伊犁鲈微卫星位点的筛选及近缘物种通用性

为开发伊犁鲈(Perca schrenkii)分子标记用于鲈属鱼类种质资源保护,以伊犁鲈为材料,应用磁珠富集法进行了微卫星标记的筛选.从伊犁鲈尾鳍提取总DNA,进行酶切、接头连接、PCR扩增,再采用生物素标记(CA)15探针及生物素标记(TG)15探针对扩增产物进行杂交富集,经再次PCR扩增及T-A克隆,成功构建了伊犁鲈基因组微卫星富集文库.采用重复序列引物筛选获得阳性克隆,随机选取48个阳性克隆进行测序,测得序列46个,其中38个克隆含有微卫星序列,41个位点的微卫星重复数在8次以上.根据测得序列设计17对微卫星引物,均能在伊犁鲈群体中扩增获得目的条带.采用该17对引物对河鲈(P.fluviatilis)及黄金鲈(P.flavescens)群体样本进行扩增,10对引物具有通用性,其中6对在河鲈中具有高度多态性(PIC>0.5),5对在黄金鲈中具有高度多态性.     

军曹鱼肌生成抑制素基因的克隆与序列分析

肌生成抑制素(Myostation,MSTN)是一种骨骼肌生长的负调控因子,其生物功能主要是抑制骨骼肌的生长。肌生成抑制素的活性降低或丧失,可使肌肉与其他组织的比例大大提高,因此在动物育种和医疗上有很大的潜在应用价值。目前包括鱼类在内的20多种脊椎动物的MSTN cDNA已经得到克隆和测序。本实验依据已知的鱼MSTN cDNA的保守区域设计一对特异引物,利用PCR技术分别从军曹鱼基因组中扩增出一个约1000bp的特异片段和300bp片段,所得目的片段回收纯化,将其酶切产物连接到pMDl8-T克隆载体上,转化入JM109感受态细胞中,挑取阳性克隆进行转化子鉴定,其质粒测序结果与文献报道的一致,证明成功地克隆了军曹鱼肌生成抑制素基因。     

大黄鱼MSTN成熟肽区基因的原核表达

根据本实验室克隆的大黄鱼肌肉生长抑制素(MSTN)基因编码序列设计引物,通过RT-PCR法扩增大黄鱼MSTN成熟肽区域的基因,将目的片段插入pMD18-T克隆质粒,并测序验证。验证后用BamHⅠ和HindⅢ限制性内切酶酶切含目的片段的pMD18-T克隆质粒和pET-28 a表达质粒,构建MSTN-pET-28 a重组表达质粒。将重组表达质粒转化至BL21菌中,经IPTG诱导后表达蛋白的大小约17 kD。     

曼氏无针乌贼GT微卫星位点的筛选

采用链霉亲和素包被的磁珠富集法筛选曼氏无针乌贼(Sepiella maindroni)微卫星位点。试验样品来自舟山六横岛,提取4个样品的DNA混合成DNA pool,用限制性内切酶Sau 3A I酶切。接上接头后构建基因组PCR文库,用生物素标记的(GT)15探针筛选。将筛选获得目的片段进行PCR扩增,连接pMD18-T载体,转入DH5α感受态大肠杆菌里,扩大培养后PCR筛选阳性克隆。总共选取278个克隆,对120个经过检测含有插入片段的克隆进行测序,发现102个克隆含有微卫星序列,阳性克隆比率为85%。除去重复测序和侧翼链不足的序列,可以设计引物的微卫星序列有64条。     

猪肌生成抑制素基因成熟蛋白编码序列的表达与纯化

猪肌生成抑制素(myostatin,MSTN)基因的cDNA去除信号肽后PCR扩增出成熟蛋白编码序列1．2kb片段,将该片段与pMD18-T载体连接。转化JM109受体菌细胞;筛选阳性克隆测序分析,结果表明与设计序列完全一致。将该克隆载体的质粒DNA用带有BamHI和Sall内切酶识别序列的另一对引物进行PCR扩增,将回收的1.2 kb PCR目的片段定向克隆到pET28a( )表达载体上。成功地构建了编码绪肌生成抑制素成熟蛋白的原核表达载体。LB液体培养基中用IPTG诱导表达,SDS—PAGE显示重组菌表达的MSTN蛋白以包涵体形式存在;经薄层扫描仪扫描分析SDS-PAGE凝胶,表达的MSTN包涵体蛋白占菌体不溶性蛋白含量的27．9％,其相对分子质量为41451．3。所构建的表达载体中含六聚组氨酸标签,HisTrap亲和柱纯化后纯度可达92．5％。该试验为获得较好的绪肌生成抑制素并制备抗体打下了良好的基础。     

鳜肌肉生长抑制素Myostatin cDNA克隆与组织表达分析

利用RT-PCR和cDNA末端快速扩增法(RACE)克隆了鳜(Siniperca chuatsi)肌肉生长抑制素(myostatin,MSTN)cDNA序列,并分析了该基因的结构特征和亲缘关系。鳜MSTN cDNA序列全长2627bp,包括5′端非翻译区117bp、3′端非翻译区1376bp和开放阅读框(ORF)1134bp,共编码377个氨基酸,含22个氨基酸的信号肽。鳜MSTN具有脊椎动物MSTN的共同序列特征,含有1个蛋白酶水解位点RARR和9个保守的半胱氨酸残基。脊椎动物MSTN氨基酸序列的亲缘关系分析表明,鳜与其他鱼类聚为一支。RT-PCR分析表明,鳜MSTN在成体不同组织中的表达情况不同,其中,卵巢、肾、眼、肌肉、心、脑、皮肤和胃中有表达,肝胰脏未见表达。     

