外源注射miR-499-5p对肉鸡腓肠肌转录组的影响

doi:10.11843/j.issn.0366-6964.2020.09.007

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (9): 2098-2108.doi: 10.11843/j.issn.0366-6964.2020.09.007

外源注射miR-499-5p对肉鸡腓肠肌转录组的影响

李志雄^1,2*, 徐亚欧², 林亚秋², 杨朝武³, 余春林³, 吴锦波⁴, 陈玲⁵

1. 西南民族大学动物科学国家民委重点实验室, 成都 610041;
2. 西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室, 成都 610041;
3. 四川省畜牧科学研究院, 成都 610066;
4. 阿坝藏族羌族自治州畜牧科学技术研究所, 阿坝 624402;
5. 安康学院现代农业与生物科技学院, 安康 725000

收稿日期:2020-03-09 出版日期:2020-09-25 发布日期:2020-09-25
通讯作者: 李志雄,主要从事家禽遗传育种研究,E-mail:lizhixiong@swun.edu.cn
作者简介:李志雄(1987-),男,山西平遥人,讲师,博士,主要从事家禽遗传育种研究,E-mail:lizhixiong@swun.edu.cn
基金资助:
四川省“十三五”畜禽育种攻关项目（2016NYZ0043）；四川省应用基础项目（20SYSX0175）；西南民族大学中央高校基本科研业务费专项资金项目（2020PTJS21003）

Effect of miR-499-5p Injection on Transcriptome in Gastrocnemius Muscle of Broiler

LI Zhixiong^1,2*, XU Yaou², LIN Yaqiu², YANG Chaowu³, YU Chunlin³, WU Jinbo⁴, CHEN Ling⁵

1. Key Laboratory of Animal Science of State Ethnic Affairs Commission, Southwest Minzu University, Chengdu 610041, China;
2. Key Laboratory of Ministry of Education for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Southwest Minzu University, Chengdu 610041, China;
3. Sichuan Animal Science Academy, Chengdu 610066, China;
4. Institute of Science and Technology of Aba Tibetan and Qiang Autonomous Prefecture, Aba 624402, China;
5. School of Modern Agriculture&Biotechnology, Ankang University, Ankang 725000, China

Received:2020-03-09 Online:2020-09-25 Published:2020-09-25

摘要/Abstract

摘要： 旨在探讨外源注射miR-499-5p对肉鸡腓肠肌肌纤维直径及基因表达的影响，完善miR-499-5p对肉鸡骨骼肌发育的分子调控机制。本研究选用14日龄健康大恒肉鸡公鸡16只，随机分为2组，每组8个重复，均正常饲喂日粮，分别在腓肠肌部位肌肉注射agomiR-NC（对照组，5 nmol）和agomiR-miR-499-5p（注射组，5 nmol），为保证试验效果，每3 d注射1次，共注射6次。注射完成后将16只肉鸡屠宰，采集腓肠肌组织进行肌纤维直径测定和转录组学（RNA-seq）分析。结果显示，注射miR-499-5p可导致肉鸡腓肠肌肌纤维直径显著减小（P<0.05）。转录组测序结果显示，注射组和对照组分别获得308 263 164和316 696 152条clean reads，与鸡参考基因组的比对率分别为90.29%和89.57%。与对照组相比，注射组共有差异表达基因18个，其中上调基因12个，下调基因6个（P<0.05）。其中差异表达最大的两个基因分别是原癌基因FOS（fos proto oncogene，FOS）和早期生长应答蛋白基因（EGR1）。GO富集分析发现，差异表达基因显著富集于跨膜受体蛋白丝氨酸/苏氨酸激酶信号通路、转录调控、脂质合成和骨骼肌再生等过程。KEGG富集分析结果显示，差异表达基因显著富集于丝裂原活化蛋白激酶（MAPK）信号通路和Wnt信号通路。利用生物信息学软件预测到miR-499-5p的靶基因共78个，GO富集分析发现，靶基因也显著富集于丝氨酸/苏氨酸激酶活性的负调控和转录因子活性等过程。本研究表明，注射miR-499-5p可改变肉鸡腓肠肌肌纤维直径从而影响肌纤维特性，差异表达基因中的FOS、EGR1、CYR61和WNT7A基因可能在miR-499-5p的作用下通过上述通路参与了脂肪沉积和肌肉发育的调控。

关键词: 肉鸡, 肌纤维直径, 转录组测序, 肌肉发育

Abstract: The objective of this study was to investigate the influence of miR-499-5p injection on gastrocnemius muscle fiber diameter and gene expression, and improve the molecular regulation mechanism of miR-499-5p in skeletal muscle development in broiler. Sixteen healthy Daheng cocks aged 14 days were randomly assigned into 2 groups with 8 replicates per group. agomiR-NC(5 nmol) were injected into the chicks in control group, and agomiR-miR-499-5p(5 nmol) were injected into the chicks in treating group, respectively. agomiR-NC and agomiR-miR-499-5p were injected once every 3 days for a total of 6 times. The gastrocnemius muscle in the 2 groups were collected after slaughter for the measurement of muscle fiber diameter and RNA-seq analysis. The results showed that the muscle fiber diameter of gastrocnemius of chicks in treating group was significantly shorter than that of chicks in control group (P<0.05). 308 263 164 and 316 696 152 clean reads were obtained from the treating group and control group, respectively. The ratios of these reads to chicken reference genome were 90.29% and 89.57%, respectively. Compared with the chicks in the control group, there were 18 differentially expressed genes in gastrocnemius muscle of miR-499-5p treated chicks, of which 12 up-regulated genes and 6 down-regulated genes (P<0.05). The top 2 differentially expressed genes were FOS proto oncogene (FOS) and early growth response gene-1 (EGR1). GO enrichment analysis showed that the differentially expressed genes were mainly enriched in the transmembrane receptor protein serine/threonine kinase signaling pathway, transcriptive regulation, lipid biosynthetic process and skeletal muscle regeneration. KEGG analysis showed that the differentially expressed genes were mainly involved in the MAPK signaling pathway and Wnt signaling pathway. A total of 78 target genes of miR-499-5p were predicted by bioinformatics software. GO enrichment analysis showed that the 78 target genes were also mainly enriched in the negative regulation of protein serine/threonine kinase activity and transcription factor activity. In conclusion, the results showed that miR-499-5p could change the gastrocnemius muscle fiber diameter and affect characteristics of muscle fiber. FOS, EGR1, CYR61 and WNT7A, which were significantly associated with the alteration of muscle fiber diameter, might be involved in the regulation of fat deposition and muscle development through the pathways mentioned above in chicks after miR-499-5p injection.

Key words: broiler, diameter of muscle fiber, transcriptome sequencing, muscle development

中图分类号:

S831.2

李志雄, 徐亚欧, 林亚秋, 杨朝武, 余春林, 吴锦波, 陈玲. 外源注射miR-499-5p对肉鸡腓肠肌转录组的影响[J]. 畜牧兽医学报, 2020, 51(9): 2098-2108.

LI Zhixiong, XU Yaou, LIN Yaqiu, YANG Chaowu, YU Chunlin, WU Jinbo, CHEN Ling. Effect of miR-499-5p Injection on Transcriptome in Gastrocnemius Muscle of Broiler[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2098-2108.

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外源注射miR-499-5p对肉鸡腓肠肌转录组的影响

Effect of miR-499-5p Injection on Transcriptome in Gastrocnemius Muscle of Broiler

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