Toll-Like Receptor 2 Gene Polymorphism and its Relationship with SCS in Dairy Cattle

Toll-like receptor 2 (TLR2) plays an important role in the innate immune response to a variety of pathogens. In this study, bovine TLR2 gene was taken as a candidate gene for mastitis resistance. Through PCR-SSCP analysis and sequencing, three missense mutations at T385 G, G398A, and G1884A were detected in the coding region that encoded extracellular domain. Altogether 240 dairy cattle of three breeds (Holstein, Simmental, and Sanhe cattle) were genotyped and allele frequencies were determined. The effects of TLR2 polymorphisms on somatic cell score (SCS) were analyzed and significant association was found between T385 G and SCS. The mean of genotype GG was significantly lower than those of genotype TT and TG. No significant associations were found with SCS for G398A and G1884A. Information provided in this research will be useful in further studies to determine the role of TLR2 gene in the mastitis resistance.     

三种海豚线粒体COI基因的序列分析

对瓶鼻海豚(Tursiops truncatus)、中华白海豚(Sousa chinensis)和糙齿海豚(Steno bredanensis)的线粒体DNA COI基因进行了测序分析。PCR产物约700bp。扩增产物直接测序,去除引物序列后分别获得643、618和618bp的核苷酸序列。碱基组成平均为,T:31·07%,C:26·13%,A:27·27%,G:15·50%,GC含量为41·63%,其中碱基G的含量明显较低。与Gen Bank中9种鲸的同源序列比对,去除部分端部序列后得到597个比对位点,包括141个简约信息位点,43个单突变子,无插入/缺失位点。12种鲸的种间序列差异较大,其序列变异度在2·1%～17·1%之间。597个比对位点编码199个氨基酸,其中有9个氨基酸发生改变,其中一个氨基酸突变可将齿鲸亚目和须鲸亚目分开。NJ系统树表明,海豚科形成单系类群,瓶鼻海豚和中华白海豚的亲缘关系较近。上述分析表明,COI基因可用于鲸类的种类鉴定和系统发育分析。     

甜菜NADH-NR基因的克隆和序列分析

利用同源序列克隆方法从标准偏高糖型甜菜品种甜研7号(Ty7)中获得氯素诱导NADH-NR基因片段,通过RACE技术克隆NADH-NR基因全长序列.该基因ORF长度2 718 bp,编码905个氨基酸,包括147 bp的5'UTR和382 bp的3' UTR,GenBank上的注册号为EU163265,基因编码蛋白的等电点为6.12,推测分子量大小为102 kD,C端(778-891 aa)有一个跨膜区域.基因编码多肽含有3个氧化还原功能区:钼辅因子功能区(eukary NR Moco,93 - 478aa),Fe-血红素结合区(Cytb5,535 -608 aa),FAD结合区(FAD binding 6,653 -760 aa).在甜研7号NR下游的氨基酸残基中含有NADH-NR特有的CGPPP-M基序,说明该蛋白以NADH为电子供体.通过比对,甜研7号的NADH-NR基因与菠菜NADH-NR基因同源性最高,为86.18％.经Southem杂变检验,甜研7号中NADH-NR基因以低拷贝数存在.经基因组克隆分析,甜研7号NADH-NR含有3个内含子,4个外显子.     

水稻黑条矮缩病毒基因组片段S7的cDNA克隆及全序列分析

应用RTPCR技术克隆了2个水稻黑条矮缩病毒 (rice blackstreaked dwarf virus,RBSDV)中国分离物,即浙江分离物和河北分离物的基因组片段S7,并测定了他们的全序列。结果表明：RBSDV浙江分离物(RBSDVZj)基因组片段S7全长2193nts(EMBL登录号为AJ297427),RBSDV河北分离物基因组片段S7全长2190nts(EMBL登录号为AJ297428),二者均含有两个开放阅读框(open reading frame,ORF),分别编码约41kD和36kD多肽,2个中国分离物核苷酸同源性高达99%,相应的ORF编码的多肽同源性分别为100%和94.4%,与日本RBSDV基因组片段S7核苷酸同源性为93.4%和93.8%,相应ORF编码的多肽同源性分别为98.1%(ORF1)、96.5%和97.8%(ORF2),与意大利MRDV S6核苷酸同源性为85.1%和85.3%,相应多肽同源性分别为92.3%(ORF1)、85.5%和86.8%(ORF2)。     

