Characterization, Imprinting Status and Tissue Distribution of Porcine GTL2 Gene

The callipyge （CLPG） phenotype, exhibiting polar overdominance （POD）, is an inherited skeletal muscle hypertrophy described in sheep. The callipyge locus maps to the distal portion of ovine chromosome 18 within the DLKI-GTL2 region and corresponds to human chromosome 14 and mouse chromosome 12. The POD phenomenon is confirmed to the homologous region of swine chromosome 7. In order to clone and investigate the expression of porcine GTL2 gene, DNA and RNA samples from 60-day-old F1 animals, generated with reciprocal crosses between Large White and Meishan breeds and their parents, were used. The authors showed that porcine GTL2 acted as a uoncoding RNA. cDNA samples exhibited maternal expression of the gene in the heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, and fat in pigs, and a unique tissue-specific expression different from that of humans and mice. These results indicated that the gene was conserved in the pig, human, mouse, and bovine. It will be of interest to further study the gene functions in muscle growth and fat deposition.     

Porcine LEM domain-containing 3: Molecular cloning,functional characterization,and polymorphism associated with ear size

Ear size exhibits remarkable diversity in pig breeds. LEM domain-containing 3(LEMD3) on chromosome 5 is considered as an important candidate for porcine ear size. This is the first study on cloning and characterization of LEMD3 c DNA. The complete c DNA contains 4 843 bp, including a 2 736-bp open reading frame(ORF), a 37-bp 5′-untranslated region(UTR) and a 2 070-bp 3′-UTR. The complete LEMD3 gene is 126 241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82–94% nucleic acid and 51–96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter of LEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms(SNPs): L964CA in the complete coding region, L4625AG in the 3′ UTR, and L-394TC in the promoter region. Genome-wide association study(GWAS) revealed that all of SNPs were shown significant association with ear size in Large White×Minzhu pig intercross population. With conditional GWAS, –log10(P-value) decreased by more than 80% when each of three SNPs was included as a fixed effect. These results suggested direct involvement of LEMD3 or close linkage to the causative mutation for ear size. The findings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds.     

Isolation, Identification and Tissue Expression Profile Analysis of OneNovel Differentially Expressed Sequence Tag in the Longissimusdorsi Muscle from Meishan, Meishan x Large White Hybridand Large White Pigs

In order to detect the molecular mechanism of heterosis in pigs, the mRNA differentialdisplay technique was performed to investigate the differences of gene expression in theLongissimus dorsi tissue from Meishan, Meishan x Large White hybrid and Large White pigswith nine 3‘-end anchored primers in combination with ten 5‘-end arbitrary primers andnearly 3000 reproducible bands were examined. One novel expressed sequence tag (EST4,GenBank accession number: AY553914) that was differentially expressed in Meishan,Meishan x Large White hybrid and Large White pigs was isolated from the Longissimus dorsimuscle tissue and identified through semi-quantitative RT-PCR. BLAST analysis revealedthat the 350 bp long EST (EST4) was not homologous to any of the known porcine genes.Tissue expression profile analyses showed that the EST4 was expressed in most of tissues.     

Sequencing, Polymorphism and Expression Profile Analysis of Porcine Hexokinase Ⅱ （HK2） Gene

Hexokinase Ⅱ has been demonstrated to play the role of a key enzyme member in the glycolysis reaction. It catalyzes the conversion of glucose to glucose-6-phosphate, thus committing glucose to the glycolytic pathway. In this paper, the partial exons and introns 10, 11, 13 and 14 of the porcine HK2 gene were cloned and sequenced by comparative genomics. Comparative sequencing of three pig breeds revealed ten putative single-nucleotide polymorphisms （SNPs）, one of which in intron 10 with differing bases （G981A） is within the restriction site for enzyme Msp I. Distribution of Msp I -RFLP genotype and allele frequencies among different pig breeds were studied. By association analysis between Msp I PCR- RFLP polymorphism （AA, AB, BB genotypes of HK2 gene intron 10） and some meat quality and carcass traits in F2 group, which was constructed by our laboratory, a significant difference of pig average backfat at rump was found between AB and BB genotypes （P〈0 05） in F2 group. In addition, the pattern of expression of ilK2 in a variety of tissues in pig was also determined using semi-quantitative RT-PCR. The expression of HK2 mRNA was detected only in pig skeletal muscle.     

