猪核糖体蛋白基因RPLP0的cDNA全长、染色体定位和多态性分析

RPLP0基因编码酸性核糖体磷蛋白大亚基P0, 是核糖体60S亚基的组成成分之一。从本室构建的猪胚胎骨骼肌cDNA文库中分离得到猪RPLP0基因的全长cDNA,并提交 GenBank 数据库。比较猪 RPLP0 基因和人及小鼠同源基因的cDNA序列和蛋白质序列,结果表明该基因在 3 个物种中具有高的相似性。用 PCR-RFLP 方法在猪 RPLP0基因cDNA 545处检测到 C→A 的单碱基突变,为 Csp6Ⅰ的酶切位点。统计分析结果表明 3 种基因型 (AA,AC,CC)在外来品种杜洛克,大约克, 长白和中国地方品种通城猪,小梅山,玉山猪中的分布各不相同。同时使用体细胞杂种板(SCHP)和辐射杂种板(IMpRH) 对 RPLP0 基因进行染色体定位,该基因被定位于 SSC 14q22-q24 并且和SW1321微卫星标志紧密连锁 (25cR, LOD = 14.54)。     

猪核糖体蛋白基因RPLP0的cDNA全长、染色体定位和多态性分析

RPLPO基因编码酸性核糖体磷蛋白大亚基P0,是核糖体60S亚基的组成成分之一。从本室构建的猪胚胎骨骼肌cDNA文库中分离得到猪RPLPO基因的全长cDNA,并提交GenBank数据库。比较猪RPLPO基因和人及小鼠同源基因的cDNA序列和蛋白质序列,结果表明该基因在3个物种中具有高的相似性。用PCR．RFLP方法在猪RPLPO基因cDNA545处检测到C—A的单碱基突变,为Csp6Ⅰ的酶切位点。统计分析结果表明3种基因型（AA,AC,CC）在外来品种杜洛克,大约克,长白和中国地方品种通城猪,小梅山,玉山猪中的分布各不相同。同时使用体细胞杂种板（SCHP）和辐射杂种板（IMpRH）对RPLPO基因进行染色体定位,该基因被定位于SSC14q22．q24并且和SW1321微卫星标志紧密连锁（25cR,LOD=14．54）。     

用原位杂交法定位猪乳铁蛋白基因于染色体2q12

本研究以非放射性标记的猪乳铁蛋白(Porcine Lactoferrin简称PLF)cDNA 为探针,通过染色体原位杂交法,对PLF基因进行了染色体定位。实验中采用金胶抗体技术并结合使用银增强系统,提高了方法的灵敏度。利用染色体的组型分析,对杂交点的分布进行统计学分析。52%(26/50)的分裂相在第2号染色体具银粒分布,实验结果表明：PLF基因定位于猪2号染色体2q¹²区域。     

生物信息学辅助定位及延伸辐射诱导未知表达序列标签

研究辐射诱导的基因表达调控对于认识细胞对辐射损伤的应激反应有重要意义.在低剂量辐射诱导新基因RIG1表达序列标签(expression sequence tag,EST)片段的基础上,通过非克隆cDNA文库和RACE(rapidamplification of cDNA end)技术获得了其3′末端.依据实验得到的这两段EST序列所提供的信息,通过生物信息学分析将RIG1基因初步定位在20号染色体.对20号染色体RIG1区基因组序列进行外显子扫描,发现预测的外显子正好与实验得到的EST相吻合.利用预测的外显子设计特异引物,成功地克隆了RIG1基因全长序列.同时,对20号染色体RIG1区的生物信息学分析表明,在RIG1基因的上游存在启动子区,从而确定了RIG1基因的基因组序列.因此,通过生物信息学辅助设计实验,快捷地定位及延伸了未知EST片段RIG1,基本完成了RIG1的全基因、基因组序列及染色体定位研究.     

生物信息学辅助定位及延伸辐射诱导未知表达序列标签

研究辐射诱导的基因表达调控对于认识细胞对辐射损伤的应激反应有重要意义.在低剂量辐射诱导新基因RIG1表达序列标签(expression sequence tag,EST)片段的基础上,通过非克隆cDNA文库和RACE(rapid amplification of cDNA end)技术获得了其3′末端.依据实验得到的这两段EST序列所提供的信息,通过生物信息学分析将RIG1基因初步定位在20号染色体.对20号染色体RIG1区基因组序列进行外显子扫描,发现预测的外显子正好与实验得到的EST相吻合.利用预测的外显子设计特异引物,成功地克隆了RIG1基因全长序列.同时,对20号染色体RIG1区的生物信息学分析表明,在RIG1基因的上游存在启动子区,从而确定了RIG1基因的基因组序列.因此,通过生物信息学辅助设计实验,快捷地定位及延伸了未知EST片段RIG1,基本完成了RIG1的全基因、基因组序列及染色体定位研究.     

