发情期和乏情期哈萨克羊垂体差异表达基因筛选及功能预测

垂体是哺乳动物重要的内分泌器官之一,主要通过调控激素合成来调节动物发情。为了研究绵羊垂体系统在发情状态和乏情状态之间的基因表达差异,本研究使用RNA-seq技术分析了发情期和乏情期哈萨克羊垂体前叶的转录组数据。通过比较转录组数据发现:在发情期和乏情期的哈萨克羊垂体中共筛选出了3 211个差异表达的基因,其中包含298个上调的和2 913个下调的基因。利用GO和KEGG富集分析这些差异表达的基因发现:它们被显著地富集在卵母细胞减数分裂和孕酮介导的卵母细胞成熟等调控发情的信号通路上,提示这些基因在调控哈萨克羊发情状态中发挥着不可或缺的作用。本研究丰富了绵羊的转录组资源,为今后深入研究绵羊季节发情机制、提高绵羊繁殖率打下了良好的基础。     

Polymorphisms in GnRHI and GnRHII genes and their association with egg production and egg quality traits in chicken

1. Two candidate genes, namely, Gonadotropin releasing hormone I (GnRHI) and Gonadotropin releasing hormone II (GnRHII) play pivotal roles in ovulation and egg production in chicken. The objective of this study was to explore polymorphism in these genes and to estimate the effects of polymorphism of these two genes on egg production and egg quality traits in White Leghorn laying hens.

2. Single strand conformation polymorphism followed by sequencing was performed to detect polymorphism in these genes.

3. The coding regions of the GnRHI and GnRHII genes were found to be polymorphic. In the GnRH1 gene, 12 haplotypes were determined, of which the h1 haplotype was predominant and the h5, h9 and h11 haplotypes were the least frequent ones. In the GnRHII gene, eight haplotypes were found, of which the h1 haplotype was the most frequent and the h6 was the least frequent haplotype in the White Leghorn population.

4. The haplogroups of GnRHI had a significant effect on body weight and egg production up to 64 weeks of age, yolk content, Haugh units and egg shell parameters. The h1h2 haplogroup of the GnRHI gene showed the highest egg production, with 211.0 ± 24.3 eggs up to 64 weeks of age, while the highest yolk content and Haugh unit was found in h3h10 haplogrouped birds. The haplogroups of GnRHII had a significant effect on age at sexual maturity (ASM) where the shortest ASM was found in the h1h4 birds (147.3 ± 5.9 d) and the longest ASM was observed in the h1h3 birds (160.6 ± 23.4 d).

5. It was concluded that GnRHI and GnRHII genes are polymorphic and have a significant effect on body weight, egg production and egg quality traits in White Leghorn laying hens.     

Identification of key microRNAs affecting melanogenesis of breast muscle in Muchuan black-boned chickens by RNA sequencing

ABSTRACT

1. Melanin content is considered an important indicator of meat quality in black-boned chickens, which have a high market value. To understand the complex physiological processes underlying muscle melanogenesis in this chicken, differentially expressed miRNAs (DEMs) were detected between black muscle (BM) and white muscle (WM) of chickens using high-throughput sequencing technology. Six small RNA libraries were constructed, and more than 16.75 million clean reads were obtained for each library.

2. A total of 582 known miRNAs and 65 novel miRNAs were identified from the six chicken sequence libraries. A total of 19 DEMs were identified between the two groups, of which nine were upregulated and 10 were downregulated. Furthermore, the DEMs were predicted to target 572 genes.

3. Certain DEMs (such as miR-204, miR-133b, and miR-12 229-3p) and their target genes may play an important role in muscle melanogenesis of chickens. These findings provide a foundation for clarifying the miRNA regulatory mechanisms involved in muscle pigmentation in avian species.     

利用基因芯片技术研究隐性白鸡不同时期肌肉组织相关差异表达基因

应用包含13319条鸡基因探针序列的表达谱芯片,对从隐性白鸡不同时期肌肉组织抽提及纯化的cRNA进行芯片杂交,并对基因表达谱进行分析,旨在筛选隐性白鸡不同时期肌肉组织相关差异表达基因,探讨造成不同时期肌肉组织生长发育的分子生物学机理。结果显示,在不同生长时期（2周和12周）共筛选出差异表达基因78条（表达上调34条,表达下调44条）,其中已知功能基因39条,主要涉及生长发育、分子代谢、免疫应答、生物合成、细胞通信以及蛋白质合成与分解等相关基因,其中包括一些尚未在GenBank上登陆的序列,推测可能是未知的新基因,它们在鸡生长发育、能量代谢等的过程所起到的作用还需进一步证明。     

Identification of differentially expressed protective genes in liver of two rainbow trout strains

