RNA-seq用于获得共轭亚油酸对贵妃鸡肌内脂肪代谢调控的关键基因

通过转录物组测序获得在贵妃鸡基础日粮中添加共轭亚油酸(CLA)对肌内脂肪代谢的差异表达基因,经生物信息学分析获得相关的信号通路及可能发挥重要作用的候选基因,为CLA对肌内脂肪沉积的分子机制奠定基础。本研究选用55日龄健康的贵妃鸡为试验动物,在基础日粮中添加CLA 0%、1%和2%,预饲期1周,正饲期6周。屠宰采集胸肌组织进行转录物组测序,对测序数据进行差异表达分析,差异表达基因GO功能和差异表达基因KEGG通路富集分析,筛选出与胸肌脂类代谢相关的差异表达基因,利用qRT-PCR对差异表达基因进行验证。结果显示,共获得1 065个差异表达基因,其中上调基因703个,下调基因362个。GO富集结果显示,差异表达基因主要富集在生物过程的细胞过程、单一生物过程、生物调节和代谢过程。KEGG信号通路富集显示,差异表达基因显著富集在黏着斑、不饱和脂肪酸生物合成、脂肪酸生物合成和类固醇生物合成等信号通路中,发现11个主要与肌内脂肪代谢相关的候选基因,分别是FADS1、FADS2、ELOVL5、ACOX2、SLC27A1、FABP5、LPL、LOC107050163、ENSGALG00000030996、ENSGALG00000005043和ENSGALG00000048882。并随机选取6个基因进行qRT-PCR验证,其相对表达量变化趋势与测序结果一致。本研究筛选到CLA影响贵妃鸡胸肌脂类代谢相关的差异表达基因,并对11个主要参与脂肪代谢相关的基因进行分析,为揭示CLA调控肌内脂肪沉积的分子机制奠定基础。     

Identification and characterization of genes related to the development of breast muscles in Pekin duck

Pekin Duck is world-famous for its fast growth, but its breast muscle development is later and breast muscle content is lower compared with other muscular ducks. Therefore, it is very important to discover the genetic mechanism between breast muscle development and relative gene expression in Pekin duck. In current study, the genes which have relationships with breast muscle development were identified by suppression subtractive hybridization. A total of 403 positive clones were sequenced and 257 unigenes were obtained. The expression of 23 genes were analyzed in the breast muscle of 2-, 4-, 6-, 8- week old Pekin ducks. The results showed that unknown clone A233, C83 and C99 showed descending tendency as age increased; KBTBD10, HSPA8, MYL1, ZFP622, MARCH4, Nexilin, FABP4 and MUSTN1 had high expression levels at 6 weeks old; WAC, NT5C3, HSP90AA1, MRPL33, KLF6, TSNAX, CDC42EP3, HSPA4, TRAK1, NR2F2, HAUS1 and IGF1 had high expression levels at 8 weeks and showed ascending tendency as age increased. Expression of these 23 genes were also analyzed in breast muscle, leg muscle, heart, kidney, liver, muscular stomach and sebum cutaneum in 4-8-week old Pekin duck and results showed that most of these genes had high expression in breast muscle, leg muscle and heart.     

Genome-wide association study reveals novel loci associated with fat-deposition and meat-quality traits in Pekin ducks

Meat-quality traits play an essential role in meat poultry production. To determine the genetic mechanisms of meat quality in Pekin ducks, we performed a large-scale GWAS to identify quantitative trait loci affecting meat quality in Pekin ducks. We measured 10 traits in 542 Pekin ducks and genotyped each duck using genotyping-by-sequencing. The genetic parameters (genomic heritability, genetic correlation) for 10 meat-quality related traits were evaluated. Based on the large genotype–phenotype dataset, we performed GWASs for all of these traits. A total of 33 significant QTL (P < 3.03 × 10⁻⁵) across 13 chromosomes were identified by loci-based analysis. Some newly identified candidate genes were discovered for fat-deposition and meat-quality traits, including PAG1 for body weight and eviscerated weight, INTU and NUP35 for abdominal fat weight and ratio, NUP3 and ARHGDIB for skin fat weight and ratio, GOLGA5 for breast muscle toughness and breast tenderness, and CTDSPL and PKP1 for breast muscle thickness. The current study is the first systematic report regarding duck meat quality.     

