Construction of Recombinant Pseudorabies Virus Expressing Canine Distemper Virus H Gene and Analysis on Its Biological Characters

[Objective] The aim was to construct a recombinant pseudorabies virus expressing canine distemper virus H gene and investigate its biological characters.[Method] H gene of canine distemper virus（CDV）strain Onderstepoort was produced by RT-PCR,inserted into pcDNA3.1（＋）vector to construct a expression cassette,which was then subcloned into transfer vector p8AA,prior to the insertion of LacZ expression cassette.The resulting new transfer vector was named as p8AAZH.Subsequently,p8AAZH was co-transfected with the genome of pseudorabies virus（PRV）Bartha-K61 into BHK-21 cells to enable gene recombination and virus package,and the virus solution was collected as cytopathic effect occurring.A series of procedures including blue plaque purification,PCR identification,observation under electron microscope and Western blot were carried out to screen the recombinant pseudorabies virus and identify the protein expression of target gene.Meanwhile,growth curve of the recombinant virus was determined in BHK-21 cells.[Result] The H gene had been inserted into the genome of Bartha-K61 strain,and RPRV-H was the same as Bartha-K61 in the one-step growth curve and cytopathic effect in BHK-21 cells.[Conclusion] The recombinant pseudorabies virus was constructed,and the insertion of H gene did not influence proliferation of recombinant virus,which laid a foundation for development of recombinant canine distemper virus vaccine.

Construction and Identification of a Goat Pox Virus Transfer Vector to Express Peste des Petits Ruminants H gene

[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vector; the recombinants were digested by Nhe Ⅰ and Hind Ⅲ, and ligated into pEGFP-N1-P7.5, yielding the recombinant vector pEGFP-N1-P7.5-H; next the expression cassette EGFP-N1-P7.5-H was first released from recombinant vector pEGFP-N1-P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ, yielding the transfer vector pUC119-TK-EGFP-P7.5-H. [Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7.5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h of transfection. [Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.

Cloning and Sequence of Glycoprotein H Gene of Duck Plague Virus

The glycoprotein H （gH） gene homologue of duck plague virus （DPV） was cloned by degenerate polymerase chain reaction （PCR） and sequenced. It was located immediately downstream from the thymidine kinase gene （TK）. In addition, the 3＇-end of the gene homologue to herpesvirus UL21 was located downstream from the gH gene. DPV gH gene open reading frame （ORF） was 2 505 bp in length and its primary translation product was a polypeptide of 834 amino acids long. It possessed several characteristics of membrane glycoproteins, including an N-terminal hydrophobic signal sequence, an external domain containing eight putative N-linked glycosylation sites, a C-terminal transmembrane domain, and a charged cytoplasmic tail. Comparison with other herpesvirus revealed identities of 20.2, 25.1, 23.0, 23.0, 26.5 and 26.0% with the gH counterparts of the human herpesvirus virus 1 （HSV1）, equine herpesvirus 4 （EHV4）, bovine herpesvirus 1 （BHV1）, pseudorabies virus （PRV）, gallid herpesvirus 2 （GHV2） and gallid herpesvirus 3 （GHV3）, respectively.

Molecular Cloning and Sequence Analysis of C4H Gene of Mangifera indica L.

Cinnamate-4-hydroxylase( C4H) is a key enzyme in phenylpropanoid pathway in plants. Its activity and abundance directly affect the biosynthesis of flavonoids and aromatic compounds. In this study,degenerate primers were designed according to previously reported C4 H gene sequences to clone C4H cDNA sequence with 3'and 5'RACE-PCR from mango( Mangifera indica L). The full-length cD NA of M. indica C4H is 1 680 bp long. Its open reading frame( ORF)is 1 518 bp,encoding a protein of 505 amino acids with a predicted molecular weight of 58. 08 kDa. The isoelectric point of the predicted protein is 9. 52. Functional prediction showed that this gene is mainly located in mitochondria. In addition,the tertiary structure of the protein was built using SWISS-MODEL,and the results showed that the protein has three possible conformations. Phylogenetic analysis based on C4H protein sequences revealed that M. indica has a close genetic relationship with olive( Canarium album) and cocoa( Theobroma cacao). By analyzing the expression level of C4H gene in three colored mango cultivars,we found that that the expression level of C4 H gene in Guifei( with red peel) was the highest,and that in Guiqi( with green peel) was the lowest. The results provide a theoretical basis for studying the molecular mechanism of anthocyanin biosynthesis and C4H's impact on the color of mango fruit.