Analysis of Genetic Diversity and Relationships of Seven Chinese Indigenous Pig Breeds and Three Exotic Pig Breeds Using the DNA Differential Display Technique

The genetic diversity and relationships of seven Chinese indigenous pig breeds （Meishan, Erhualian, Hezuo, Bamei, Qingping, Tongcheng, and Huainan） and three exotic pig breeds （Large White, Landrace, and Duroc） were analyzed using the DNA differential display technique by means of eight primer combinations. A total of 123 reproducible bands were used to calculate mean Nei＇s gene diversity, and mean Shannon＇s information index for each pig population. Based on these the Nei＇s standard genetic identity and distance were estimated, which was used to construct a dendrogram tree for the 10 pig breeds. The experimental results obtained and the method used in this study for evaluating the genetic diversity and relationships of pigs were also discussed.     

Determination and Difference Analysis of DNA Methylation Content Both in Blood and Muscle Tissue of Pigs

In this experiment, DNA samples from parental lines Large White, Landrace, and Meishan pigs, and their hybrids Large White × Landrace, Landrace × Large White, Large White × Meishan, and Meishan × Large White pigs were used for the determination of DNA methylation content in both blood and muscle tissue. The differences about DNA methylation content between parental lines and their hybrids were analyzed. These will offer theoretical support from molecular level for heterosis. High performance liquid chromatography （HPLC） was firstly used to detect DNA methylation content. The average DNA methylation content in 163 DNA samples of muscle tissue was 16.92%, whereas, the average DNA methylation content in 182 samples of blood was 6.49%, the difference between which was especially prominent （P 〈 0.01）. In blood, the methylation content was lower than 10%, with the highest level of 9.93% in Landrace x Large White and the lowest of 2.97% in Landrace. The difference between Large White and Landrace was highly significant （P 〈 0.01）, but not significant between Large White and Meishan; and the differences between reciprocal cross hybrids in both hybrid systems were significant （P 〈 0.05）. In muscle tissue, the differences in methylation content among three parents were not significant （P 〉0.05）; and the differences between reciprocal cross hybrids in both hybrid systems were not significant （P 〉 0.05）, but between different hybrid systems, the hybrids had a significant difference （P 〈0.05）. The average methylation content in muscle samples was higher than that in blood samples, and the methylation in different tissues was different.     

Genome-wide association study for rib eye muscle area in a Large White×Minzhu F_2 pig resource population

Rib eye muscle area(REMA) is an economically important trait and one of the main selection criteria for breeding in the swine industry. In the genome-wide association study(GWAS), the Illumina Porcine SNP60 Bead Chip containing 62 163 single nucleotide polymorphisms(SNPs) was used to genotype 557 pigs from a porcine Large White×Minzhu intercross population. The REMA(at the 5th–6th, 10th–11th and the last ribs) was measured after slaughtered at the age of(240±7) d for each animal. Association tests between REMA trait and SNPs were performed via the Genome-Wide Rapid Association using the Mixed Model and Regression-Genomic Control(GRAMMAR-GC) approach. From the Ensembl porcine database, SNP annotation was implemented using Sus scrofa Build 10.2. Thirty-three SNPs on SSC12 and 3 SNPs on SSC2 showed significant association with REMA at the last rib at the chromosome-wide significance level. None of the SNPs of REMA at the 5th–6th rib and only a few numbers of the SNPs of REMA at the 10th–11th ribs were found in this study. The Haploview V3.31 program and the Haplo.Stats R package were used to detect and visualize haplotype blocks and to analyze the association of the detected haplotype blocks with REMA at the last rib. A linkage analysis revealed that 4 haplotype blocks contained 4, 4, 2, and 4 SNPs, respectively. Annotations from pig reference genome suggested 2 genes(NOS2, NLK) in block 1(266 kb), one gene(TMIGD1) in block 2(348 kb), and one gene(MAP2K4) in block 3(453 kb). A functional analysis indicated that MYH3 and MYH13 genes are the potential genes controlling REMA at the last rib. We screened several candidate intervals and genes based on the SNPs location and the gene function, and inferred that NOS2 and NLK genes maybe the main genes of REMA at the last ribs.     

