中外两品种鸡胸肌组织差异表达基因的研究

运用mRNA差异显示技术对北京油鸡和AA肉鸡胸肌组织基因的差异表达进行研究, 从分子水平分析导致两品种肌肉组织基因差异表达的机制。通过反向Northern dot blot技术验证共筛选出差异表达基因7条, 经与GenBank数据库进行相似性比对, S1与HMGN3基因有较高同源性; S3与ChEST294a8有很高同源性, 但功能未知; S4和S5与鸡的磷酸葡萄糖变位酶Ⅰ同源性很高; S6和S2与已有核酸数据库中的基因克隆或EST具有较高同源性, 为已知的EST, 但功能未知; 序列S7未在数据中发现同源序列, 可以确定其在AA肉鸡中特异表达,确定为新发现的EST(GenBank登录号: EU594549)。为进一步研究中外两品种鸡胸肌组织基因差异表达机制奠定了基础。     

猪脂肪组织表达序列标签(ESTs)大规模测序及分析

利用大规模DNA序列测定的方法,对猪脂肪组织进行了表达序列标签(Expressed Sequence Tag,EST)序列测定,获得高质量EST共7790个,并对此进行了初步分析。使用STACK-PACK软件进行聚类分析,得到4354个基因聚类,包括3609个单拷贝基因和745个多拷贝基因;将候选基因序列用BlastN与nr库进行比较(e=1e-10),从4354个候选基因中得到2712个已知基因,其中单基因为1987个,多拷贝基因为725(3694克隆)个;未知功能基因和新EST有2109个克隆。根据BlastN结果,利用基因组文库添加序号(GenBank Accession No．)为索引,构建了猪脂肪组织已知功能基因表达谱。从基因表达谱可以看出,在猪脂肪组织中参与代谢的基因所占比例最高,在某些方面也显示了脂肪组织旺盛的代谢活性。同时发现在猪脂肪组织中主组织相容性抗原(Major Histocompatibility Complex,MHC)或与MHC相关的基因表达丰度很高。其中单拷贝基因181个,多拷贝基因44个,共计257个克隆,占细胞机体防御(cell and organism defense)总数的44．9％。占总已知基因数的5.4％。提取出全部与MHC相关的EST序列(257个克隆),发现所有EST的部分序列(长约200个碱基),几乎分布在每一个已知猪BAC的所有编码序列上。据此推测,构成MHC的这些EST序列中,有一段长约200个碱基(200bp)的碱基序列高度保守,MHC基因中每一段编码序列都包含有这一段序列。这些MHC序列虽然在不同的BAC上其蛋白的域不同,但均为高度保守区域,并且都与免疫功能密切相关。猪脂肪中如此大量表达的MHC部分保守序列,由于与免疫功能高度相关,在MHC基因的传递过程中,可以反复复制,并能够稳定遗传。     

北京油鸡与AA肉鸡脂肪组织差异表达基因的研究

运用mRNA差异显示技术对AA肉鸡和北京油鸡脂肪组织基因的差异表达进行研究,从分子遗传学角度分析导致两品种脂肪组织差异表达的原因,对了解性状形成的遗传基础和调控机理是十分必要的。通过反Northern杂交验证共筛选出脂肪组织差异表达基因10条,经与GenBank数据库进行相似性比对,XF1 与已知基因有较高同源性,该基因是人类cDNA全长开放阅读框（ORF）的一段; XF2、YF1、YF2及YF4经与nr数据库进行同源性比对,均可找到同源性较高的基因,但功能未知;XF4 与克隆人类胎盘CL0BA010ZF08基因的一段cDNA序列同源性为83%;YF3与预测原鸡MLL5 (LOC417712)基因有一定的同源性,目前尚无功能报道;XF5和YF5 与原鸡高迁移率族蛋白（HMGN3）有较高同源性;XF3 在nr库中未找到同源序列,确定为新发现的EST,提交数据库获得GenBank登录号(Accession number: EU594549)。为进一步研究北京油鸡与AA肉鸡脂肪组织差异基因的功能与脂肪发育的关系奠定基础。     

