中国农业科学 ›› 2016, Vol. 49 ›› Issue (4): 754-764.doi: 10.3864/j.issn.0578-1752.2016.04.014

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

北京油鸡和来航鸡脾脏差异表达microRNA的鉴定与分析

耿立英,潘素敏,陈娟,朱文进,巩元芳,刘铮铸,彭永东,赵书雨,张传生,李祥龙   

  1. 河北科技师范学院,河北昌黎 066600
  • 收稿日期:2014-11-19 出版日期:2016-02-16 发布日期:2016-02-16
  • 通讯作者: 张传生,E-mail:cszhang1976@126.com。李祥龙,E-mail:lixianglongcn@yahoo.com
  • 作者简介:耿立英,E-mail:rosegengly@126.com;Tel:0335-2039629
  • 基金资助:
    国家青年科学基金(31001003)、河北省教育厅优秀青年科学基金(Y2011118)、河北省自然科学基金(C2012407009)、河北省现代农业公关计划(15226302D)、河北省高等学校创新团队领军人才培育计划(LJRC004)和河北省现代农业产业技术体系蛋鸡产业创新团队项目

Identification and Bioinformatics Analysis of Differential Expression MicroRNAs in the Spleen Between Beijing Fatty Chickens and Leghorns Chickens

GENG Li-ying, PAN Su-min, CHEN Juan, ZHU Wen-jin, GONG Yuan-fang, LIU Zheng-zhu, PENG Yong-dong, ZHAO Shu-yu, ZHANG Chuan-sheng, LI Xiang-long   

  1. Hebei Normal University of Science and Technology, Changli 066600, Hebei
  • Received:2014-11-19 Online:2016-02-16 Published:2016-02-16

摘要: 【目的】明确北京油鸡和来航鸡脾脏重量、组织结构及其miRNA表达谱的差异,筛选与两个鸡种免疫应答能力差异相关的候选基因,为家禽抗病育种研究奠定基础。【方法】选取1日龄同期孵化的北京油鸡和来航鸡母雏各45只作为试验材料,在相同营养水平和环境条件下饲养,42日龄测量体重和脾脏重。每个品种随机选取3只个体(体重接近群体均值),取其一半脾脏组织制作石蜡切片,利用H.E.染色,显微镜下观察脾脏组织结构、脾小体的数量和动脉周围淋巴鞘厚度。提取另一半脾脏组织中的小RNA,等量混合组成2个RNA池,构建cDNA文库,利用Solexa平台进行高通量测序。测序结果与参考基因组数据库比对获得表达基因。利用Mireap软件预测新miRNAs基因。利用RPKM(reads per kb per million reads)方法计算基因的表达量,根据FDR(false discovery rate)<0.001和|log2 ratio(T/CK)|≥1的标准筛选差异表达的基因。利用TargetScan和Pictar软件预测差异表达miRNA的靶基因,并对其进行GO和KEGG数据库功能注释。利用DAVID软件对靶基因蛋白进行富集分析。将预测靶基因与已知脾脏重和脾脏指数相关QTL区域注解的基因取交集。【结果】在42日龄,北京油鸡的体重和脾脏重均显著高于来航鸡(P<0.01)。组织学观察显示,两个鸡种的脾脏组织结构发育完整,可见清晰的白髓、红髓、脾小结和动脉周围淋巴细胞鞘。与来航鸡相比,北京油鸡脾小体的数量更多、动脉周围淋巴鞘更厚,鞘内淋巴细胞也更为密集。高通量测序在两组样本共鉴定出486种已知miRNA 、130个候选miRNA 和216个共表达miRNA 。两组间显著差异表达的miRNA共计35种,其中在北京油鸡中有8个表达上调,27个表达下调,它们的变化倍数在1.002—10.41之间。差异基因表达丰度在前5位的miRNA是miR-2954miR-6606-5pmiR-146b-5pmiR-21-3pmiR-21-5p,对其2 767个靶基因的生物信息学分析结果显示,它们参与了T和B淋巴细胞的分化、免疫器官的发育、蛋白质磷酸化和细胞形态发生等生物学过程,并在泛素介导的蛋白水解、凋亡和磷脂酰肌醇信号系统等14个通路中显著富集。miR-6606-5pmiR-146b-5p的共同靶基因BLMmiR-2954的靶基因IRF8均定位于脾重或脾指数相关的QTL区域。【结论】(1)42日龄北京油鸡和来航鸡脾重、组织结构及其miRNA表达谱的确存在一些差异。(2)某些重要的差异高丰度表达miRNA(如miR-2954miR-6606-5pmiR-146b-5p miR-21-3pmiR-21-5p ) 可能通过对靶基因调控,来参与调节T、B淋巴细胞分化、增殖、激活等过程,进而影响脾脏的重量、组织结构及其免疫应答能力。