Analysis of gene co-expression networks and function modules at different developmental stages of chicken breast muscle

The development of poultry muscle fibers after hatching is closely related to meat quality and production efficiency. It is necessary to identify functional modules (groups of functionally related genes) related to muscle development at different developmental stages, and to investigate their relationships based on the weighted gene co-expression network analysis (WGCNA) methods. Accordingly, we investigated the co-expression associations between genes related to chicken breast muscle at four different developmental stages (between 2 and 14 weeks of age), and systematically analyzed the network topology in Jinmao Hua chicken. As a result, 2341 differentially expressed genes were identified and subjected to co-expression analysis. Four modules were identified to be related to a particular growth stage for the development of breast muscle. A series of genes with the highest connectivity were identified in the pink (2 weeks), yellow (6 weeks), green (10 weeks) and black modules (14 weeks), respectively, and visualized by Cytoscape. These hub genes (FGF, MAPKAPK5, NRG1, SCD, ACSL1, PPAR etc.) were mainly enriched in 15 pathways, such as MAPK signaling pathway, NRG/ErbB signaling pathway, and insulin signaling pathway. They shared biological functions related to development of breast muscle and adipogenesis. This is the first study of gene network with different stages of muscle development in Jinmao Hua chicken to observe co-expression patterns. It may contribute to the underlied molecular mechanisms of chicken breast muscle development.     

Transcriptional profiling of skeletal muscle tissue from two breeds of cattle

We used a 9.6 K cattle muscle/fat cDNA microarray to study gene expression differences between the longuissimus dorsi (LD) muscle of Japanese Black (JB) and Holstein (HOL) cattle. JB cattle exhibit an unusual ability to accumulate intramuscular adipose tissue with fat melting points lower than that in other breeds. The LD biopsies from three JB (Tajima strain) and three HOL animals were used in this breed comparison. Seventeen genes were identified as preferentially expressed in LD samples from JB and seven genes were found to be expressed more highly in HOL. The expression of six selected differentially expressed genes was confirmed by quantitative real-time PCR. The genes more highly expressed in JB are associated with unsaturated fatty acid synthesis, fat deposition, and the thyroid hormone pathway. These results are consistent with the increased amounts and proportions of monounsaturated fatty acids observed in the muscle of JB animals. By discovering as yet uncharacterized genes that are differentially regulated in this comparison, the work may lead us to a better understanding of the regulatory pathways involved in the development of intramuscular adipose tissue.     

Identification and Profiling of MicroRNAs in the Embryonic Breast Muscle of Pekin Duck

MicroRNAs (miRNAs) regulate gene expression by fully or partially binding to complementary sequences and play important roles in skeletal muscle development. However, the roles of miRNAs in embryonic breast muscle of duck are unclear. In this study, we analyzed the miRNAs profiling in embryonic breast muscle of Pekin duck at E13 (the 13^th day of hatching), E19, and E27 by high-throughput sequencing. A total of 382 miRNAs including 359 preciously identified miRNAs 23 novel miRNA candidates were obtained. The nucleotide bias analysis of identified miRNAs showed that the miRNAs in Pekin duck was high conserved. The expression of identified miRNAs were significantly different between E13 and E19 as well as between E27 and E19. Fifteen identified miRNAs validated using stem-loop qRT-PCR can be divided into three groups: those with peak expression at E19, those with minimal expression at E19, and those with continuous increase from E11 to E27. Considering that E19 is the fastest growth stage of embryonic Pekin duck breast muscle, these three groups of miRNAs might be the potential promoters, the potential inhibitors, and the potential sustainer for breast muscle growth. Among the 23 novel miRNAs, novel-miRNA-8 and novel-miRNA-14 had maximal expression at some stages. The stem-loop qRT-PCR analysis of the two novel miRNAs and their two targets (MAP2K1 and PPARα) showed that the expression of novel-mir-8 and PPARα reached the lowest points at E19, while that of novel-mir-14 and MAP2K1 peaked at E19, suggesting novel-miRNA-8 and novel-miRNA-14 may be a potential inhibitor and a potential promoter for embryonic breast muscle development of duck. In summary, these results not only provided an overall insight into the miRNAs landscape in embryonic breast muscle of duck, but also a basis for the further investigation of the miRNAs roles in duck skeletal muscle development.     

Investigation of key microRNAs associated with hepatocellular carcinoma using small RNA-seq data

To identify key microRNAs (miRNAs) associated with hepatocellular carcinoma (HCC) using small RNA-seq data. Small RNA-seq data for two HCC samples and two normal samples were downloaded from NCBI Gene Expression Omnibus. MiRNAs were identified through database search. Differentially expressed miRNAs were screened out with t test and their target genes were retrieved. Functional enrichment analysis was performed to uncover their biological functions. Regulatory networks and core metabolic networks were also constructed to present the global patterns. In addition, new miRNAs and their target genes were predicted. A total of 59 differentially expressed miRNAs were obtained, 12 up-regulated and 47 down-regulated. A total of 3,306 target genes were retrieved for eight miRNAs. Pathway enrichment analysis for the target genes showed that “pathways in cancer” and “MAPK signaling pathway” were significantly over-represented. Functional enrichment analysis found that “biological regulation” and “macromolecule modification” were significantly related to the target genes. Two regulatory networks were constructed for up- and down-regulated differentially expressed miRNAs with information from Ingenuity Pathway Analysis database. Two metabolic networks were also established based upon “pathways in cancer” and “MAPK signaling pathway”, consisting of miRNAs, target genes, compounds and others genes. Moreover, a number of new miRNAs and relevant target genes were predicted. Our study discloses a number of miRNAs as well as genes which may be involved in the development of HCC and these findings are beneficial in guiding future researches.     