黄瓜抗黑星病相关基因的差异表达分析

以抗黑星病黄瓜材料HX1为试材,接种黑星病菌（Cladosporium cucumerinum）2h、8h、20h、32h和72h的叶片作为试验方（Tester）,相应的未接种叶片作为对照方（Driver）,利用SSH技术,构建了黑星病菌侵染初期的正向和反向cDNA-SSH文库。用巢式引物PCR检测插入片段,获得了200个阳性克隆,通过测序,除去重复序列,共得到105个Unique ESTs。与非冗余蛋白数据库进行BLASTx比对,结果显示,17条ESTs未找到同源序列,88条非重复序列和已知基因的同源性较高,占全部ESTs序列的83.8%,其中86条ESTs与非冗余蛋白数据库已知功能的蛋白具有高度的相似性。结合高密度点阵膜杂交差异筛选,阳性率为75.0%。经初步分析这些序列的功能,差异表达的ESTs功能涉及能量和基础代谢、信号转导、蛋白和核酸代谢、光合作用及逆境中特异表达的基因等方面。为研究黄瓜抗黑星病基因提供了依据。     

中国地方品种香猪的肌肉特异组织表达序列标签(ESTs)的

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列.在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs.对这10个已知、未知ESTs进行开放阅读框预测并进行B1ast分析,没有找到高度同源的氨基酸序列.对上述EST所对应的基因功能分析结果表明,除去27.27%的EST未能分类外,克隆到的EST大多来自与基因/蛋白的表达调控相关的基因(占45.46%).来自具有其他功能的基因的EST依次是细胞代谢占10.10%、细胞结构/迁移占10.10%、细胞/机体防御占5.05%和细胞信号/传导占2.02%.没有发现和细胞分裂相关的已知功能基因.本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础.     

中国地方品种香猪的肌肉特异组织表达序列标签（ESTs）的分析

通过构建香猪肌肉组织cDNA文库,并在文库中随机挑选克隆进行测序的方法,获得了131个香猪肌肉EST序列。在这131个EST序列所代表的109个单一克隆中,有99个为人类及其他物种的同源序列,3个为已知的猪的ESTs,7个为未知ESTs。对这10个已知、未知ESTs进行开放阅读框预测并进行BlastX分析,没有找到高度同源的氨基酸序列。对上述EST所对应的基因功能分析结果表明,除去27．27％的EST未能分类外,克隆到的EST大多来自与基因／蛋白的表达调控相关的基因（占45．46％）。来自具有其他功能的基因的EST依次是细胞代谢占10．10％、细胞结构／迁移占10．10％、细胞／机体防御占5．05％和细胞信号／传导占2．02％。没有发现和细胞分裂相关的已知功能基因。本研究结果为中国地方品种香猪提供了第一个骨骼肌的基因表达谱,为今后寻找猪肌肉生长和肉用品质的候选基因奠定了基础。     

泌乳前后家猪乳腺基因表达谱的构建以及不同猪种和不同发育时期差异表达基因的确定

构建了不同猪种、不同发育时期的非标准化家猪乳腺cDNA文库, 并从中获得28941条高质量ESTs (expressed sequence tags). 利用序列拼接软件CAP3, 这些EST序列被拼接成2212条重叠群和5642条单一序列. 这些序列在经过功能注释后被聚类成6857个基因聚类, 其中2072条无相应功能注释的序列被认为来自于新基因. 按照标准基因词汇体系的分类标准, 已经有功能注释的基因进行了聚类分析. 通过比较基因表达谱, 确定了几组在猪种间和猪种内不同发育时期乳腺中差异表达的基因, 这些基因中某些可能与家猪的繁殖特性相关, 另外则在乳汁合成、分泌和乳腺复旧过程中发挥重要的作用. 这些来源于家猪乳腺特定发育时期的基因表达谱和一些功能未知的EST序列为进一步的研究提供了重要资源.     

白叶枯病菌胁迫下云南普通野生稻SSH文库的构建及抗病相关基因分析

以云南普通野生稻为材料,利用抑制差减杂交技术（SSH）,构建了白叶枯病菌胁迫的云南普通野生稻特异表达基因的差减文库.通过对文库所有阳性单克隆进行测序,聚类分析后共获得494条高质量的表达序列标签（EST）.经过BlastN分析,有417条与已知功能的序列有较高同源性;经BlastX分析,有104条EST与未知功能蛋白或假定蛋白有较高相似性,49条EST未能找到同源匹配,341条EST与已知功能蛋白有较高同源性.初步分析发现,这些基因主要涉及能量代谢、蛋白质代谢、核酸代谢、防御与抗逆应答反应、信号转导、光合作用及膜运输等代谢过程.使用半定量RT-PCR研究了7个可能与白叶枯病抗性相关的EST序列在云南普通野生稻对照和白叶枯病菌处理的叶片中的表达情况,并获得这些基因的表达谱.结果发现,克隆编号为OR7,OR68和OR826的EST受白叶枯病菌胁迫诱导上调表达,其中OR826 EST在蛋白数据库中无同源序列,可能是一类新的白叶枯病抗性基因,而组成型表达的OR143 EST在对照和接菌处理的叶片中均能检测到其mRNA的表达,但其表达量在白叶枯病菌胁迫48 h后逐渐增强,推测这些基因直接参与了云南普通野生稻抗病防御反应.本研究为从云南普通野生稻中发掘和克隆新的白叶枯病抗性基因提供了理论依据,为进一步研究云南普通野生稻抗白叶枯病的分子机制奠定了基础.     

