基于转录组测序研究绿光影响鹅胚心脏早期发育的候选基因

doi:10.11843/j.issn.0366-6964.2024.05.016

摘要/Abstract

摘要： 旨在通过分析鹅胚心脏在绿光光照刺激下的基因表达差异，挖掘心脏发育的候选基因。本研究将512枚鹅种蛋，随机均分在正常黑暗孵化以及补充绿光的两台孵化机中，每台孵化机内放置4层(64枚·层^-1)，绿光孵化机提供15 min开灯和15 min关灯的间歇光照。在孵化第13、16、20、24、28和30天时，每组随机挑选种蛋8枚，称取胚胎重量，分离胚胎心脏组织并称重；采集孵化第13天的胚胎心脏组织(每组各3)，进行转录组测序(RNA-Seq)，筛选差异表达基因(DEGs)并进行功能富集分析，鉴定与绿光促进心脏发育相关的候选基因。并通过荧光定量PCR(qRT-PCR方法)验证测序结果的可靠性。结果表明：绿光显著提高16和30胚龄胚重比例(胚胎重/入孵蛋重×100)(P<0.05)，以及13和16胚龄心脏指数(心脏重/胚胎重×100)(P<0.05)。心脏转录组测序分析共筛选出1 643个DEGs。随机选择 7个DEGs 进行 RT-qPCR 验证，表达趋势与测序结果一致。功能富集分析表明，DEGs 主要涉及PI3K-Akt信号通路、AMPK信号通路等通路，最终筛选出GATA4、GATA5、Smad4和GHR这4个候选基因。对它们在不同胚龄阶段心脏组织中的mRNA表达量进行分析，其表达模式进一步表明了其在绿光调控心脏发育过程中的作用。本研究发现，鹅蛋孵化期绿光处理促进了胚胎和心脏发育，进一步通过心脏组织转录组数据分析，鉴定到了4个鹅胚心脏发育候选基因 GATA4、GATA5、Smad4和GHR，为鹅胚心脏组织发育的分子调控机制提供了线索，并为绿光应用于鹅种蛋孵化提供了理论基础。

关键词: 鹅, 绿光, 孵化, 心脏, 转录组, GATA4

Abstract: The aim of this study was to identify candidate genes for heart development in goose embryos by analyzing the gene expression differences in embryonic hearts tissue under green light stimulation. The 512 goose eggs were randomly divided equally into green light and dark incubators, with 4 layers and 64 eggs per layer placed in each incubator. During the entire incubation period, the green light incubator provided intermittent light regimen of 15 minutes on and 15 minutes off. On the 13, 16, 20, 24, 28, and 30 day of incubation, 8 eggs were randomly selected from each group, the embryo weight was measured, and the embryonic heart tissue was separated and weighed. The embryonic heart tissues were collected at day13 of incubation, and transcriptome sequencing (RNA-Seq) were performed. Differentially expressed genes (DEGs) were screened, and subjected to functional enrichment analysis to identify candidate genes related to green light promoting heart development. And the reliability of the sequencing results was validated using fluorescence quantitative PCR (qRT-PCR). The green light significantly increased the proportion of embryo weight at 16 and 30 embryonic ages (embryo weight/hatched egg weight×100) (P<0.05), and the heart index (heart weight/embryo weight×100) at 13 and 16 embryonic ages in the green light group were significantly higher than individuals in the dark group (P<0.05). A total of 1 643 DEGs were screened from the green light group and the dark group, 7 DEGs were randomly selected for RT-qPCR validation, and the expression trend was consistent with the sequencing results. Enrichment analysis showed that DEGs were mainly enriched in the PI3K-Akt signaling pathway, AMPK signaling pathway, and other pathways. Four candidate genes: GATA4, GATA5, Smad4 and GHR were ultimately screened out. Their expression in heart tissues at different embryonic ages were analyzed to further validated their regulatory role in heart development. Through the analysis of transcriptomic data of heart tissue, we identified GATA4, GATA5, Smad4 and GHR as candidate genes for goose embryonic heart development, which provide clues to the molecular regulatory mechanism of green light on embryonic heart development, and provide a theoretical basis for the application of green light in goose egg hatching.

Key words: goose, green light, incubation, heart, transcriptome, GATA4

中图分类号:

S835.2

陈哲, 曲小露, 郭彬彬, 孙雪峰, 闫乐艳. 基于转录组测序研究绿光影响鹅胚心脏早期发育的候选基因[J]. 畜牧兽医学报, 2024, 55(5): 1978-1988.

