鸡骨骼肌中天然反义lncRNA VGLL2-AS的鉴定及其与VGLL2的关系研究  

Identification of Natural Antisense lncRNA VGLL2-AS in Chicken Skeletal Muscle and Its Relationship with VGLL2

作　　者：李文雅[1] 牛欣然 任团辉 蔡含芳 韩瑞丽[1] 田亚东[1] 刘小军[1] 康相涛[1] 李转见[1] LI Wenya;NIU Xinran;REN Tuanhui;CAI Hanfang;HAN Ruili;TIAN Yadong;LIU Xiaojun;KANG Xiangtao;LI Zhuanjian(College of Animal Science and Technology,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物科技学院,郑州450046

出　　处：《畜牧兽医学报》2023年第1期122-132,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(U1804107);中原青年拔尖人才(ZYYCYU202012156);河南省高校科技创新人才支持计划(22HASTIT038)。

摘　　要：旨在探究鸡骨骼肌中VGLL2基因天然反义链转录本VGLL2 AS lncRNA(VGLL2-AS)与VGLL2的关系。本研究首先采用PCR和测序技术验证VGLL2-AS是否存在;之后分别采集不同周龄固始蛋鸡(1日龄、6周龄、16周龄、22周龄、30周龄,每个周龄各6只)组织样,采用荧光定量PCR分析VGLL2基因和其反义链转录本VGLL2-AS的表达谱;采用双向转录试验和核酸酶保护试验检测VGLL2和VGLL2-AS是否可以双向转录且两者之间关系;体外分离培养原代成肌细胞(11胚龄固始鸡胚),然后用2μg·mL^(-1)的放线菌素D处理成肌细胞(对照组不做处理),收取处理不同时间点细胞(0~8 h,每组各个时间点各做3个重复),检测VGLL2-AS与VGLL2的半衰期;分离鸡成肌细胞的细胞核和细胞质,通过RT-qPCR方法确定两者的细胞定位;利用PCR扩增及测序对VGLL2的转录本进行验证;最后利用RT-qPCR检测它们在固始蛋鸡胸肌组织(1日龄、6周龄、16周龄、22周龄、30周龄每个周龄各6只)中的时空表达规律和相关性。结果表明,VGLL2-AS在鸡的转录组中真实存在;VGLL2与VGLL2-AS均在肌肉中特异高表达(P<0.05);VGLL2-AS与VGLL2可以进行双向转录且二者之间可以形成双链RNA;VGLL2-AS半衰期较VGLL2长;在成肌细胞中,VGLL2-AS和VGLL2主要定位于细胞质中(P<0.001);VGLL2只存在VGLL2-mRNA、VGLL2-X2和VGLL2-X3转录本;VGLL2-mRNA和VGLL2-X3与VGLL2-AS表达趋势一致,且VGLL2-mRNA、X3和VGLL2-AS的表达呈现极强的正相关(r分别为0.943和0.935,P<0.01)。综上所述,VGLL2-AS作为VGLL2的反义链编码的lncRNA定位于细胞质中,可能通过与VGLL2形成的双链RNA之间的相互作用,然后参与调节VGLL2的表达并维持其稳定性,最终在鸡的早期肌肉发育中发挥重要作用。本研究的结果扩展了鸡中关于NATs的研究,并为鸡VGLL2基因与其天然反义链转录本VGLL2-AS在鸡骨骼肌发育中的生物学功能的研究奠定了基础,对于提高禽类的生长发育具有一定的意义。The purpose of the study was to explore the relationship between VGLL2-AS and VGLL2 in chicken skeletal muscle. PCR and sequencing techniques were used to verify the existence of VGLL2-AS;The tissue samples of Gushi layers at different weeks of age(1 day old, 6 weeks old, 16 weeks old, 22 weeks old and 30 weeks old, 6 for per week-old) were collected respectively, and the expression profiles of VGLL2 gene and its antisense chain transcript VGLL2-AS were analyzed by real-time quantitative PCR. Bidirectional transcription test and nuclease protection test were used to detect whether VGLL2 and VGLL2-AS could be transcribed in two directions and the relationship between them. Then, the primary myoblasts of 11 embryo-old Gushi chicken embryos were isolated and cultured in vitro, and then treated with 2 μg·mL^(-1)actinomycin D(the control group was not treated), and the cells were collected at different time points(0-8 h, 3 repeats in each time point). The half-life of VGLL2-AS and VGLL2 were measured. The nucleus and cytoplasm of chicken myoblasts were isolated, and their cellular localization was determined by real-time quantitative PCR. The transcripts of VGLL2 were verified by PCR amplification and sequencing;Finally, RT-qPCR was used to detect their spatiotemporal expression and correlation in the breast muscle tissues of Gushi layers(6 birds at 1 day, 6 weeks, 16 weeks, 22 weeks and 30 weeks, respectively). The results showed that VGLL2-AS existed in the chicken transcriptome. VGLL2 and VGLL2-AS were highly expressed in muscle(P<0.05). VGLL2-AS and VGLL2 could conduct bidirectional transcription and form double stranded RNA between them. VGLL2-AS had a longer half-life than VGLL2;In myoblasts, VGLL2-AS and VGLL2 were mainly located in the cytoplasm(P<0.001). VGLL2 only contained VGLL2-mRNA, VGLL2-X2 and VGLL2-X3 transcripts. The expression trend of VGLL2-mRNA and VGLL2-X3 was consistent with that of VGLL2-AS, and the expression of VGLL2-mRNA, VGLL2-X3 and VGLL2-AS showed a strong positive correlation(r=0.943 an

关 键 词：NATs VGLL2-AS VGLL2基因 骨骼肌 

分 类 号：S831.2[农业科学—畜牧学]