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

doi:10.11843/j.issn.0366-6964.2023.01.012

Abstract

Abstract: 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 and 0.935, respectively, P<0.01). To sum up, VGLL2-AS, as the antisense chain encoding lncRNA of VGLL2, is located in the cytoplasm, possibly through the interaction with the double stranded RNA formed by VGLL2, and then participates in regulating the expression of VGLL2 and maintaining its stability, finally playing an important role in the early muscle development of chickens. The results of this study expand the research on NATs in chickens, and lay a foundation for the study of biological function of chicken VGLL2 gene and its natural antisense chain transcript VGLL2-AS in chicken skeletal muscle development, which has certain significance for improving the growth and development of poultry.

Key words: NATs, VGLL2-AS, VGLL2 gene, skeletal muscle

CLC Number:

S831.2

LI Wenya, NIU Xinran, REN Tuanhui, CAI Hanfang, HAN Ruili, TIAN Yadong, LIU Xiaojun, KANG Xiangtao, LI Zhuanjian. Identification of Natural Antisense lncRNA VGLL2-AS in Chicken Skeletal Muscle and Its Relationship with VGLL2[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 122-132.

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

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