转录组测序筛选克氏原螯虾卵巢发育、免疫和生长相关基因

为发掘克氏原螯虾卵巢发育、免疫和肌肉生长的重要功能基因,采用Illumina HiSeqTM 2 500高通量测序平台对克氏原螯虾的卵巢、肝胰腺和肌肉组织进行了转录组测序.所得序列经质控、组装后比对到NR、Swiss-Prot、pfam、COG、GO和KEGG数据库中注释,并进行差异基因聚类分析.结果 显示,测序共获得...     

慢性盐度胁迫下金钱鱼卵巢转录组分析

为了阐明盐度胁迫对金钱鱼(Scatophagus argus)代谢和生殖的影响,采用RNA-seq技术对低盐组(5)、对照组(25)和高盐组(35)处理40 d后的2龄性成熟金钱鱼卵巢进行转录组分析。结果发现,金钱鱼卵巢转录组测序获得raw reads共398681318,clean reads共396910398。从低盐胁迫相对对照组(5 vs 25)和高盐胁迫相对对照组(35 vs 25)中分别筛选到373个和874个差异表达基因(DEGs)。与对照组相比,低盐胁迫后氨基酸代谢相关基因(sds、bhmt)和脂肪酸代谢相关基因(pnpla2)显著下调;高盐胁迫后pgr、cyp17a1和ers1等生殖相关基因显著下调。KEGG通路富集分析发现,低盐胁迫组相对对照组显著富集与代谢相关的通路为半胱氨酸和甲硫氨酸代谢,缬氨酸、亮氨酸和异亮氨酸生物合成代谢,脂肪酸生物合成,脂肪酸代谢,皮质醇的合成和分泌,醛固酮的合成和分泌,脂肪细胞脂解的调节等;高盐胁迫组相对对照组中显著富集的与生殖相关的通路为雌激素信号传导通路。这些结果表明,氨基酸和脂肪酸的代谢调控可能在金钱鱼卵巢的低渗透压调节中起重要作用...     

大黄鱼高温适应的转录组学分析

为了探究大黄鱼高温胁迫条件下基因表达水平的变化,实验利用Illumina Hiseq 2500的125 pair-ended测序模式分别对大黄鱼高温处理组和常温对照组进行了转录组测序。对照组(3个生物学重复)和高温处理组(3个生物学重复)分别获得15.28和13.92 Gb测序数据,GC含量平均值约为51%。过滤后的高质量测序reads使用Bowtie2软件比对到大黄鱼参考转录组序列上估计基因表达量,进而进行基因表达差异统计学检验。以常温对照组为参比,高温处理组中阈值设为|log2(FC)|2和FDR0.05,共检测到1 259条显著差异表达基因。其中,821条基因为高表达,438条基因为低表达。随机选取12条差异表达基因,运用实时荧光定量PCR(qRT-PCR)进行了验证,结果证实转录组分析可靠。进一步将所有差异表达的基因进行GO功能注释和KEGG通路富集分析,结果发现大量差异表达基因的功能与氧化还原反应、蛋白质折叠和去折叠、糖和脂代谢以及某些疾病的发生相关。该研究结果为下一步深入研究大黄鱼高温适应的调控机制提供了重要的参考材料。     

基于转录组测序筛选乌苏里白鲑肌肉生长关键候选基因研究

为了挖掘调控乌苏里白鲑(Coregonus ussurinsis Berg)肌肉生长的关键候选基因,本研究对不同生长速度乌苏里白鲑的肌肉组织进行转录组测序,以期为乌苏里白鲑群体选育提供基础数据。首先,在同等条件下(从混合池中)随机选择乌苏里白鲑F2个体进行实验分组(快长组和慢长组)。接着分别从快长组[体重为(219.20±38.66) g]和慢长组[体重为(74.30±17.86) g]中随机选取10尾样本,取其背部肌肉进行转录组测序。以FDR (false discovery rate)<0.05且|log2(FC)|>1 (FC, fold change)为条件筛选差异表达基因并对其进行GO (gene ontology)和KEGG (Kyoto encyclopedia of genes and genomes)富集分析,并通过qPCR验证转录组数据的准确性。转录组测序结果显示,共筛选出2 211个差异表达基因,与慢长组相比,快长组中583个差异基因表达上调及1 628个基因表达下调。GO功能注释结果显示,差异基因主要参与细胞过程和结合过程,差异基因显著富集到251条KEGG通路(P<0.05),其中,MAPK信号通路(MAPK signaling pathway)、PI3K-Akt信号通路(PI3K-Akt signaling pathway)、紧密连接(tight junction)、胰岛素信号通路(insulin signaling pathway)、糖酵解/糖异生(glycolysis/gluconeogenesis)和PPAR信号通路(PPAR signaling pathway)参与细胞生长。之后结合功能注释结果和KEGG,鉴定出肌浆/内质网钙ATP酶基因atp2a1和atp2a2、葡萄糖-6-磷酸脱氢酶基因g6pc、生长因子结合蛋白1基因igfbp1以及肌球蛋白重链基因myh1、myh4、myh6、myh7、myh9和myh13等可能与肌肉生长密切相关的基因。本研究共筛选出10个可能与乌苏里白鲑肌肉生长相关的关键候选基因,为今后乌苏里白鲑分子标记辅助育种提供了基础数据。     

iTRAQ‐based identification of differentially expressed proteins related to growth in the swimming crab,Portunus trituberculatus