新疆哈萨克族H型高血压与MTHFR基因多态性及叶酸水平的相关性研究

目的:探讨哈萨克族H型高血压与同型半胱氨酸(Hcy)代谢相关基因MTHFR多态性和叶酸的关联,为H型高血压发病机制提供理论依据。方法:应用新疆哈萨克族H型高血压现况调查数据库,采用病例-对照研究,随机选取H型高血压患者100例、单纯高血压患者100例、高Hcy血症患者100例,健康人群100例作为对照组。应用Sanger测序法对所有研究对象的MTHFR基因rs1801133、rs1801131位点进行基因型检测。叶酸采用电化学发光法进行检测。采用χ^(2)检验进行Hardy-Weinberg平衡检验;应用二分类非条件Logistic回归分析H型高血压与MTHFR基因位点以及叶酸之间的关联。结果:以健康组为对照,高水平叶酸是哈萨克族H型高血压的保护因素(OR=0.33,95%CI:0.15-0.72),rs1801133-(CT+TT)基因型是危险因素(OR=6.37,95%CI:2.97-13.68);高水平叶酸是哈萨克族高Hcy血症的保护因素(OR=0.45,95%CI:0.23-0.86),rs1801133-(CT+TT)基因型是危险因素(OR=2.92,95%CI:1.52-5.62)。结论:MTHFR基因的rs1801133-T等位基因和rs1801133-(CT+TT)基因型是H型高血压的危险因素;高水平叶酸是H型高血压的保护因素。

茎莴苣F6′H家族基因鉴定及其与鲜切莴苣褐变的关系初探

为深入探究阿魏酰-CoA-6′-羟化酶(feruloyl-CoA 6′-hydroxylase, F6′H)在鲜切莴苣褐变过程中的作用及机制,本研究通过Blastp结合结构域筛选的方法从茎莴苣基因组中搜索鉴定F6′H家族成员,并对其进行生物信息学分析及褐变过程中的表达分析,同时测定了鲜切莴苣贮藏过程中褐变度和总酚含量的变化。结果表明,共鉴定出11个茎莴苣F6′Hs基因,其编码蛋白均为定位于细胞质中的亲水蛋白,氨基酸数在181~840个之间,分子量在20.74~93.35 kDa之间,等电点在5.16~8.36之间,脂肪系数在82.28~102.98之间,不稳定指数为26.13~55.39。鲜切莴苣贮藏过程中LsF6′H9基因明显上调表达,LsF6′H3和LsF6′H11在贮藏4~6 d上调表达,其余基因下调表达或变化较小;褐变度逐渐升高,总酚含量先升高后降低,贮藏6 d时最高。相关性分析结果显示,总酚含量与褐变度呈显著正相关,表明酚类的积累可能是导致鲜切莴苣发生褐变的重要因素;褐变度及总酚含量均与LsF6′H1、LsF6′H2、LsF6′H3、LsF6′H4、LsF6′H9、LsF6′H10和LsF6′H11的表达量呈正相关,特别是总酚含量与LsF6′H9表达量呈极显著正相关,褐变度与LsF6′H9表达量呈显著正相关,表明LsF6′H9可能在鲜切莴苣褐变中扮演重要角色。本研究结果可为深入探究F6′H在鲜切莴苣褐变过程中的作用及机制提供理论依据。

拟南芥TRX-h5基因的克隆和表达分析

硫氧还蛋白(TRX-h)是一类催化二硫键还原的小分子蛋白,在调控氧化应激响应中发挥重要作用。为了研究硫氧还蛋白的结构及其功能,文章以野生型拟南芥(Arabidopsis thaliana)Columbia(Col-0)为研究材料,通过反转录-聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)技术获得TRX-h5基因的编码序列(coding sequence,CDS),体外构建pET28a(+)-TRX-h5及pET28a(+)-TRX-h5M(第39位和第42位半胱氨酸位点突变)的原核表达载体,转化至大肠杆菌感受态细胞BL21(DE3)中,诱导后经镍柱纯化得到带有6个His标签的融合蛋白。通过SDS-PAGE电泳技术检测到14 kDa处的目的条带,与理论值预测一致。运用生物信息学分析得出,TRX-h5基因编码118个氨基酸,相对分子质量为13122.32,理论等电点为5.19,该蛋白的不稳定参数为26.74,属于稳定性蛋白。点突变分析发现,TRX-h5的第39位和第42位半胱氨酸位点决定了该蛋白的催化活性,为进一步探究TRX-h5的蛋白功能和高等植物氧化还原系统的研究提供了新思路。