Evolutionary Analysis for Functional Divergence of the Toll-Like Receptor Gene Family and Altered Functional Constraints

The Toll-like receptor (TLR) gene family consists of type 1 transmembrane receptors, which play essential roles in both innate immunity and adaptive immune response by ligand recognition and signal transduction. Using all available vertebrate TLR protein sequences, we inferred the phylogenetic tree and then characterized critical amino acid residues for functional divergence by detecting altered functional constraints after gene duplications. We found that the extracellular domain of TLR genes showed higher functional divergence than that of the cytoplasmic domain, particularly in the region between leucine-rich repeat (LRR) 10 and LRR 15 of TLR 4. Our finding supports the concept that sequence evolution in the extracellular domain may be responsible for the broad diversity of TLR ligand-binding affinity, providing a testable hypothesis for potential targets that could be verified by further experimentation.     

鹟科五种鸟线粒体DNA序列变化与亲缘关系的研究

首次对鹟科（Muscicapidae）画眉亚科三种鸟----画眉（Garrulax canorus）、红嘴相思鸟（Leiothrix lutea）、棕头雅雀(Paradoxornis webbiana);鸫亚科的乌鸫（Turdinae merula）线粒体DNA的12S rRNA基因片段的DNA序列进行了测定,并与北美画眉亚科弯嘴鹛属（Pomatostomus）的灰冠弯嘴鹛（P.temporalis）同源序列进行分析比较并构建分子进化树。实验结果与传统形态学论述存在一定的差异,与同工酶研究结果相同：画眉与红嘴相思鸟的亲缘关系最近,棕头鸦雀较乌鸫与画眉亚科的关系更近,为将其置于画眉亚科提供了分子证据。 Abstract：The classfication on Muscicapinae disturbed us for a long time.The important thing is to find an appropriate character to resolve it.It is the first time that we testified the mitochondrial 12S rRNA gene fragment sequence of four birds (G.canorus,L.lutea,P.webbianus and T.merula-owing to Muscicapinae) and aligned with the same sequence of P.temporalis.Using the mitochondrial 12S rRNA sequences date,we built phylogenetic trees.The results indicated that that the relationship between Garrulax canorus and Leiothrix lutea in the four birds was closer which is congruent with morphology.According to the results of isoenzyme and DNA sequencing data,we can draw a conclusion that Paradoxornis webbianus has closer relationship to Timaliinae than that of Turdus merula,and that putting Paradoxonithinae to Timaliinae was reasonable.     

新疆哈萨克族脑梗塞与ICAM-1基因G241R多态性的关系

目的：探讨新疆哈萨克族脑梗死与细胞黏附分子1（ICAM-1）G241R基因多态性的关系。方法：采用多聚酶链式反应法及限制性内切酶片段长度多态性技术,对新疆哈萨克族100例脑梗死患者及110例健康者（对照组）进行ICAM-1基因G241R多态性检测,比较不同基因型与哈萨克族脑梗塞发病风险的关系。结果：脑梗塞患者ICAM-1基因G41R多态性的基因型频率和等位基因频率与健康对照组相比无明显差异。结论：ICAM-1基因G214R多态性可能不是新疆哈萨克族脑梗塞发病的遗传学危险因素。     

雌激素受体a基因多态性与原发性肝癌的相关性研究

目的探讨西南地区雌激素受体a(estrogen receptor a,ERa)基因多态性与原发性肝癌关系.方法选择西南地区100名原发性肝癌患者为实验组,100名非肝病人群作为正常对照组.应用分子生物学的方法分析ERa基因1号内含子内切酶PvuⅡ,XbaⅠ限制性片段长度多态性(restriction fragment length polymorph-ism,RFLP),观察ERa基因多态性基因型在实验组与对照组中的基因型分布.RFLP用PP、Pp、pp(PvuⅡ)和XX、Xx、xx(XbaⅠ)来表示.结果 P基因型频率实验组为32%,对照组为49%,OR值:0.490.X基因型频率实验组为33.5%,对照组为20.5%,OR值:1.954;PvuⅡ和XbaⅠ限制性片段长度多态性在两组中均呈多态性分布.结论 ERa基因多态性与原发性肝癌有关,P等位基因可能是其保护因素;X等位基因可能是其危险因素.     