仔猪腹泻抗性相关特异表达ESTs的筛选

以久仰香猪、剑白香猪和长白猪3个品种的脾脏为材料,以品种间同一组织不同的发病状态为对照,将DDRT—PCR技术与银染法相结合进行差异显示研究,筛选与仔猪腹泻抗性相关的特异表达ESTs。在脾脏未发病RNA池中检测到5条特异表达ESTs,其中一条与Nck转导蛋白同源,一条与长散布元件反转录酶同源,一条为相似性较高的未知功能序列,两条为新序列。     

成人视网膜假定蛋白基因ARHP的克隆及生物信息学分析

从UniGene库中选取编号为BG2 2 2 62 4来自人鼻咽组织的表达序列标签 (EST )序列 ,联网到NCBI调用Blast服务器分析 ,发现该EST序列是一个代表新基因的未知序列 .利用Blast检索GenBank的nr数据库和EST数据库 ,构建EST重叠群 ,联网到NCBI的ORFfinder服务器 ,分析发现该EST重叠群具有完整的阅读框架 .分别在cDNA序列阅读框架的起始密码子和终止密码子的两侧设计引物 ,以人胎脑cDNA文库为模板 ,进行PCR扩增 ,测序确定该基因的cDNA全长序列 .该基因cDNA序列全长为 1672bp ,阅读框架位于第 3 0 4～ 1557位之间 ,编码由 417个氨基酸组成 ,分子质量为 46 58ku的蛋白质 ,其理论 pI为 4 2 1.将蛋白质序列通过NCBI的Blast服务器进行序列相似性分析 ,发现该基因编码的蛋白质和成年小鼠视网膜未知蛋白 (BAB3 2 2 14 )同源 .经与国际人类基因组命名委员会协商定名为成人视网膜假定蛋白 (adultretinahypotheticalprotein ,ARHP) ,GenBank登录号为AY174896.生物信息学分析表明 ,该蛋白质可能为一参与转录调控的核蛋白 .ARHP基因定位在染色体 5q3 5,跨越 3 5163bp ,含 4个外显子和 3个内含子 .在基因的 5′非翻译区有 2个CpG岛     

Assignment of MHC in swine to chromosome 7 by in situ hybridization and serological typing

Mapping of the MHC in swine (SLA) was achieved by direct in situ hybridization to chromosome preparations. We took advantage of the fact that the cDNA probe coding for class I HLA-B7 antigen cross-hybridizes with swine genomic DNA. By nick-translation, 35S nucleotides were incorporated to a specific activity of 2,7 10(7) cpm/ug. Analysis of 91 randomly selected labeled metaphases revealed highly significant labeling on chromosome 7. The SLA complex is most probably located at the proximal half of the long arm, as indicated in families carrying a modified chromosome 7 and heterozygous for SLA. The abnormal chromosome was always inherited with a specific haplotype whereas the other parental haplotypes were found in association with the normal 7.     

Difference in number of loci of swine leukocyte antigen classical class I genes among haplotypes

The structure of the entire genomic region of swine leukocyte antigen (SLA)-the porcine major histocompatibility complex--was recently elucidated in a particular haplotype named Hp-1.0 (H01). However, it has been suggested that there are differences in the number of loci of SLA genes, particularly classical class I genes, among haplotypes. To clarify the between-haplotype copy number variance in genes of the SLA region, we sequenced the genomic region carrying SLA classical class I genes on two different haplotypes, revealing increments of up to six in the number of classical class I genes in a single haplotype. All of the SLA-1(-like) (SLA-1 and newly designated SLA-12) and SLA-3 genes detected in the haplotypes thus analyzed were transcribed in the individual. The process by which duplication of SLA classical class I genes was likely to have occurred was interpreted from an analysis of repetitive sequences adjacent to the duplicated class I genes.     