基于H&E图像和基因表达数据的多模态深度学习模型预测胃癌生存风险

胃癌作为高发恶性肿瘤,其致死率近年来居高不下,因此精准预测胃癌患者的生存风险对于治疗至关重要。文章提出了一种基于多模态深度学习的预测模型,旨在评估胃癌患者的生存风险。该模型整合了H&E(Hematoxylin-Eosin staining)染色图像和基因表达数据,首先,采用ResNet18卷积神经网络模型提取深层H&E图像信息,将其编码为一维特征向量。其次,采用多模态紧凑型双线性池化方法,将图像特征与基因表达数据进行融合,用于预测胃癌患者的风险分数。在TCGA的胃癌样本中,该模型的一致性指数(c-index)为0.70。在测试集上进行的Kaplan-Meier分析结果显示,模型成功地区分出高风险群和低风险群。结果表明,该模型在区分胃癌患者风险层次方面表现出色,具有显著优势。

Cloning, Characterization and Chromosome Localization of Two Powdery Mildew Resistance-Related Gene Sequences from Wheat

Reverse_transcription Polymerase Chain Reaction (RT_PCR) was performed using cDNAs as templates from wheat_ Haynaldia villosa 6VS/6AL translocation line and 'Yangmai 5' induced with fungus Erysiphe graminis , and degenerate primers designed based on the conserved amino acid sequences of known plant disease_resistance genes. The cDNA sequences encoding cyclophilin_like and H +_ATPase_like genes were first isolated and characterized in wheat. The putative amino acid sequences of the two clones showed that they were highly homologous to those of cyclophilin proteins and H +_ATPases isolated from other plants. Thus they were designated as Ta_Cyp and Ta_MAH . The obvious expression differences could be observed between wheat_ H. villosa 6VS/6AL translocation line and susceptible wheat cultivar 'Yangmai 5', implying that the two genes may be related with the resistance of wheat_ H. villosa 6VS/6AL translocation line to disease. Southern blot indicated that the wheat genome contained 2-3 copies of Ta_Cyp gene and one copy of the Ta_MAH gene. Chinese Spring nulli_tetrasomic line analysis located the Ta_Cyp homologous genes on wheat chromosome 6A, 6B and 6D. Southern blot using Ta_Cyp clone as a probe showed that the polymorphic bands existed among the H. villosa , amphiploid of Triticum durum _ H. villosa , wheat_ H. villosa 6VS/6AL translocation line and 'Yangmai 5', suggesting that Ta_Cyp homologies exist in wheat genome as well as on the short arm of chromosome 6V in H. villosa .

Screening an Na^+/H^+ Antiporter Gene from the Halophiles Colonizing in the Dagong Ancient Brine Well of Zigong City,China

[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.

HBA2:c.2T>C和HBA2:c.2delT两例罕见突变引起血红蛋白H病家系分析

目的:分别对HBA2:c.2T>C和HBA2:c.2delT两种罕见HBA2基因起始密码子突变复合东南亚型α-地贫的血红蛋白H病病例及其家系成员进行致病基因分析,了解HBA2:c.2T>C和HBA2:c.2delT突变与临床表型的关系。方法:采集家系成员外周血进行血细胞分析及毛细管电泳血红蛋白分析,缺口PCR(Gap-PCR)、反向点杂交法(RDB)检测ɑ-地贫基因常见类型突变,Sanger测序法对HBA1和HBA2基因序列进行分析。结果:检测出两个先证者基因型分别为--SEA/αα复合HBA2:c.2T>C和--SEA/αα复合HBA2:c.2delT,家系成员中检出HBA2:c.2T>C/WT和HBA2:c.2delT/WT,均表现为小细胞低色素性贫血。结论:HBA2:c.2T>C和HBA2:c.2delT为杂合突变时机体可出现静止型α-地贫的表型,当其复合轻型α-地贫时可使机体出现血红蛋白H病的临床表现,本研究为遗传咨询提供依据。