The Effects of Mx1 Locus on Immunity Tracts Components in Large White×Meishan F2 Offspring

The mouse Myxovirus resistance protein 1 (Mx1) is known to be sufficient to confer resistance to influenza viruses, and genes encoding Myxovirus resistance protein 1 (Mx1) is, therefore, an interesting candidate gene for disease resistance in farm animals. The porcine Mxl gene has already been identified and characterized based on its homology with mouse Mxl; the full-length coding region of the pig Mxl gene spans 2 545 bp (M65087) and is organized into 17 exons compared with the human ortholog mRNA. In this study, the exons 9, 10, 11 and introns 6, 9 of the porcine Mxl gene were cloned and sequenced; two SNPs were identified in exons 9, 10, 11 but none of the SNPs led to an amino acid exchange, and the other eleven variants were detected in introns 6 and 9, respectively. Differences in allele frequency among Meishan, Large White, Tibetan, Tongcheng, Huainan, and Duroc pigs were observed within intron 9, of which an A → G substitution at position 186 was detected as an Msp Ⅰ PCR-restriction fragment length polymorphism (PCR-RFLP). The association analysis using the Large White×Meishan F2 offspring suggested that the Mxl genotype was associated with variation in several immunity traits that are of interest in pig breeding. However, further investigations in more populations are needed to confirm the above concept.     

猪繁殖候选基因ERK2的克隆、表达及基因多态分析

 【目的】ERK2基因在细胞增殖和分化调控以及启动卵巢排卵的分子信号等过程中发挥重要作用,是影响猪繁殖性状的重要候选基因。本试验对猪ERK2基因序列、基因结构、基因多态性及其表达规律进行初步研究。【方法】以大白猪为材料,采用RT-PCR方法克隆了猪ERK2基因,Real-Time PCR测定该基因在猪各组织器官中的分布,并对该基因的结构和多态性进行分析。【结果】从猪卵巢组织中克隆获得ERK2基因部分cDNA序列,长1 888 bp,包括一个1 080 bp的开放阅读框,编码359个氨基酸与预测的猪ERK2基因、已报道的人和小鼠等的ERK2基因高度相似;猪ERK2基因在各组织中表达广泛,其中脾脏是表达量最高的组织,在脂肪组织、前后腿肌中基本不表达;猪ERK2基因定位于14号染色体,全长在22 kb以上,包含9个外显子和8个内含子;对第2—9外显子及内含子外显子交界处的内含子序列进行序列突变检测,共检测到11个SNPs和1个插入缺失突变,但绝大部分的变异都是在内含子区域,仅有1个SNP发生于3′UTR区域。【结论】猪ERK2基因cDNA为1 888 bp,编码359个氨基酸残基;在猪各组织中广泛分布,以脾脏的表达量最高;该基因由9个外显子和8个内含子组成,基因保守性较高,共检测到11个SNP和1个插入缺失突变均不在编码区中。     

华山新麦草蔗糖:蔗糖1-果糖基转移酶基因Ph-1-SST的克隆及序列分析

蔗糖:蔗糖1-果糖基转移酶基因1-SST在植物逆境胁迫反应中起重要作用。为了挖掘华山新麦草(Psathyrostachys huashanica Keng)抗非生物胁迫关键功能基因,以华山新麦草叶片为材料,利用RT-PCR结合RACE技术克隆1-SST基因的全长cDNA,命名为Ph-1-SST,登录号为KX761897。通过PCR法扩增其gDNA序列,测序结果表明该基因的gDNA和cDNA序列长度分别为3 344bp和2 001bp,编码666个氨基酸残基,序列结构分析结果表明该基因含4个外显子3个内含子,预测该蛋白相对分子质量为72.9ku,理论等电点为4.87。序列比对显示,该基因与大麦1-SST基因编码蛋白的相似性最高(90%),为糖基水解酶32家族成员。对1-SST氨基酸序列的系统进化树分析显示,Ph-1-SST及其同源蛋白位于不同分支,初步推断此全长cDNA是华山新麦草中编码蔗糖:蔗糖1-果糖基转移酶的一个新基因。结果为作物非生物胁迫改良提供重要基因资源。     