关键词: 脾脏, microRNA, Solexa测序技术, 差异表达基因

Abstract: 【Objective】 To investigate the differences of spleen weight, histological structure and miRNA expression profile in Beijing Fatty Chicken and Leghorn Chicken, we screened the candidate genes related with the immune response capacity difference in two kind of chickens, so as to lay a foundation for poultry breeding for disease resistance. 【Method】 45 one-day-age Beijing Fatty Chickens and 45 female Leghorn pullets hatched in the corresponding period were selected as the experimental materials, fed in the same nutritional and environmental condition. The body weight and spleen weight were measured in 42 days. Three individuals were randomly selected for each species (body weight was close to the average value of the group). Half of the spleen was made into the paraffin sections. The spleen histological structure, number of splenic corpuscles and thickness of the periarterial lymphatic sheath were observed using HE staining. The small RNA was extracted from other half of the spleen tissue and composed into 2 RNA pools through balanced mix. The cDNA library was constructed. The high-throughput sequencing was conducted using a Solexa platform. The expressed genes were obtained through comparing the sequencing result and reference genome database. The new miRNAs gene was predicted using Mireap software. The gene expression was calculated using RPKM (reads per kb per million reads) method. The differentially expressed genes were screened according to the criteria of FDR(false discovery rate)<0.001 and |log2 ratio (T/CK)| ≥1. The target genes differentially expressing miRNA were predicted using TargetScan and Pictar softwares, the GO and KEGG database functions were annotated. The enrichment analysis of the target gene protein was conducted using DAVID software. The predicted target genes were intersected with the known QTL region annotation genes related with spleen weight and spleen index. 【Result】The body weight of the Beijing Fatty Chicken and spleen weight were significantly higher than those of Leghorns (P<0.01). Histological observation showed that the tissue structure of the spleen was integrated in the two chicken breeds. The white pulp, red pulp, splenic corpuscle and periarterial lymphatic sheath could be clearly observed. Compared with the Leghorn chicken, the number of splenic corpuscles was more, periaterial lymphatic sheaths were thicker and intrathecal lymphocytes were more intensive in the Beijing Fatty Chicken. 486 known miRNAs, 130 candidate miRNAs and 216 coexpressed miRNAs were identified in the two groups of samples using high-throughput sequencing. There were 35 kinds of miRNAs differentially expressed between two groups. Among them, 8 kinds were up-regulated and 27 kinds were down-regulated in the Beijing Fatty Chicken. Their fold change was 1.002-10.41. The first five expressive abundances of differentially expressed genes were miR-2954, miR-6606-5p, miR-146b-5p, miR-21-3p and miR-21-5p. The bioinformatics analysis of 2767 target genes showed that they were involved in the biological processes of T, B lymphocyte cell differentiation, immune organ development, protein phosphorylation and cell morphogenesis. They were significantly enriched in ubiquitin mediated proteolysis, apoptosis, phosphatidylinositol signaling system etc. 14 pathways. The miR-6606-5p and miR-146b-5p common target genes- BLM and miR-2954 target gene IRF8 were localized in the QTL region related with spleen weight or spleen index. 【Conclusion】(1) There were some differences in spleen weight, histological structure and miRNA expression profile of the 42-day-old Beijing Fatty Chickens and Leghorn Chickens. (2) The differential high abundance miRNA (miR-2954, miR-6606-5p, miR-146b-5p, miR-21-3p and miR-21-5p) might be involved in the regulation of T, B lymphocytes differentiation, proliferation, activation and other processes through regulating target gene, so as to affect the spleen weight, organizational structure and immune response capacity.

Key words: spleen, microRNA, Solexa sequencing, differentially expressed genes