怀化地区犬瘟热病毒H、F基因遗传多样性及其遗传进化分析

【目的】探究怀化地区犬瘟热病毒的基因变异与遗传多样性情况,阐明其遗传进化关系。【方法】从怀化地区收集30株犬源犬瘟热病毒样本,用PCR法扩增其H、F基因序列。利用DNAStar软件分析H、F基因序列碱基组成;利用Clustal X、MegAlign软件进行变异位点和遗传多样性分析;运用Clustal X、PhyML 3.0软件,采用最大似然法(ML)对怀化地区犬瘟热病毒进行遗传进化分析,并用FigTree v 1.3.1软件构建遗传进化树。【结果】30株怀化地区犬瘟热病毒均为Asia-Ⅰ型,其H、F基因序列长度分别为1778和1850 bp,其AT含量分别为56.8%~57.9%和54.9%~56.1%,GC含量分别为42.2%~43.0%和44.1%~44.9%。H基因的种内差异为0~3.1%,种间差异为51.36%~60.15%;F基因的种内差异为0~2.4%,种间差异为39.60%~53.09%。基于H、F基因序列所构建的遗传进化树发现,来源于怀化地区的30株犬源犬瘟热病毒全部位于遗传进化树的同一分支上。【结论】来源于怀化地区犬瘟热病毒之间虽存在一定程度的基因变异和遗传多样性,但它们之间的亲缘关系较近。且其H、F基因序列的种内差异小、种间差异大,是研究犬瘟热病毒基因变异、遗传多样性和遗传进化的重要遗传标记。

吉林地方鸡H-FABP基因多态性及其与屠宰性能、肉品质的关联分析

为研究吉林地方鸡H-FABP基因多态性及其对屠宰性能和肉质性状的影响,试验通过基因组PCR测序鉴定出H-FABP基因部分片段SNP位点,并采用竞争性等位基因特异性PCR(KASP)技术对特定多态位点在吉林芦花鸡、矮脚芦花鸡和吉林黑鸡3个亲本群体及正反交6个群体多态性进行了检验,最后对上述9个群体的肉品质及屠宰性能进行了相关性分析。结果显示:H-FABP基因后段存在8个单碱基SNP位点和1处碱基插入位点,其中g.2892 C>T位点位于外显子3,并导致编码氨基酸由丝氨酸(TCG)突变为亮氨酸(TTG)。g.3173G>A和g.3234~3254位于外显子4中,但所处区域为基因3′UTR区,其余SNP均位于内含子区。KASP技术对不同群体g.2980G>A位点多态性分析发现不同群体三种基因型频率差异较大,但卡方检验分析发现仅在3个群体(LL,AA和HA)该位点处于Hardy-Weinberg不平衡状态。进一步针对不同群体屠宰性能和肉品质进行相关分析发现,不同群体中该位点对不同屠宰性能和肉品质相关性存在差异。本试验证实吉林地方鸡H-FABP基因存在丰富的遗传多样性,且个别位点与屠宰及肉质性状存在一定的相关性。

Research of AtNHX1 Gene Transformation in Brassica napus L. by Agrobacterium tumefaciens

[Objective] The aim was to investigate AtNHX1 gene transformation in Brassica napus L. mediated by Agrobacterium tumefaciens. [Method] By using Agrobacterium-mediated method and cre/lox plant expression vector,the transformation of AtNHX1 gene of Na＋/H＋ antiporter in Brassica napus was studied. [Result] The regeneration rate of cotyledon with petiole was much higher than that of hypocotyl,thus,the cotyledon with petiole was selected as the recipient for transformation. After the cotyledon with petiole was soaked in bacterial solution （OD600=0.4） for 8-10 min,kanamycin-resistant green seeding percentage could reach 3.75%. [Conclusion] The PCR detection of kanamycin-resistant plants proved that NHX1 gene had been inserted into Brassica napus genome. And this research could provide a new way to improve the salt tolerance of Brassica napus.

Expression analysis of a Na^+/H^+ antiporter gene PeNHX1 from Populus euphratica

Na+/H+ antiporters play an important role in the salt tolerance of a wide variety of plants.Using the rapid amplification of cDNA ends method,a Na+/H+ antiporter gene (PeNHX1) was isolated from Populus euphratica.The deduced amino acid sequence contained 528 amino acid residues with a conserved amiloride-binding domain (77LFFIYLLPPI86) and shared more than 68% identity with that of AtNHX1 from Arabidopsis thaliana.PeNHX1 can confer resistance to Na+,as well as Li+,to (EP432) an Escherichia coli strain deficient in both nhaA and nhaB,thus proving that it is a functional Na+/H+ antiporter.PeNHX1 expression profile in EP432 reflected pH independent manner.PeNHX1 expression was regulated by salt at the transcriptional level.Meanwhile,results demonstrated that transcripts of PeNHX1 in P.euphratica calli showed a salt dependent response,and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response in P.euphratica.