两种弧菌感染大黄鱼免疫相关基因的SNP位点分析

为了探究大黄鱼(Larimichthys crocea)免疫相关基因的SNP与弧菌抗性关系,分别利用鳗弧菌(Vibrio anguillarum)和副溶血弧菌(Vibrio parahaemolyticus)人工感染大黄鱼。对感染前后抗感群体的转录组进行高通量测序、筛选并分析其抗病差异:(1)筛选氨基酸的非同义突变SNP位点在抗鳗弧菌组有17个,而抗副溶血弧菌组的有28个;(2)一代测序验证结果发现,染色体NW_011323507.1上白细胞介素6受体基因(IL-6R)第91 196位碱基G突变为C,导致缬氨酸突变为亮氨酸,该位点G/C在抗鳗弧菌组、对照组样本之间突变基因型CC频率分别为12.5%和0,呈显著性差异(P0.05);(3)补体C1q/肿瘤坏死因子相关蛋白9(CTRP9)基因在染色体NW_011323975.1上的35 665位碱基突变(A-G),在副溶血弧菌抗感易感群体中突变位点基因型GG频率分别为37.5%和0,呈极显著性差异(P0.01)。结果表明,IL-6R-91196-G/C位点突变与大黄鱼抗鳗弧菌有关联,CTRP9-35665-A/G位点突变与大黄鱼抗副溶血弧菌有关联,这为大黄鱼抗弧菌群体的选育提供了理论依据。     

五华鸡Mx基因A2032G变异位点与禽白血病关联分析

Mx基因是具有抗禽流感病毒作用的基因。为探讨Mx基因A2032G变异位点是否与禽白血病有关,本试验采用PCR-RFLP方法,检测了99只56周龄五华鸡Mx基因多态性,并与禽白血病进行关联分析。检测群体Mx基因2032位点存在2个等位基因A、G,频率分别为3.03%和96.97%;两种基因型AG、GG,频率分别为6.06%和93.94%。卡方适合性检验显示,该群体Mx基因2032位点的分布偏离Hardy-Weinberg平衡。分析这2种基因型与禽白血病阳性率的相关性显示,AG型群体禽白血病阳性率为16.67%低于GG型阳性率58.06%。基于上述结论,则阴性群体中AG型个体多于GG型,以40只1日龄禽白血病阴性鸡群为对照,相同方法检测Mx基因2032位点突变率,共检测到3种基因型AA、AG、GG,其频率分别为2.50%、5.00%和92.50%,与上述结论不符。推测五华鸡GG型为禽白血病易感群体,禽白血病阳性检出率可能随日龄而发生变化,且AG基因型的禽白血病易感率低于GG基因型。     

偏肿革裥菌漆酶基因克隆及启动子序列分析

以优化后的漆酶培养基为基础,通过RT-PCR和RACE技术相结合,从偏肿革裥菌 Lenzites gibbosa 菌株中获得编码漆酶基因的cDNA及Genomic DNA的全长序列,Genomic DNA大小为2 165 bp.通过比较该漆酶基因的cDNA和Genomic DNA的全长序列,发现该基因包含11个外显子和10个内含子.cDNA序列的全长为1 873 bp,其中包含一个完整的ORF,长度为1 563 bp,编码520个氨基酸.序列在氨基酸水平上与彩绒革盖菌 Trametes versicolor 的相似性评价最高,相似性达83%.通过SEFA-PCR的方法,扩增得到漆酶基因起始密码子上游长986 bp的启动子序列.分析表明,该启动子区域上除分布有TATA-box、CAAT-box以及AP2等基本的转录起始元件外,还存在有多个潜在的顺式作用元件序列位点,包括7个MRE元件、2个STRE元件、1个HSEs元件、7个氮因子结合位点等.这些结果表明,不同的外源诱导物可以调节偏肿革裥菌漆酶基因的表达.     