新疆哈萨克族脑梗塞与ICAM-1 基因G241R 多态性的关系

目的:探讨新疆哈萨克族脑梗死与细胞黏附分子1(ICAM-1)G241R基因多态性的关系。方法:采用多聚酶链式反应法及限制性内切酶片段长度多态性技术,对新疆哈萨克族100例脑梗死患者及110例健康者(对照组)进行ICAM-1基因G241R多态性检测,比较不同基因型与哈萨克族脑梗塞发病风险的关系。结果:脑梗塞患者ICAM-1基因G41R多态性的基因型频率和等位基因频率与健康对照组相比无明显差异。结论:ICAM-1基因G214R多态性可能不是新疆哈萨克族脑梗塞发病的遗传学危险因素。     

人B组轮状病毒vp7基因的克隆和序列分析

利用逆转录—聚合酶链反应(RT-PCR)技术,扩增了人B组轮状病毒WH—2vp7基因,连接到克隆载体pUCm—T,并对其基因序列进行了分析。WH—2与ADRV的同源性达98％,与印度加尔各达分离株CAL—1达92％,而与动物B组轮状病毒同源性差别较大,如与IDIR(鼠)同源性仅为58％,与WD653(牛)B组轮状病毒同源性为63％,与ATI(牛)同源性为61％。对vp7基因的二级结构分析发现其mRNA折叠形成多达18个发卡环状结构。VP7蛋白是249个氨基酸组成,分子：量为28．4kDa,含有3个潜在的N连接的糖基化位点和多个磷酰化位点,从氨基酸序列的同源性来看,WH—2与ADRV的同源性达99％,与CAL—1达95％,而IDIR仅为51％,说明了WH—2和ADRV的起源相同。此研究对了解B组轮状病毒基因的进化和变异规律具有重要意义,也将为B组轮状病毒预防性疫苗的研制提供科学的依据。     

IL-10基因启动子-627位点多态性与过敏性哮喘

目的：探讨白细胞介素-10（IL-10）启动子-627C／A基因多态性和等位基因频率与过敏性哮喘血清IgE、IL-10浓度以及病情严重程度的相互关系。方法：从哮喘病人DNA文库中选择青岛地区过敏性哮喘病人518例和健康志愿者501例,采用PCR—RFLP方法对IL．10基因启动子．627位点多态性进行观察,比较两组基因型和等位基因的分布频率,同时测定血清中总IgE、IL-10浓度和肺功能检查（FEV1、FVC、FEV1／FVC）。结果：轻度和中一重度哮喘组AA、CA和CC基因型所占比例分别为38．1％、46．0％、15．9％和45．6％、46．2％和8．2％（P=0．0168,X2=8．232,df=2）。A等位基因与哮喘病轻的严重程度有明显相关性（P〈0．05）。AA基因型哮喘病人血清的IgE浓度显著升高（P〈0．01）,但其血清IL-10浓度比CC基因型携带者明显降低（P〈0．01）。结论：IL-10基因启动子-627位点多态性与过敏性哮喘的发生有一定的相关性,等位基因A是哮喘患病的风险基因,而等位基因C则是哮喘病的保护基因。     

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

IL-10基因启动子-627位点多态性与过敏性哮喘

目的:探讨白细胞介素-10(IL-10)启动子-627C/A基因多态性和等位基因频率与过敏性哮喘血清IgE、IL-10浓度以及病情严重程度的相互关系。方法:从哮喘病人DNA文库中选择青岛地区过敏性哮喘病人518例和健康志愿者501例,采用PCR-RFLP方法对IL-10基因启动子-627位点多态性进行观察,比较两组基因型和等位基因的分布频率,同时测定血清中总IgE、IL-10浓度和肺功能检查(FEV1、FVC、FEVl/FVC)。结果:轻度和中-重度哮喘组AA、CA和CC基因型所占比例分别为38.1%、46.0%、15.9%和45.6%、46.2%和8.2%(P=0.0168,X~2=8.232,df=2)A等位基因与哮喘病轻的严重程度有明显相关性(P<0.05)。AA基因型哮喘病人血清的IgE浓度显著升高(P<0.01),但其血清IL-10浓度比CC基因型携带者明显降低(P<0.01)。结论:IL-10基因启动子-627位点多态性与过敏性哮喘的发生有一定的相关性,等位基因A是哮喘患病的风险基因,而等位基因C则是哮喘病的保护基因。     

