-DQA外显子2中发现了7个新等位基因,共18个核苷酸多态位点,10个氨基酸多态位点。合作猪SLA-DQA外显子2具有较丰富的多态性,群体内可能蕴藏着更加丰富的遗传资源;合作猪SLA-DQA外显子2基因最初可能由一个等位基因突变分化成一大类基因;合作猪SLA-DQA外显子2序列与各个猪种的SLA-DQA外显子2序列具有较高的同源性,预示着这些猪种的SLA-DQA外显子2基因最早可能来源于其分歧之前的共同祖先原始序列;新发现的7个SLA-DQA外显子2等位基因,可能由遗传关系较近的两个等位基因突变产生。     

牛科动物HSL基因序列分析及其分子进化研究

在对牛科中4种动物即牦牛、瘤牛、普通牛和水牛HSL基因外显子Ⅰ部分核苷酸序列进行测定的基础上,与Gen-Bank中其他物种相应基因核苷酸序列、氨基酸序列进行了比对分析,并构建了牦牛与其他物种间分子系统进化树。结果表明:牦牛与普通牛、瘤牛、水牛、猪、人、小鼠、大鼠7个物种HSL基因外显子Ⅰ部分核苷酸序列间保守性较高,同源性大小依次为99.8%、99.6%、97.4%、90.6%、88.4%、83.5%、82.3%。相应氨基酸序列间保守性更高,同源性分别为100%、100%、98.2%、94.0%、92.2%、89.8%、89.8%。牦牛与各物种该基因部分核苷酸序列间碱基变异类型主要表现为碱基转换和颠换,无碱基插入和缺失发生,碱基转换的频率高于颠换的频率;在核苷酸水平上的多数碱基替换都是同义替换;序列间单碱基变异位点大多出现在同一位点,多发生在密码子第3位,其次是第1位,最少发生在第2位,符合分子进化的中性学说。HSL基因外显子Ⅰ部分核苷酸序列进行多序列对位排列构建的各物种间分子系统进化树结果表明,普通牛和瘤牛首先聚为一类,再分别与牦牛、水牛、猪、人聚类,最后与大鼠、小鼠聚为一类。该聚类结果与动物学上的分类结果一致,表明HSL基因外显子Ⅰ部分核苷酸序列适合于构建物种间分子系统进化树。研究表明,牦牛、普通牛和瘤牛3个物种间的遗传距离大小相近,牦牛和水牛间的遗传距离与普通牛、瘤牛和水牛间的遗传距离大小相当。牦牛、普通牛和瘤牛3个物种间的遗传距离远小于它们各自与水牛这一物种的遗传距离,它们三者之间的亲缘关系也相对于它们各自与水牛间的亲缘关系都较近,故将牦牛、普通牛和瘤牛划分在同一个属——牛属(Bos)更为合理。     

鸡二价金属转运蛋白1 cDNA的克隆与序列分析

鸡二价金属转运蛋白1（divalent metal transporter 1, DMT1）在动物胃肠道锰吸收过程中起重要作用.根据哺乳动物Dmt1同源蛋白氨基酸序列的保守性设计引物,应用3′RACE(rapid amplification of cDNA ends)技术,扩增并克隆获得鸡小肠Dmt1 cDNA 3′端1 289bp和1 092bp的2种片段,发现其3′端翻译区和非翻译区存在差异. 根据鸡Dmt1 cDNA 3′端片段的测序结果设计引物,扩增获得1个与3′端片段部分重叠的鸡Dmt1 cDNA 5′端907 bp片段,并对其进行了克隆测序. 根据鸡小肠Dmt1 3′RACE片段和5′RACE片段序列信息进行拼接,从而获得鸡小肠Dmt1 cDNA全序列信息.结果表明,鸡小肠Dmt1 cDNA有2种形式,1种全长为1 972个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 695个核苷酸,3′非翻译区为173个核苷酸,编码1个含564个氨基酸残基的蛋白质;另1种形式为1 775个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 593个核苷酸,3′非翻译区为78个核苷酸,编码1个含530个氨基酸残基的蛋白质.据鸡Dmt1 cDNA推测出的2种形式蛋白质的氨基酸序列与人、大鼠和小鼠的Dmt1蛋白具有高度同源性,它们的同源性分别为82%、82%、80%,和 84%、84%、83%. 对推测氨基酸序列进行疏水性和跨膜区分析表明,Dmt1蛋白为1种跨膜整合蛋白,具有膜转运蛋白糖基化位点和底物结合位点的保守序列.     

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.