Cloning of TaCYP707A 1 Gene that Encodes ABA 8′-Hydroxylase in Common Wheat（Triticum aestivum L.）

The plant hormone abscisic acid （ABA） regulates many important physiological and developmental processes in plants. The objective of this study was to clone the ABA 8′-hydroxylase gene in common wheat. In the present study, we used the eDNA sequence of barley HvCYP707A1 gene （GenBank accession no. AB239299） as a probe for BLAST search against the common wheat （Triticum aestivum L.） EST database in GenBank. All wheat ESTs sharing high similarity with the reference gene were subjected to contig assembly. Primers were designed based on the constructed contigs to clone the wheat CYP707A1 gene, designated as TaCYP707A1. The genomic DNA sequence of TaCYPTO7A1 gene comprised five exons and four introns, with a size of 2225 bp. The corresponding cDNA sequence of TaCYP707A1 was 1737 bp, containing an open reading frame （ORF） of 1431 bp, a 42-bp 5′-untranslated region （UTR） and a 264-bp 3′UTR, with 94.9% of identical sequences to HvCYP707A1 gene （AB239299）. The neighbor joining tree indicated that the deduced amino acid sequences of TaCYP707A1 gene was highly similar to those of barley and rice. The TaCYP707A1 gene was located on chromosome 6BL using a set of Chinese Spring nullisomic-tetrasomic lines and ditelosomic line 6BS. These results will be of high importance in understanding of molecular mechanism of ABA catabolism.     

猪LPAR3基因克隆及其在子宫内膜细胞的表达

【目的】获得猪LPAR3基因完整mRNA及基因结构,研究其启动子活性;探究LPAR3基因在子宫内膜的转录调控及可能影响母猪产仔的机制。【方法】应用5'RACE和3'RACE技术获取LPAR3基因完整mRNA序列;预测5'调控区潜在的启动子转录因子结合位点及CpG岛,构建不同长度的启动子双荧光素酶报告基因重组载体,与pRL-TK质粒共同转染至猪子宫内膜细胞,检测启动子活性;应用RT-qPCR比较LPAR3基因在妊娠第12天的二花脸猪和长大二元猪子宫内膜的相对表达量;应用亚硫酸氢钠修饰后测序比较LPAR3基因在妊娠第12天的二花脸猪和长大二元猪子宫内膜的甲基化状态。【结果】猪LPAR3 mRNA全长为2 127 bp,其中5'UTR和3'UTR的长度分别为202和860 bp,CDS区为1 065 bp。克隆获得包括LPAR3转录起始位点上游3 080 bp (–2 430/+650bp)的5'调控序列,分析预测显示该调控区不存在TATA box,存在GC元件、CPBP及糖皮质激素受体IR3等调控因子结合位点,且在–190/–84和–44/+651 bp处存在2个潜在CpG岛。成功构建9个不同长度的5'缺失报告重组载体并转染猪子宫内膜细胞。双荧光素酶活性检测结果显示,启动子P4(+454/+80 bp)的转录活性最高,其次是P6(–123/+80 bp)。RT-qPCR结果显示,妊娠第12天二花脸猪LPAR3基因在子宫内膜的表达量高于在其他组织的表达量,且极显著高于在妊娠第12天长大二元猪子宫内膜的表达量,LPAR3在2个猪种子宫内膜均处于低甲基化状态且差异不显著。【结论】猪LPAR3 mRNA全长为2 127 bp,妊娠第12天LAPR3基因在二花脸猪子宫内膜表达高于其在长大二元猪子宫内膜的表达,显示LPAR3可能参与了猪早期妊娠并影响产仔数。     

藏猪生长激素（pGH）基因多态性研究

猪生长激素（pGH）是猪生长发育性状重要的候选基因之一。采用PCR RFLP技术检测藏猪pGH基因+159～+734 bp片段的Dra I、Apa I和Msp I酶切多态性。结果显示,藏猪群体中Dra I酶切全部切开,无多态;Apa I酶切具有4个等位基因,出现5种基因型,其中CC基因型（299A/432C）频率最高,为0.7857;藏猪群体Msp I酶切出现3种基因型,杂合性CT基因型频率最高,为0.5089,等位基因C和T频率分别为0.5580和0.4420。经比较,藏猪pGH基因Dra I酶切位点与其他猪种一样无多态性,而Apa I酶切出现了特异的432突变位点,Msp I酶切等位基因分布相对均衡,表现出与其他猪种的差异。