Proteomic and transcriptomic analyses of differential stress/inflammatory responses in mandibular lymph nodes and oropharyngeal tonsils of European wild boars naturally infected with Mycobacterium bovis

Differential stress/inflammatory responses were characterized at the mRNA and protein levels in mandibular lymph nodes (MLN) and oropharyngeal tonsils of European wild boars (Sus scrofa), naturally infected with Mycobacterium bovis. Suppression-subtractive hybridization combined with immunohistochemistry and/or quantitative real-time RT-PCR were used to identify and characterize abundant stress/inflammatory gene sequences differentially expressed in tuberculous (TB+) wild boars. Genes identified in MLN and tonsils corresponded to serum amyloid A, arginase I, osteopontin, lysozyme, annexin I, and heat shock proteins, respectively. Global protein patterns in MLN and tonsils were compared between TB+ and nontuberculous (TB-) boars by 2-DE and MALDI-TOF MS. Five proteins, including stress/inflammatory proteins annexin V, serum albumin, and apolipoprotein A1 were found at lower levels in MLN of TB+ boars. Manganese superoxide dismutase was found up-regulated in MLN of TB+ boars. Five proteins, including creatine kinase and MHC class II antigens were found up-regulated in tonsils of TB+ boars. These results demonstrated differential stress/inflammatory responses in wild boars naturally infected with M. bovis and suggest possible markers of tuberculosis in this species that may prove useful for future studies of host-pathogen interactions and for diagnostics and vaccine development.     

Molecular characterization of swine leucocyte antigen class I genes in outbred pig populations

The highly polymorphic swine leucocyte antigen ( SLA ) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation success. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to type alleles at the three classical SLA class I loci ( SLA-1 , SLA-3 and SLA-2 ) using the PCR-sequence-specific primer (PCR-SSP) strategy. This typing system relies on 47 discriminatory PCR primer pairs designed to amplify the SLA class I alleles by groups that have similar sequence motifs. We applied this low-resolution group-specific typing method to characterize the SLA class I alleles present in three outbred pig populations ( n =  202). Alleles from 24 class I allele groups corresponding to 56 class I genotypes were detected. We also identified 23 low-resolution SLA class I haplotypes in these pigs and found haplotypes Lr-1.0 ( SLA-1 *01XX- SLA-3 *01XX- SLA-2 *01XX) and Lr-4.0 ( SLA-1 *04XX- SLA-3 *04XX- SLA-2 *04XX) in all three pig populations with a high prevalence. Over 80% of the pigs examined ( n  =   162) were found to bear at least one of these haplotypes, resulting in a combined haplotype frequency of nearly 50%. This PCR-SSP-based typing system demonstrates a reliable and unambiguous detection of SLA class I alleles, and can be used to effectively investigate the SLA diversity in outbred pig populations. It will help to identify the role of SLA antigens in disease-resistant pigs and may facilitate the development of effective vaccines.     

Molecular characterization of swine leukocyte antigen gene diversity in purebred Pietrain pigs

The porcine major histocompatibility complex (MHC) harbors the highly polymorphic swine leukocyte antigen (SLA) class I and II gene clusters encoding glycoproteins that present antigenic peptides to T cells in the adaptive immune response. In Austria, the majority of commercial pigs are F ₂ descendants of F ₁ Large White/Landrace hybrids paired with Pietrain boars. Therefore, the repertoire of SLA alleles and haplotypes present in Pietrain pigs has an important influence on that of their descendants. In this study, we characterized the SLA class I ( SLA‐1 , SLA‐2 , SLA‐3 ) and class II ( SLA‐DRB1 , SLA‐DQB1 , SLA‐DQA ) genes of 27 purebred Pietrain pigs using a combination of the high‐resolution sequence‐based typing (SBT) method and a low‐resolution (Lr) PCR‐based method using allele‐group, sequence‐specific primers (PCR‐SSP). A total of 15 class I and 13 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotype Lr‐43.0 ( SLA‐1 *11XX– SLA‐3 *04XX– SLA‐2 *04XX) was identified in 11 animals with a frequency of 20%. For SLA class II, the most prevalent haplotype, Lr‐0.14 [ SLA‐DRB1 *0901– SLA‐DQB1 *0801– SLA‐DQA *03XX], was found in 14 animals with a frequency of 26%. Two class II haplotypes, tentatively designated as Lr‐Pie‐0.1 [ SLA‐DRB1 *01XX/be01/ha04– SLA‐DQB1 *05XX– SLA ‐ DQA*blank] and Lr‐Pie‐0.2 [ SLA‐DRB1 *06XX– SLA‐DQB1 *03XX– SLA‐DQA *03XX], appeared to be novel and have never been reported so far in other pig populations. We showed that SLA genotyping using PCR‐SSP‐based assays represents a rapid and cost‐effective way to study SLA diversity in outbred commercial pigs and may facilitate the development of more effective vaccines or identification of disease‐resistant pigs in the context of SLA antigens to improve overall swine health.     