人天然免疫基因BCL10的猪同源物的识别、克隆与初步表达分析

采用电子克隆方法克隆到大小为925 bp的人天然免疫蛋白BCL10的猪同源基因完整cDNA序列(GenBank登录号：EU088132), 并利用RT-PCR方法从猪的全血中扩增出包含702 bp的完整开放读码框架(ORF)的cDNA片段。经核酸测序, 证明与电子克隆结果相符。利用NCBI BLAST分析该cDNA包含3个大小为57 bp、289 bp和356 bp的外显子, 并且定位于猪的4号染色体上。采用半定量PCR技术检测基础水平猪各组织BCL10基因mRNA表达丰度, 并将该基因构建到带有绿色标签的真核表达载体pEGFP-C1中, 采用脂质体转染法将该基因转入PK-15细胞, 通过绿色荧光标记和RT-PCR方法检测实验组的BCL10蛋白表达。研究结果表明, BCL10基因mRNA在脾脏中表达最高; 胸腺、大脑和淋巴结表达次之, 而肝脏只有微量表达, 肾脏没有检测到表达; 同时BCL10基因在PK-15细胞中得到了有效表达。     

转基因猪染色体原位杂交外源生长激素基因定位

近些年来随着原位杂交技术的不断改进,该技术已广泛用于染色体的基因定位。非放射性标记探针的应用使基因定位变得更加简单易行,从而有可能对动物的转基因进行定位研究。本文首次采用胶体金标记药盒（Anti-digoxigenin-gold)和银加强试剂（Silver enhance-ment reagents)的非同位素原位杂交技术对转基因猪外基因进行了定位研究。如Fig.1所示：表达质粒pSMTPGH含有载体pUC19,羊启动子MT011和猪生长激素PGH基因。选5头带有pSMTPGH的转基因猪,分别制备含有染色体DNA的杂交膜。用BglII和Smai对pSMTPGH进行完全酶切,收集0．9kb片段作为探针,以dig-11-dUTP进行标记。探针与DNA杂交后,用光学显微镜检查。选择分散良好、显影银颗粒清楚的玻片进行摄影记录（Fig.2)。对染色体上的显影银颗粒进行统计分析,参照家猪的染色体标准带型,确定外源PGH基因整合位点。Fig.3为4104号转基因猪染色体上的银颗粒分布情况。对5头转基因猪外源PGH基因定位的结果见Table1。探针的合理设计是外源基因定位研究成功的关键。本实验所用探针必须地与外源PGH基因杂交,而不受内源PGH基因的影响。我们设计的探针符合这一要求。采用dig11－dUTP标记探针,抗体金显色,银加强试剂放大杂交信号,在光学显微镜下可以直接观察杂交位点处的显影银颗粒,但于对实验进行统计分析。估计数据表明：转基因猪的外源PGH基因随机整合在所有染色体上,但在13号染色体上的机率略高。     

用人/啮齿类体细胞杂种系及荧光原位杂交将人类新基因ZNF191定位于染色体18q12.1

本文以锌指蛋白ZNF191全长cDNA为探针,与人/啮齿类体细胞杂种系DNA杂交,将这个新的人类锌指基因定位于18号染色体。又用该cDNA筛选人基因组DNA lambda/DASH文库,以获得的DNA片段为探针,进行人染色体荧光原位杂交(FISH)分析,将ZNF191基因精细定位在染色体18q 12.1区带。依据有关遗传连锁分析和等位基因荧光原位杂交(FISH),将ZNF191精确定位于人染色体18q12.1。通过遗传连锁及染色体杂合性丢失分析.目前已知多种遗传病和肿瘤与这个区域相关。因此,ZNF191基因可作为这些疾病或肿瘤的候选相关基因。     