老年女性H型高血压患者亚甲基四氢叶酸还原酶C677T基因多态性与清晨血压的关系

目的探讨老年女性H型高血压患者亚甲基四氢叶酸还原酶(MTHFR)C677T基因多态性与清晨血压的关系。方法选取2021年2月至2022年2月我院收治的老年H型高血压患者310例,根据有无清晨高血压发生分为清晨高血压组232例和无清晨高血压组78例。清晨高血压组又分为女性清晨高血压组146例,男性清晨高血压组86例;无清晨高血压组又分为女性无清晨高血压组38例,男性无清晨高血压组40例。所有患者进行动态血压监测,记录24 h、昼间、夜间收缩压和舒张压,检测各组血浆同型半胱氨酸(Hcy)水平、MTHFR C677T基因型分布及等位基因频率,进行多元logistic回归分析。结果清晨高血压组血清Hcy、肌酐、尿酸及尿微量白蛋白水平明显高于无清晨高血压组(P<0.01)。清晨高血压组MTHFR C677T基因TT基因型和T等位基因频率明显高于无清晨高血压组(P<0.05,P<0.01)。女性清晨高血压组和男性清晨高血压组TT基因型和T等位基因频率明显高于女性无清晨高血压组,差异有统计学意义(P<0.05,P<0.01)。多元logistic回归分析显示,MTHFR C677T位点TT基因型、T等位基因携带者是老年女性H型高血压患者清晨血压增高的独立危险因素(OR=3.025,95%CI:1.526~5.996,P=0.002;OR=2.305,95%CI:1.363~3.897,P=0.002)。结论老年女性H型高血压患者MTHFR C677T基因多态性与清晨高血压密切相关,TT基因型和T等位基因携带者可能影响清晨高血压发生。

Genetic Effects of H-FABP Gene on Some Pig Economic Important Traits in a F2 Resource Population

The F2 design was used in construction of the pig resource population, and 14 economic important traits of 119 F2 offspring were measured. The polymorphisms of heart fatty acid binding protein gene (H-FABP) were detected by PCR-RFLP. The effects of different H-FABP genotypes were analyzed by a fixed model. The results showed that, the carcass composition traits were affected by H-FABP gene significantly, backfat thickness alive, carcass backfat thickness between 6th - 7th rib, and average carcass backfat thickness, of different H-FABP genotypes were significantly different (P<0. 05). The dominant effects on the traits demonstrated that the gene affected the carcass composition traits overdominantly. The results also showed that H-FABP gene affected the growth traits and meat quality traits, and the pH, of different H-FABP genotypes was significantly different.

Cloning of Na^+/H^+ antiport gene from Dunaliella salina

1 Introduction Dunaliella Salina,which taxi Dunaliella,Volvocales,Chlorophyceae Chlorophyta,is unicell algae with double flagllum at top,and cup shaped chloroplast without cell wall.Dunaliella Salina is the most salt tolerance eucaryotes.It can grow at the range of salt concentration

MTHFR C677T基因多态性与H型高血压及其靶器官损害的相关研究进展

H型高血压患者占原发性高血压的75%,同型半胱氨酸(Hcy)作为一种含巯基的毒性氨基酸,在H型高血压人群中,高Hcy与高血压协同对心脑肾等靶器官造成损害,严重危害患者的生命健康和生活质量,故关注H型高血压的病因,对其进行提前预防和治疗至关重要。既往对H型高血压病因的研究更多关注于吸烟、高龄、饮食等环境因素。近年来,随着科研技术水平的提高,亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)基因尤其是C677T位点成为人们研究的热点。MTHFR作为Hcy代谢的关键酶,其基因突变与H型高血压的发病及靶器官的损害有着密不可分的关系。但由于基因的分布具有地区与种族的差异性,故其同一疾病在不同地区与种族的相关性研究结果可能不一致。现将MTHFR C677T基因多态性与H型高血压及其靶器官损害的相关性研究进展进行综述。

Cloning of plasma membrane H^+-ATPase gene in Populus euphratica Oliv.

For this paper, the plasma membrane （PM） H^＋-ATPase gene has been cloned from Populus euphratica Oliv. through a ho- mology based strategy. The isolated 3,210 bp cDNA contains a single 2,862 bp open reading frame （ORF） which encodes a putative H^＋-ATPase protein of 953 amino acid residues, with a significant homology to plasma membrane H^＋-ATPase of Prunus persica, Phaseolus vulgaris, Sesbania rostrata and Daucus carota. The predicted protein has a molecular weight of 104,553 Da. The copy number analysis revealed multiple copies of the PM H^＋-ATPase in the P. euphratica genome after digestion of their genomic DNA by the restriction enzymes EcoRI, NdeI, FbaI and Bg/Ⅱ, and Southern blot.