Since 1975, the rainbow trout strain BORN (Germany) has been bred in brackish water from a coastal form imported from Denmark. Accompanying phenotypic monitoring of the adapted BORN trout until now revealed that this selection strain manifested a generally elevated resistance towards high stress and pathogenic challenge including lower susceptibility towards Aeromonas salmonicida infections in comparison to other trout strains in local aqua farms. We focus on the elucidation of both, genetic background and immunological basis for the increased survivorship to infections. A first comparison of gene expression profiles in liver tissue of healthy rainbow trout from the local selection strain BORN and imported trout using a GRASP 16K cDNA microarray revealed six differentially expressed genes evoking pathogen and wounding responses, LEAP2A (encoding for liver-expressed antimicrobial peptide), SERPINA1 (alpha-1 antitrypsin), FTH1 (middle subunit of ferritin), FGL2 (fibroleukin), CLEC4E (macrophage-inducible C-type lectin), and SERPINF2 (alpha-2 antiplasmin). Since the latter gene is not described in salmonid species so far, our first aim was to characterize the respective sequence in rainbow trout. Two trout SERPINF2 genes were identified, which share only 48% identical amino acid residues and a characteristic SERPIN domain. Second, we aimed to analyse the expression of those genes after temperature challenge (8 °C and 23 °C). Only FTH1 was upregulated in BORN and import trout after increase of temperature, while SERPINA1 and FGL2 were only elevated in import trout. Third, the expression of all named genes was analyzed after pathogen challenge with A. salmonicida subsp. salmonicida. As a main finding, we detected a comparably faster regeneration of LEAP2A mRNA abundance in BORN trout following bacterial infection. Ingenuity Pathways Analysis suggested a functional interplay among the mentioned factors and the pro-inflammatory cytokine TNF, whose stronger expression was validated in liver of BORN trout. This data indicate that the examined genes contribute to an improved first barrier against invading pathogens in BORN trout.     

Use of bioinformatic SNP predictions in differentially expressed genes to find SNPs associated with Salmonella colonization in swine

Asymptomatic Salmonella-carrier pigs present a major problem in preharvest food safety, with a recent survey indicating >50% of swine herds in the United States have Salmonella-positive animals. Salmonella-carrier pigs serve as a reservoir for contamination of neighbouring pigs, abattoir pens and pork products. In addition, fresh produce as well as water can be contaminated with Salmonella from manure used as fertilizer. Control of Salmonella at the farm level could be through genetic improvement of porcine disease resistance, a potentially powerful method of addressing preharvest pork safety. In this research, we integrate gene expression profiling data and sequence alignment-based prediction of single nucleotide polymorphisms (SNPs) to successfully identify SNPs in functional candidate genes to test for the associations with swine response to Salmonella. A list of 2527 genes that were differentially regulated in porcine whole blood in response to infection with Salmonella enterica serovar Typhimurium were selected. In those genes, SNPs were predicted using ANEXdb alignments based on stringent clustering of all publically available porcine cDNA and expressed sequence tag (EST) sequences. A set of 30 mostly non-synonymous SNPs were selected for genotype analysis of four independent populations (n = 750) with Salmonella faecal shedding or tissue colonization phenotypes. Nine SNPs segregated with minor allele frequency ≥15% in at least two populations. Statistical analysis revealed SNPs associated with Salmonella shedding, such as haptoglobin (HP, p = 0.001, q = 0.01), neutrophil cytosolic factor 2 (NCF2 #2, p = 0.04, q = 0.21) and phosphogluconate dehydrogenase (p = 0.066, q = 0.21). These associations may be useful in identifying and selecting pigs with improved resistance to this bacterium.     

鸡脾脏组织抑制消减cDNA文库构建及其差异表达基因的分析

为分离与鸡抗病性密切相关的差异表达基因,并进行功能分析,利用抑制性消减杂交技术,以大骨鸡和海兰褐商品代蛋鸡20周龄时的脾脏组织为试验材料构建消减cDNA文库。文库的插入片段集中在600 bp左右,挑取760个克隆进行PCR筛选获得663个阳性克隆,经过点杂交筛选后选择了531个阳性克隆,从中随机挑取100个阳性克隆进行测序。经过同源性比对归并后得到37个差异表达基因或ESTs序列,其中,32个是已知基因,包括一般抗病性或免疫性能、特异抗病相关基因、细胞信号分子、膜蛋白质、转录因子等差异表达基因;5个为功能尚未确定的基因。并对4个可能影响鸡抗病性能的差异表达基因进行了RT-PCR半定量检测鉴定。该试验结果为进一步研究这些差异表达基因在抗病过程中的重要功能及其调控作用机理奠定了基础。     

Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands

Cells of the adaptive immune system express Toll-like receptors (TLRs) and are able to respond to TLR ligands. With this in mind, the goal of the current study was to determine the expression of antiviral response genes in the cells of the chicken bursa of Fabricius (BF) to stimulation with TLR ligands. We investigated initially the response of bursal B cells to CpG-ODN, lipopolysaccharide (LPS) and poly(I:C) treatment. The expression level of type I interferons (IFNs) and interferon regulatory factor 7 (IRF7) did not differ between CpG-ODN and LPS treated groups compared to the non-stimulated cells. Poly(I:C) was the only TLR ligand, which has induced significant expression of antiviral innate immune response genes from bursal cells. Further in vitro and in vivo studies need to examine the efficacy of these antiviral responses against avian viruses.     