马铃薯StPSKRs基因的克隆及其亚细胞定位分析

该研究采用同源克隆与PCR扩增方法,从马铃薯品种‘Desiree’中克隆植物磺肽素受体基因StPSKR1和StPSKR2的全长cDNA,并对其进行生物信息学分析及亚细胞定位分析,为深入研究StPSKR1和StPSKR2基因在马铃薯生长发育和生物胁迫中的作用提供理论依据。结果发现:(1)通过同源克隆与PCR扩增获得StPSKR1和StPSKR2的全长cDNA片段,并将其克隆到pGWB5-GFP载体;测序结果显示这2个基因编码的蛋白质与数据库给定的蛋白质序列保持一致,表明成功克隆到StPSKR1和StPSKR2基因。(2)StPSKR1位于马铃薯1号染色体上,cDNA全长3 042 bp,编码1 013个氨基酸,预测蛋白相对分子质量为112.16 kD,理论等电点6.27;StPSKR2位于7号染色体,cDNA全长3 135 bp,编码1 044个氨基酸,相对分子量为114.99 kD,理论等电点6.19。(3)生物信息学分析显示,StPSKR1和StPSKR2都属于跨膜蛋白。(4)亚细胞定位结果显示,StPSKR1和StPSKR2均定位于细胞膜上。     

利用杂种细胞克隆板将猪CDC16基因定位于11号染色体

运用比较基因组学的方法,根据人的CDC16基因序列设计引物,从大围子猪和宁乡猪基因组DNA分离了CDC16基因内含子4和内含子8（GenBank收录号为AY880670和DQ206823）,通过扩增体细胞杂种克隆板上27个样品和辐射杂种克隆板上118个样品,确定了CDC16基因在猪染色体上的物理位置,首次将CDC16基因物理定位于猪SSC11 q11-17.该基因与微卫星SW1452标记紧密连锁,LOD值为16.08,存留率为22%,在放射杂交图谱上的图距为62 cR.CDC16基因区间定位结果与精细定位结果相一致,也与比较定位结果相一致,进一步验证了猪11号染色体和人13号染色体大部分片段存在同源性,这为该基因的克隆及和功能研究打下坚实基础.     

Mapping of the human Voltage-Dependent Anion Channel isoforms 1 and 2 reconsidered

Eukaryotic porins or VDACs (Voltage-Dependent Anion-selective Channels) are integral membrane proteins forming large hydrophilic pores. Three functioning genes for VDAC isoforms have been detected in mouse and the corresponding cDNAs are known also in humans. Tissue-specific VDAC isoform 1 (HVDAC1) deficiency in human skeletal muscle is responsible of a rare mitochondrial encephalomyopathy, fatal in childhood. Since coding sequences are not affected in the patient, we focused our interest in the gene structure. HVDAC1 and 2 have been previously mapped at chromosomes Xq13-21 and 21, respectively. Screening of an human chromosome X cosmid library resulted only in the isolation of processed pseudogenes, finely mapped at Xq22 and Xp11.2. Here, we report the mapping of HVDAC1 to chromosome 5q31 and HVDAC2 to chromosome 10q22 by FISH. Exon/intron probes, designed on the basis of the mouse gene structures, were obtained by long extension PCR amplification using the whole genomic DNA as a template. The sequence of the probe extremities clearly pointed to a genuine VDAC genomic sequence. Human and mouse regions where VDAC 1 and 2 genes were mapped are known to be synthetic, thus reinforcing the mapping of the human homologues.     

Linkage and physical mapping of prolactin to porcine chromosome 7

Comparative mapping studies between human and pig have shown that there is conserved synteny between human chromosome 6 and pig chromosomes 1 and 7, but some gene locations are not well established. Prolactin ( PRL ), an anterior pituitary hormone, has been mapped to human chromosome 6, and has tentatively mapped to pig chromosome 7 using Southern-RFLP analysis with a limited number of meioses. To confirm the assignment of prolactin to porcine chromosome 7 by physical and linkage analysis, pig cDNA and human genomic DNA sequences were used to design pig-specific PCR primers. The primers amplified a fragment of ≈2·8 kb. Two polymorphic restriction sites were identified within this fragment with the restriction endonuclease Bst UI. Prolactin was significantly linked to six markers on the published PiGMaP map of pig chromosome 7. Prolactin was physically mapped using a pig × rodent somatic cell hybrid panel. An analysis of these data placed PRL on pig 7p1·1–p1·2 with 100% concordance and was in complete agreement with the linkage data. Both mapping techniques placed PRL in a conserved order with the loci in the syntenic region of human chromosome 6.     

Mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) gene to pig chromosome 15

The natural resistance-associated macrophage protein 1 (NRAMP1) was mapped in the pig for study as a potential candidate gene in controlling pig resistance to Salmonella infection. Primers were designed from the pig cDNA to amplify a 1·6 kb fragment between exons 1 and 3. By using a pig-rodent somatic cell hybrid panel, NRAMP1 was mapped to pig chromosome 15 (SSC15) with 100% probability, and the regional assignment was SSC15q23-26 with 87% concordance. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker was developed by using the Hinf I enzyme and three alleles were identified from a population including 11 breeds. Linkage analysis confirmed the physical assignment by using the PiGMaP reference families. Pig NRAMP1 was linked to SSC15 markers S0088, S0149 and S0284 (LOD > 3). A small population study revealed large allele frequency differences among tested breeds. An A allele is only observed in dam (white) lines whereas a similar exclusivity of the C allele was seen in sire (colored) breeds.     

