Transcriptome Sequencing Analysis Reveals the Regulation of the Hypopharyngeal Glands in the Honey Bee, <i>Apis mellifera carnica</i> Pollmann

<div><p>Transcriptome sequencing has become the main methodology for analyzing the relationship between genes and characteristics of interests, particularly those associated with diseases and economic traits. Because of its role of functional food for humans, commercial royal jelly (RJ) and its production are major research focuses in the field of apiculture. Multiple lines of evidence have demonstrated that many factors affect RJ output by activating or inhibiting various target genes and signaling pathways. Available coding sequences from the Honey Bee Genome Sequencing Consortium have permitted a pathway-based approach for investigating the development of the hypopharyngeal glands (HGs). In the present study, 3573941, 3562730, 3551541, 3524453, and 3615558 clean reads were obtained from the HGs of five full-sister honey bee samples using Solexa RNA sequencing technology. These reads were then assembled into 18378, 17785, 17065, 17105, and 17995 unigenes, respectively, and aligned to the DFCI Honey Bee Gene Index database. The differentially expressed genes (DEGs) data were also correlated with detailed morphological data for HGs acini.</p></div

Table_3_Comprehensive Transcriptome Analysis Reveals Competing Endogenous RNA Networks During Avian Leukosis Virus, Subgroup J-Induced Tumorigenesis in Chickens.XLS

<p>Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that induces myeloid tumors and hemangiomas in chickens and causes severe economic losses with commercial layer chickens and meat-type chickens. High-throughput sequencing followed by quantitative real-time polymerase chain reaction and bioinformatics analyses were performed to advance the understanding of regulatory networks associated with differentially expressed non-coding RNAs and mRNAs that facilitate ALV-J infection. We examined the expression of mRNAs, long non-coding RNAs (lncRNAs), and miRNAs in the spleens of 20-week-old chickens infected with ALV-J and uninfected chickens. We found that 1723 mRNAs, 7,883 lncRNAs and 13 miRNAs in the spleen were differentially expressed between the uninfected and infected groups (P < 0.05). Transcriptome analysis showed that, compared to mRNA, chicken lncRNAs shared relatively fewer exon numbers and shorter transcripts. Through competing endogenous RNA and co-expression network analyses, we identified several tumor-associated or immune-related genes and lncRNAs. Along transcripts whose expression levels significantly decreased in both ALV-J infected spleen and tumor tissues, BCL11B showed the greatest change. These results suggest that BCL11B may be mechanistically involved in tumorigenesis in chicken and neoplastic diseases, may be related to immune response, and potentially be novel biomarker for ALV-J infection. Our results provide new insight into the pathology of ALV-J infection and high-quality transcriptome resource for in-depth study of epigenetic influences on disease resistance and immune system.</p

Venn analysis of the DEGs.

<p>The number of DEGs in the five samples named 2 vs. 1, 3vs. 1, 4vs. 1, and 5vs. 1. Numbers stands for genes expressed in each class. Up- and down- arrow represents the up and down regulated genes, respectively (FDR≤0.001).</p

Abundance distribution of unigenes.

<p>The expression level ranges are as follows: [0, 10): low-expression genes; [10, 500): moderate-expression genes; and [500, ∞): high-expression genes. Large differences in abundance were observed for samples 1 and 5.</p

GO enrichment analysis.

<p>The GO terms were classified into 3 categories from the GO database and are ranked by p-value. Green indicates biological processes, red indicates molecular functions, and blue indicates cellular components. (A) Sample 2 vs. 1, (B) Sample 3 vs. 1, (C) Sample 4 vs. 1, (D) Sample 5 vs. 1.</p

Cluster analysis of DEG levels.

<p>Each column represents an experimental condition relative to sample 1, and each row represents a gene. Expression differences are shown in different colors. Red and green indicate up-regulation and down-regulation, respectively.</p

Table_2_Comprehensive Transcriptome Analysis Reveals Competing Endogenous RNA Networks During Avian Leukosis Virus, Subgroup J-Induced Tumorigenesis in Chickens.XLS

<p>Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that induces myeloid tumors and hemangiomas in chickens and causes severe economic losses with commercial layer chickens and meat-type chickens. High-throughput sequencing followed by quantitative real-time polymerase chain reaction and bioinformatics analyses were performed to advance the understanding of regulatory networks associated with differentially expressed non-coding RNAs and mRNAs that facilitate ALV-J infection. We examined the expression of mRNAs, long non-coding RNAs (lncRNAs), and miRNAs in the spleens of 20-week-old chickens infected with ALV-J and uninfected chickens. We found that 1723 mRNAs, 7,883 lncRNAs and 13 miRNAs in the spleen were differentially expressed between the uninfected and infected groups (P < 0.05). Transcriptome analysis showed that, compared to mRNA, chicken lncRNAs shared relatively fewer exon numbers and shorter transcripts. Through competing endogenous RNA and co-expression network analyses, we identified several tumor-associated or immune-related genes and lncRNAs. Along transcripts whose expression levels significantly decreased in both ALV-J infected spleen and tumor tissues, BCL11B showed the greatest change. These results suggest that BCL11B may be mechanistically involved in tumorigenesis in chicken and neoplastic diseases, may be related to immune response, and potentially be novel biomarker for ALV-J infection. Our results provide new insight into the pathology of ALV-J infection and high-quality transcriptome resource for in-depth study of epigenetic influences on disease resistance and immune system.</p

Validation of selected genes by qRT-PCR.

<p>Gene IDs refer to the corresponding cDNAs from Genbank. qRT-PCR was performed on each sample in triplicate. <i>β-actin</i> was used an endogenous control. Ratio-RPKM used for comparing the difference of gene expression among samples and validation by qRT-PCR to show a certain consistent between the two methods except several inconsistencies. It may be attributed to the experimental errors which caused by the biological replicas deficiency. Only one pooled RNA sequenced for each sample due to the limitations of specimen collection that might cause the experimental errors unpredictably. We will sequence more samples to eliminate the errors in the further research.</p

Sample statistics.

*<p>Agilent Bioanalyzer 2100 RNA integrity number.</p>**<p>The total unigene number in the DFCI Honey Bee Gene Index database is 25007; <a href="http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=honeybee" target="_blank">http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=honeybee</a>.</p>***<p>The unique matched genes from all five samples cover the DFCI database completely.</p

Additional file 1: of The dopamine β-hydroxylase gene in Chinese goose (Anas cygnoides): cloning, characterization, and expression during the reproductive cycle

Primers used in this study. (DOCX 20 kb