Myostatin基因打靶载体的构建及猪胎儿成纤维细胞Myostatin基因的敲除

目的:用缺口修复等技术构建Myostatin(肌肉生长抑制素,MSTN)基因打靶载体,并对大白猪胎儿成纤维体细胞进行转染,获得基因敲除细胞。方法与结果:首先构建用于MSTN基因同源长臂(LA)的抓捕载体,然后在大肠杆菌内利用Red同源重组系统介导的缺口修复,从含大白猪MSTN基因座的细菌人工染色体上亚克隆9.9 kb的LA到抓捕载体上,经过部分序列测定,同源性为100%;通过PCR获得1.4 kb的同源短臂(SA);将LA和SA连入载体pLOXP,构建含有neo和tk正负筛选标记基因的MSTN基因打靶载体pLOXP-MSTN-KO;将线性化的pLOXP-MSTN-KO通过电转染整合到大白猪胎儿成纤维细胞基因组中,利用G418和丙氧鸟苷进行药物筛选,获得抗性细胞克隆890个,通过PCR和DNA测序鉴定获得基因敲除的细胞克隆4个。结论:构建了有效的MSTN基因打靶载体,通过转染获得基因敲除细胞,为利用体细胞核移植制备MSTN基因敲除猪奠定了基础。     

锌指核酸酶技术制备肌肉生长抑制素基因敲除的五指山小型猪成纤维细胞

敲除猪肌肉生长抑制素(Myostatin, MSTN)基因可能提高猪瘦肉率, MSTN基因敲除猪也可作为相关疾病的动物模型。文章利用锌指核酸酶(Zinc-finger nucleases, ZFNs)技术敲除五指山小型猪胎儿成纤维细胞MSTN基因, 为制备MSTN基因敲除猪奠定基础。ZFNs质粒或编码ZFNs的mRNA均能高效敲除MSTN基因, 使用ZFNs mRNA能直接得到MSTN+/-和MSTN-/-两种基因型的细胞克隆。DNA序列测定与分析发现, 细胞克隆的突变类型多为ZFNs作用靶位点处不大于10 bp的碱基插入或缺失(92.18 %); 氨基酸预测发现, 突变型MSTN基因的终止密码子常常提前出现。将MSTN基因敲除的细胞进行体细胞核移植(Somatic cell nuclear transfer, SCNT)发现, 胚胎体外早期发育潜力与野生型无显著差异, 表明这些细胞可用于后续MSTN基因敲除猪的制备。     

细梢小卷蛾微卫星富集文库的构建与分析

目的:构建细梢小卷蛾Rhyacionia leptotubula微卫星富集文库.方法:提取细梢小卷蛾基因组DNA,经限制性内切酶Rsa Ⅰ酶切,用(CT)10和(GT)10生物素探针与其杂交,利用磁珠富集含有微卫星的DNA序列,并对其进行PCR扩增,将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星富集文库:结果:对100个克隆进行随机测序,获得98个微卫星序列,其中具有5次及以上碱基重复次数的微卫星克隆占26%,最高碱基重复次数为33次,非完美型占12%,说明构建的细梢小卷蛾微卫星富集文库是一个高质量的文库.结论:该文库的建立为后续筛选具高多态性的微卫星标记引物研究细梢小卷蛾的种群遗传结构、迁移扩散规律等奠定了基础.     

黄河裸裂尻鱼肌肉生长抑制素基因克隆及表达分析

肌肉生长抑制素(myostatin,MSTN)属于转化生长因子β(TGF-β)超家族中的一个成员,是骨骼肌生长发育的负调控因子。该文以黄河裸裂尻鱼肌肉总RNA为模板,采用RT-PCT、5’-RACE和3’-RACE法获得MSTN基因全长cDNA序列为2180bp,包含长为1128bp的开放阅读框,编码375个氨基酸。以肌肉总DNA为模板,通过PCR法进一步获得了MSTN基因的2个内含子序列,分析表明,黄河裸裂尻鱼MSTN基因与其他脊椎动物具有相似的基因结构(包括3个外显子和2个内含子)。黄河裸裂尻鱼MSTN具有脊椎动物MSTN的共同序列特征,含有1个蛋白酶水解位点RXXR和8个位于TGF-β功能区域保守的半胱氨酸残基。氨基酸序列同源性分析表明,黄河裸裂尻鱼MSTN序列与其他鲤科鱼类MSTN具有较高的同源性;而与哺乳动物和禽类的MSTN同源性较低。系统发育分析表明,黄河裸裂尻鱼MSTN与其他鲤科鱼类聚于同一进化支。RT-PCR分析表明,该基因在黄河裸裂尻鱼9个被检组织中均有表达,但在心、肾、肠、精巢中表达量较高。Real-TimePCR分析显示,MSTN基因在胚胎中的相对表达量,随胚胎发育阶段的不同而有所差异,暗示MSTN的功能可能并不局限在对肌肉生长发育的负调控作用,可能还有其他功能。     

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.     

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     

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.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various