Conserved synteny and gene order difference between human chromosome 12 and pig chromosome 5.

A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs). These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2). Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison. Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1. These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases. In addition, the human YACs were FISH mapped on pig chromosomes. This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel. While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig. In addition, it was also possible to determine the position of the synteny breakpoint.     

Isolation and mapping of two porcine skeletal muscle myosin heavy chain isoforms

The present paper describes the isolation and linkage mapping of two isoforms of skeletal muscle myosin heavy chain in pig. Two partial cDNAs (pAZMY4 and pAZMY7), coding for the porcine myosin heavy chain-2B and -β respectively, have been isolated from a pig skeletal muscle cDNA library. Four RFLPs were detected with the putative porcine skeletal myosin heavy chain-2B probe (pAZMY4) and one RFLP was identified with the putative myosin heavy chain-β probe (pAZMY7). Two myosin heavy chain loci were mapped by linkage analysis performed with the five RFLPs against the PiGMaP linkage consortium ResPig database: the MYH1 locus, which identifies the fast skeletal muscle myosin heavy chain gene cluster, was located at the end of the map of porcine chromosome 12, while the MYH7 locus, which identifies the myosin heavy chain-α/-β gene cluster, was assigned to the long arm of porcine chromosome 7.     

Characterization of a cDNA encoding a rice mitochondrial voltage-dependent anion channel and its gene expression studied upon plant development and osmotic stress

The voltage-dependent anion channel (VDAC) of mitochondria forms a large pore in the outer envelope membrane. Here, the full Oryza sativa OSVDAC1 cDNA was sequenced and is shown to belong to a small multigene family in the rice genome. This cDNA is 1093 bp long and codes for a protein of 274 amino acids. Expression studies of the osvdac1 gene show a regulation of its level in function of the plantlets maturation and organs. In contrast with several bacterial porins, osmotic stress does not have any effect on the plant osvdac1 gene expression.     

Mapping of growth hormone releasing hormone receptor to swine chromosome 18

The growth hormone releasing hormone receptor (GHRHR) was mapped in the pig for study as a potential candidate gene in controlling pig quantitative growth and carcass characteristics. Primers were designed from the pig GHRHR sequence to amplify a 1·65-kb intronic fragment between exons 6 and 7. By using a pig–rodent somatic cell hybrid panel, GHRHR was mapped to pig chromosome 18 (SSC18) with 100% concordance, and the regional assignment was SSC18q24 with 89% concordance. The polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) with Mse I and Taq I were developed to confirm this assignment with linkage analysis by using the European Pig Gene Mapping Project (PiGMaP) reference families. Pig GHRHR was mapped with strong linkage to SSC18 markers S0062 and S0120 (lod > 8). The GHRHR and IGFBP3 were found to map near to each other on human chromosome 7 (HSA7), and the pig IGFBP3 gene has been mapped to SSC18 by others. Our mapping of pig GHRHR increases the comparative information available on the SSC18 maps and further confirms the synteny conservation between HSA7 and SSC18.     

鸡gga-miR-7b靶基因VDAC1-3’UTR双荧光素酶报告基因质粒的构建

本研究以鸡gga-miR-7b作为研究对象,为了明确与肿瘤形成相关的关键基因VDAC1(Voltage-dependent anion channel 1)是否为gga-miR-7b的靶基因,深入研究gga-miR-7b对VDAC1基因的调控作用,本研究运用TargetScan和miRBD生物学软件预测了gga-miR-7b与VDAC1 m RNA的3’端非编码区(3’UTR)存在种子结合位点(GTCTTCC),并使用PCR克隆以及同源重组突变的方法构建VDAC1-3’UTR野生型和突变型双荧光素酶报告基因重组质粒,经SacⅠ和XbaⅠ双酶切鉴定及测序结果显示,片段大小符合且序列正确;成功构建了VDAC1-3’UTR野生型和突变型双荧光素酶报告基因重组质粒并命名为pmirGLOVDAC1-wt3’UTR和pmirGLO-VDAC1-mut3’UTR。本研究结果进一步为gga-miR-7b候选靶基因VDAC1的鉴定以及功能研究提供理论依据。