茶足柄瘤蚜茧蜂蛹滞育过程中胰岛素信号通路及其相关途径的初探

本实验通过探索胰岛素信号通路及其相关途径对茶足柄瘤蚜茧蜂蛹滞育的影响,从而方便寻找胰岛素替代物,为害虫防治提供新思路。利用RNA-Seq,对滞育组与非滞育组的茶足柄瘤蚜茧蜂进行转录组测序,结合生物信息学方法对转录组中胰岛素信号通路及其相关途径的差异表达基因进行了分析。与胰岛素信号通路相关差异表达基因共31个,重点分析的PI3K-Akt, FoxO, MAPK三条途径,差异表达基因分别为55, 21和28个。这些滞育关联基因呈现不同程度的上调或下调表达,发现Sos, FASN, TSC1, PRKAB等基因与茶足柄瘤蚜茧蜂滞育密切相关,共同影响茶足柄瘤蚜茧蜂的滞育。胰岛素信号通路及其相关途径对茶足柄瘤蚜茧蜂的滞育起着非常重要的作用,主要体现在影响虫体能量代谢、脂质积累、细胞增殖等方面。     

Does overfeeding enhance genotype effects on energy metabolism and lipid deposition in breast muscle of ducks?

We evaluated the effects of genotype (Muscovy, Pekin and their crossbreed hinny and mule ducks) and feeding levels (overfeeding between 12 and 14 weeks of age vs ad libitum feeding) on energy metabolism and lipid deposition in breast muscle of ducks. Samples of breast muscle (Pectoralis major) were collected at 14 weeks of age from 8 birds per group. Overfeeding induced an accumulation of lipids in breast muscle (1.5- to 1.7-fold, depending on genotype) mainly induced by triglyceride deposition. It also induced a considerable increase in the amounts (expressed as g/100 g of tissue) of saturated and mono-unsaturated fatty acids (SFA, MUFA), while the amounts of poly-unsaturated fatty acids (PUFA) remained unchanged in hinny and Muscovy ducks or slightly increased in Pekin and mule ducks. In breast muscle, overfeeding decreased the activity of the main enzymes involved in lipogenesis from glucose (glucose-6-phosphate dehydrogenase, G6PDH, malic enzyme, ME, acetyl CoA carboxylase, ACX). Lipoprotein lipase (LPL) activity in Pectoralis major muscle was also significantly decreased (-21%). The ability of muscle tissues to catabolize long-chain fatty acids, as assessed by beta-hydroxyacyl CoA dehydrogenase (HAD) activity, was increased in Pectoralis major muscle, as was cytochrome-c oxidase (COX) activity. Hybrid and Pekin ducks exhibited higher levels of ACX and LPL activity in Pectoralis major muscle than Muscovy ducks, suggesting a greater ability to synthesise lipids in situ, and to take up circulating lipids. Total lipid content in breast muscle of hybrid and Pekin ducks was higher than in that of Muscovy ducks. In hybrid and Pekin ducks, lipid composition of breast muscle was characterized by higher amounts of triglycerides, SFA and MUFA than in Muscovy ducks. Finally, oxidative metabolism was greater in Pectoralis major muscles of hybrid and Pekin ducks than in Muscovy ducks, suggesting an adaptative strategy of muscle energy metabolism according to lipid level.     

Characterization of gene expression in double-muscled and normal-muscled bovine embryos

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth. Cattle with mutations that inactivate myostatin exhibit a remarkable increase in mass of skeletal muscle called double muscling that is accompanied by an equally remarkable decrease in carcass fat. Although a mouse knockout model has been created which results in mice with a 200% increase in skeletal muscle mass, molecular mechanisms whereby myostatin regulates skeletal muscle and fat mass are not fully understood. Using suppressive subtractive hybridization, genes that were differentially expressed in double-muscled vs. normal-muscled cattle embryos were identified. Genetic variation at other loci was minimized by using embryonic samples collected from related Piedmontese x Angus dams or Belgian Blue x Hereford dams bred to a single sire of the same breed composition. Embryos were collected at 31-33 days of gestation, which is 2-4 days after high-level expression of myostatin in the developing bovine embryo. The suppressive subtraction resulted in 30 clones that were potentially differentially expressed, 19 of which were confirmed by macroarray analysis. Several of these genes have biological functions that suggest that they are directly involved in myostatin's regulation of skeletal muscle development. Furthermore, several of these genes map to quantitative trait loci known to interact with variation in the myostatin gene.