Rapid assignment of swine leukocyte antigen haplotypes in pedigreed herds using a polymerase chain reaction-based assay

We present a simple assay to determine the swine leukocyte antigen (SLA) haplotypes of animals within two experimental populations of MHC defined miniature pigs. The Yucatan miniature pigs have four founder haplotypes ( w, x, y, z) and one recombinant haplotype ( q). The NIH miniature pigs have three founder haplotypes ( a, c, d) and two recombinant haplotypes ( f, g). Because most crossovers occur between the class I and class II regions, haplotypes can be assigned by typing one class I locus and one class II locus for practical purposes. We have previously characterized these seven founder haplotypes by sequencing the cDNA of three SLA class I loci, designated as SLA-1, SLA-3 and SLA-2 and four SLA class II loci, SLA-DQA1, SLA-DQB1, SLA-DRA1 and SLA-DRB1. These sequences were used to design allele-specific primers to amplify one MHC class I and one MHC class II gene for each haplotype. Primers were tested for specificity in homozygous and heterozygous animals. Positive control primers were also designed to amplify a portion of the E-selectin or alpha-actin gene and multiplexed with the allele-specific primers to check for false negatives. This combination of allele-specific and positive control primers produced specific and robust PCR-site-specific primer assays for assigning SLA haplotypes in the two populations.     

Genomic sequence analysis of the 238-kb swine segment with a cluster of TRIM and olfactory receptor genes located, but with no class I genes, at the distal end of the SLA class I region

Continuous genomic sequence has been previously determined for the swine leukocyte antigen (SLA) class I region from the TNF gene cluster at the border between the major histocompatibility complex (MHC) class III and class I regions to the UBD gene at the telomeric end of the classical class I gene cluster (SLA-1 to SLA-5, SLA-9, SLA-11). To complete the genomic sequence of the entire SLA class I genomic region, we have analyzed the genomic sequences of two BAC clones carrying a continuous 237,633-bp-long segment spanning from the TRIM15 gene to the UBD gene located on the telomeric side of the classical SLA class I gene cluster. Fifteen non-class I genes, including the zinc finger and the tripartite motif (TRIM) ring-finger-related family genes and olfactory receptor genes, were identified in the 238-kilobase (kb) segment, and their location in the segment was similar to their apparent human homologs. In contrast, a human segment (alpha block) spanning about 375 kb from the gene ETF1P1 and from the HLA-J to HLA-F genes was absent from the 238-kb swine segment. We conclude that the gene organization of the MHC non-class I genes located in the telomeric side of the classical SLA class I gene cluster is remarkably similar between the swine and the human segments, although the swine lacks a 375-kb segment corresponding to the human alpha block. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL, and GenBank databases under accession numbers AB158486 and AB158487     

Establishment of a resource population of SLA haplotype-defined Korean native pigs

The highly polymorphic porcine major histocompatibility complex (MHC), or the swine leukocyte antigens (SLA), has been repeatedly associated with variations in swine immune response to pathogens and vaccines as well as with production traits. The SLA antigens are also important targets for immunological recognition of foreign tissue grafts. We recently established a resource population of Korean native pigs as models for human transplantation and xenotransplantation research. In this study, 115 animals derived from three generations of the Korean native pigs were genotyped for three SLA class I (SLA-2, SLA-3 and SLA-1) and three SLA class II loci (DRB1, DQB1, DQA) using PCR with sequence-specific primers (PCR-SSP) at the allele group resolution. A total of seven SLA haplotypes (Lr-5.34, Lr-7.23, Lr-31.13, Lr-56.23, Lr-56.30, Lr-59.1, Lr-65.34), comprising six unique class I and five unique class II haplotypes, were characterized in the founding animals. Class I haplotype Lr-65.0 and class II haplotype Lr-0.34 were novel; and together with Lr-56.0 these haplotypes appeared to be breed-specific. In the progeny population, Lr-7.23 and Lr-56.30 appeared to be the most prevalent haplotypes with frequencies of 34.7% and 31.6%, respectively; the overall homozygosity was 27.4%. This resource population of SLA-defined Korean native pigs will be useful as large animal models for various transplantation and xenotransplantation experiments, as well as for dissecting the roles of SLA proteins in swine disease resistance and production traits.