黑白花牛INHA基因多态性与超数排卵性状关系

为了研究黑白花牛抑制素α基因(INHA)的遗传多样性和超数排卵效果的关联分析,找到一种能够作为黑白花牛超数排卵预测的候选基因,本试验以50头黑白花牛为实验材料,对其进行了超排处理并且采集了黑白花牛的血液样本提取基因组。采用PCR-SSCP技术,在INHA基因的第一外显子和第二外显子各设计了一对引物进行基因型检测,然后与超排性状进行关联分析。结果表明:黑白花牛INHA基因的177 bp处(+1为转录起始位点)存在一个A>G突变的多态性位点,AA和AG基因型的频率分别为0.62和0.38。与超排性状关联分析,结果显示AG基因型黑白花牛的排卵数和鲜胚数均显著多于AA基因型(p<0.05),这说明黑白花牛个体的超排性状可能与其本身的INHA基因的遗传特性有关。     

Design and synthesis of tetrahydropyridopyrimidine based Toll-Like Receptor (TLR) 7/8 dual agonists

In a continuing effort to discover novel TLR agonists, herein we report on the discovery and structure–activity relationship of novel tetrahydropyridopyrimidine TLR 7/8 agonists. Optimization of this series towards dual agonist activity and a high clearance profile resulted in the identification of compound 52a1. Evaluation in vivo revealed an interferon stimulated response (ISG) in mice with limited systemic exposure and demonstrated the potential in antiviral treatment or as a vaccine adjuvant.     

Molecular Characterization,Polymorphism, and Association of Porcine GADD45G Gene

Growth arrest and DNA-damage-inducible gamma (GADD45G) is a reproduction related gene. In this study, the full-length cDNA sequence of porcine GADD45G gene was cloned through rapid amplification of cDNA ends (RACE) method. The porcine GADD45G gene encodes a protein of 159 amino acids that shares high homology with the GADD45G of nine species: chimpanzee (97%), sumatran orangutan (97%), white-tufted-ear marmoset (97%), northern white-cheeked gibbon (97%), cattle (97%), human (97%), rhesus monkey (97%), rat (96%), and mouse (95%). This novel porcine gene was assigned to GeneID: 100152997. Phylogenetic analysis revealed that the porcine GADD45G gene has a closer genetic relationship with the GADD45G gene of cattle. Computer-assisted analysis indicated that porcine GADD45G gene is structured in four exons and three introns. PCR-Rsa I-RFLP was established to detect an A/G mutation on the position of 294-bp of coding sequence and eight pig breeds display obvious genotype and allele frequency differences at this mutation locus. Association of this SNP with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and result demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White and Landrace sows (P < 0.01). Therefore, porcine GADD45G gene could be a useful candidate gene in selection for increasing the litter size. These data serve as a foundation for further insight into this novel porcine gene.     

野猪CAPN7基因的克隆、表达和变异分析

钙蛋白酶是细胞质中主要的蛋白水解酶,在肌肉生长、蛋白转化及嫩化过程中发挥复杂的作用。本研究利用RT-PCR、生物信息学方法克隆野猪（Sus scrofa ussuricus）CAPN7 cDNA并进行序列分析,利用相对定量RT-PCR方法研究其组织表达情况和利用PCR-SSCP方法对其进行变异分析。结果表明,野猪CAPN7基因编码区全长2 442 bp,预期编码813个氨基酸残基,多肽链中存在着calpain家族的催化结构域和催化活性中心;野猪CAPN7不同程度地表达于所检测的12种组织中,在6、9月龄野家杂交猪肌肉组织中的相对表达量高于同一时期的大白猪肌肉组织;所检测到的3种基因型在野猪、民猪和杜洛克中的分布存在着极显著差异。本研究为进一步揭示calpain7的功能提供了分子生物学基础。