鸡输卵管表达的重组人溶菌酶的鉴定

用直接结晶法从重组载体注射鸡蛋清中提取重组人溶菌酶（rhLYZ），并对其理化及生物学特性进行了鉴定。结果显示，rhLYZ能被hLYZ特异抗体识别；纯化的rhLYZ和天然hLYZ的热稳定性无显著差异，二者在pH3．0～11．0范围内均具有活性，pH为7．0时活性最强。采用最小抑菌浓度法测定了含rhLYZ蛋清和纯化rhLYZ对12种常见细菌的溶菌活性，二者对除有鞭毛伤寒杆菌和鳖嗜水气单胞菌以外的参试茵均有不同程度的抑制作用。表明，表达在重组栽体注射鸡蛋清中的rhLYZ具有与天然hLYZ十分相似的分子质量、热稳定性、pH活性范围和溶菌谱。     

基于RNA-Seq筛选禽致病性大肠杆菌损伤雏鸡小肠差异表达基因

为筛选禽致病性大肠杆菌(APEC)感染雏鸡后其小肠损伤的差异表达基因,本研究利用APEC菌株经腿部肌肉途径接种两周龄雏鸡,对照组鸡以相同的途径注射相同体积的生理盐水,选取病变严重的肠道与对照组肠道,利用高通量RNA-Seq技术筛选出雏鸡小肠受APEC损伤后的差异表达基因,对其进行生物信息学GO功能分类和KEGG信号通路分析,并利用荧光定量PCR验证部分差异表达基因。结果显示差异变化在2倍以上的基因共有131个,其中74个上调表达,57个下调表达。GO功能分类结果显示,这些基因主要涉及到蛋白结合、免疫反应、转运活动等功能。KEGG分析表明这些差异表达基因参与PPAR、新陈代谢、细胞因子受体相互作用、Jak-STAT、RIG-I-样受体、吞噬体等信号通路。本研究为进一步研究APEC的致病作用及宿主抗病机制奠定了基础。     

鸡输卵管表达的重组人溶菌酶的鉴定

为了对鸡输卵管特异表达载体表达的重组人溶菌酶(rhLYZ)的理化及生物学特性进行鉴定,用直接结晶法从重组载体注射鸡蛋清中提取溶菌酶,以兔抗hLYZ血清为一抗,HRP-标记的羊抗免IgG为二抗,进行Western blotting检测,结果表明表达在鸡蛋清中的rhLYZ能被hLYZ特异抗体识别;将纯化的rhLYZ和天然hLYZ在不同温度的水浴中孵育30min,用RBB-R染料标记比色法检测溶菌酶活性的变化,结果显示二者的热稳定性无显著差异;将纯化的rhLYZ和天然hLYZ用不同PH的溶液稀释,40C孵育30min后进行酶的活性测定,结果显示两者在pH3.0～11.0范围内具有活性,最强活性pH为7.0;以12种常见细菌为试验对象,用最小抑菌浓度法测定含rhLYZ蛋清和纯化rhLYZ的溶菌活性,结果显示两者对鲫鱼嗜水气单胞菌、变形杆菌、枯草杆菌、无鞭毛伤寒杆菌、白色念珠菌和柠檬色葡萄球菌具有很强的抑制作用,对痢疾杆菌、白色葡萄球菌、金黄色葡萄球菌和大肠杆菌具有较强的抑制作用,对有鞭毛伤寒杆菌和鳖嗜水气单胞菌无明显的抑制作用.这些试验结果显示,表达在重组载体注射鸡蛋清中的rhLYZ具有与天然hLYZ十分相似的相对分子质量、热稳定性、pH活性范围和溶菌谱.     

DNA polymorphism in the chicken growth hormone gene: Association with egg production

Restriction fragment length polymorphic markers in the chicken growth hormone (GH) gene were tested for their association with egg production traits. Alleles of GH were determined in 3 non‐inbred strains from different genetic bases. The frequency of PS1 (+) allele in the three strains was very low in contrast to the layer strain previously reported. Results of the present study indicate that the frequency of PS (+) is related to egg production and that the PS1 (+) allele may be used as a genetic marker for selection in egg production.