The swimming crab, Portunus trituberculatus, is an important farmed species in China, and it has been studied extensively, which requires more information on its genetic background. To date, information on the proteomics of P. trituberculatus is scarce. In this study, we used isobaric tags for relative and absolute quantitation (iTRAQ)‐based proteomics to investigate growth regulation in P. trituberculatus. Total proteins were isolated from five tissues (eyestalk, gill, heart, hepatopancreas and muscle). Equal quantities of protein from each tissue were pooled at the proteome level using the iTRAQ method. A total of 961 proteins were identified using liquid chromatography coupled with tandem mass spectrometry and de novo sequencing data. Using a 1.2‐fold change in expression as a physiologically significant benchmark, 30 differentially expressed proteins (DEPs) were found to undergo differential expression related to the growth of P. trituberculatus, and most of the growth‐related proteins, including those involved in metabolism, immune responses, DNA duplication and protein synthesis, were upregulated, indicating that conservation of energy is an important strategy to cope with growth. There was a high consistency between the expression levels determined using iTRAQ and mRNA, highlighting the high reproducibility of our proteomic approach and its great value in elucidating the molecular mechanisms of P. trituberculatus.     

A global view of hepatopancreas and intestinal reveals the potential influencing mechanism of aflatoxin B1 on nutrition and metabolism in Litopenaeus vannamei

The present study was to determine the time‐dependent alterations in growth performance, histomorphology, digestive enzymes activities and the genes expression related to target of rapamycin (TOR) signalling pathway involved in hepatopancreas and intestine of Litopenaeus vannamei at 3, 6, 12, 18, 24 and 30 days after Aflatoxin B1 (AFB1) challenge. The results manifested that AFB1 could induce a significant decrease in growth performance and visible variations of the hepatopancreas and intestine structures in shrimp. Meanwhile, the digestive enzymes activities and genes expression level were significantly lower in the experimental group than in the control group and showed a time‐dependent decrease (p < 0.05). The expression levels of tor and s6k were significantly higher in the experimental group than in the control group from days 6 to 24 and showed a tendency to increase first and then decrease as a whole (p < 0.05). Meanwhile, we analysed candidate pathways involved in hepatopancreas and intestine of shrimp in metabolic response to AFB1. The mainly disturbed pathways related to metabolism in hepatopancreas were involved in pyrimidine, glycosylphosphatidylinositol, glycosaminoglycan, vitamin and amino acid biosynthesis and metabolism, while the mainly disturbed metabolic pathways in intestine were glycosaminoglycan, vitamin and amino acid biosynthesis and metabolism.     

Mucus piRNAs profiles of Vibrio harveyi-infected Cynoglossus semilaevis: A hint for fish disease monitoring

The half-smooth tongue sole, Cynoglossus semilaevis, is an important cultured flatfish species. Vibrio harveyi is a common pathogen to this fish, which may result in great economic loss to C. semilaevis culture industry. piRNAs, a non-coding RNAs with 26–32 nt, have been regarded as promising biomarkers for cancer diagnosis and fish diseases. Here, we extracted the RNA from mucus of C. semilaevis and constructed the differential expression profiles of piRNAs between the sick fish (MS) and healthy fish (MC). We identified 45,696 differentially expressed piRNAs including 22,735 up-regulated piRNAs and 22,961 down-regulated piRNAs in MS group compared with MC group. The GO enrichment and KEGG pathway enrichment analyses of the differential piRNAs were carried out. The result showed immunity-related target genes mainly involved in immune system process, response to stimulus, cell killing, immune system, infectious diseases and cell growth and death. The 10 most differentially expressed piRNAs were chosen to perform the qRT-PCR, while only seven piRNAs were consistent with the sequence result. Compared with MC group, the expression levels of piR-mmu-72173>piR-rno-62831>piR-xtr-704880, piR-dme-15546979, piR-mmu-49941660, piR-mmu-29283297 and piR-mmu-1758399 were significantly lower, and piR-gga-10574 and piR-gga-134812 were significantly higher in MS group. These piRNAs may be potential biomarkers during the V. harveyi infection of C. semilaevis. This study could provide a new method to identify the infection status of C. semilaevis and understand better about the innate and adaptive immune system in C. semilaevis during bacterial infection.     

Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate

Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F₁ hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.     

SNP discovery and gene annotation in the surf clam Mesodesma donacium

The main objective of this research was to identify single‐nucleotide polymorphisms (SNPs) from an Expressed Sequences Tags (EST) data set generated by 454 pyrosequencing in the soft clam Mesodesma donacium. A total of 180 159 ESTs were yielded from a M. donacium cDNA library. De novo assembly was performed using stringent calling parameters, producing 10 178 contigs and 41 765 singletons. Here, a total of 2594 SNPs were discovered related to 613 consensus sequences, achieving a frequency of 1 SNPs per 260 bp. SNP variants showed that A/G, A/T and C/T were the most abundant among the identified polymorphisms. We validated a total of 12 SNPs loci by HRMA for annotated genes such as heat shock protein‐70 and the translation elongation factor 1‐alpha. The Gene Ontology analysis regarding molecular function level revealed that sequences with SNPs were mainly classified to protein and nucleotide binding, as well hydrolase activity, ion binding and oxidoreductase activity. Further, biological processes like cellular and metabolic process, biogenesis, localization and biological regulation were highly annotated. The most expressed genes were related to the mitochondrial electron transport chain, senescence‐associated protein, ubiquitin and actin. Interestingly, some relevant genes related to immune response and biomineralization showed a high abundance, such as tumor necrosis factor (TNF)‐alpha‐receptor‐like protein, serine protease inhibitor, heat shock protein, aragonite‐binding protein and ferritin. This study contributes to relevant genes associated with functional polymorphisms and gives an overview for future genetic investigations.