CHEN Zhe, QU Xiaolu, GUO Binbin, SUN Xuefeng, YAN Leyan. Study on Candidate Genes for Green Light Affecting Early Development of Goose Embryo Heart Based on Transcriptome Sequencing[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1978-1988.

参考文献

[1] 陈哲,陈永霞,于建宁,等.光照在禽蛋孵化期的应用和研究进展[J].中国家禽,2018,40(4):1-6.
CHEN Z,CHEN Y X,YU J N,et al.Application and research progress of illumination during avian hatching process[J].China Poultry,2018,40(4):1-6.(in Chinese)
[2] 泮进明,王小双,蒋劲松,等.家禽规模养殖LED光环境调控技术进展与趋势分析[J].农业机械学报,2013,44(9):225-235.
PAN J M,WANG X S,JIANG J S,et al.Advancement and trend of LED light environment control technology for intensive poultry production[J].Transactions of the Chinese Society for Agricultural Machinery,2013,44(9):225-235.(in Chinese)
[3] LI X J,RATHGEBER B,MCLEAN N,et al.Providing colored photoperiodic light stimulation during incubation:2.
Effects on early posthatch growth,immune response,and production performance in broiler chickens[J].Poult Sci,2021,100(9):101328.
[4] ALI S,SHERZADA S,USMAN M,et al.Impact of different light intensities to Japanese quail eggs on hatching results and post-hatch performance[J].Trop Anim Health Prod,2023,55(6):387.
[5] ROZENBOIM I,HUISINGA R,HALEVY O,et al.Effect of embryonic photostimulation on the posthatch growth of turkey poults[J].Poult Sci,2003,82(7):1181-1187.
[6] 张林,张海军,武书庚,等.单色光间歇性刺激胚蛋对肉仔鸡胸肉生长及肉品质的影响[J].中国农业科学,2012,45(5):951-957.
ZHANG L,ZHANG H J,WU S G,et al.Effect of intermittently monochromatic light stimuli during the embryogenesis on breast muscular growth and meat quality in male broiler chicks[J].Scientia Agricultura Sinica,2012,45(5):951-957.(in Chinese)
[7] TANG W Y,TONG Q,LI B M,et al.Effects of different light-emitting diode light on hatch performance,embryo development,eye structure,and plasma melatonin in layer incubation[J].Poult Sci,2023,102(10):102977.
[8] HUTH J C,ARCHER G S.Effects of LED lighting during incubation on layer and broiler hatchability,chick quality,stress susceptibility and post-hatch growth[J].Poult Sci,2015,94(12):3052-3058.
[9] COOPER C B,VOSS M A,ARDIA D R,et al.Light increases the rate of embryonic development:implications for latitudinal trends in incubation period[J].Funct Ecol,2011,25(4):769-776.
[10] MAURER G,PORTUGAL S J,CASSEY P.Review:an embryo’s eye view of avian eggshell pigmentation[J].J Avian Biol,2011,42(6):494-504.
[11] DISHON L,AVITAL-COHEN N,ZAGURI S,et al.In ovo green light photostimulation during the late incubation stage affects somatotropic axis activity[J].Poult Sci,2021,100(2):467-473.
[12] WANG P L,SUN Y Y,LI Y L,et al.Monochromatic green light stimulation during incubation shortened the hatching time via pineal function in White Leghorn eggs[J].J Anim Sci Biotechnol,2021,12，17.
https://doi.org/10.1186/s40104-020-00539-x.
[13] CHEN Z,QU X L,FENG C G,et al.Monochromatic green light stimulation during incubation alters hepatic glucose metabolism that improves embryonic development in Yangzhou goose eggs[J].Int J Mol Sci,2023,24(1):405.
[14] WITTIG J G,MVNSTERBERG A.The early stages of heart development:insights from chicken embryos[J].J Cardiovasc Dev Dis,2016,3(2):12.
[15] TONG Q,MCGONNELL I M,DEMMERS T G M,et al.Effect of a photoperiodic green light programme during incubation on embryo development and hatch process[J].Animal,2018,12(4):765-773.
[16] GIMENO M A,ROBERTS C M,WEBB J L.Acceleration of rate of the early chick embryo heart by visible light[J].Nature,1967,214(5092):1014-1016.
[17] 陈雪娇,刘会杰,臧蕾,等.鸡胚心脏组织转录组数据鉴定雪域白鸡高原低氧适应性关键基因[J].畜牧兽医学报,2023,54(10):4154-4163.
CHEN X J,LIU H J,ZANG L,et al.Transcriptome data from chicken embryo heart tissue identified key genes for altitude hypoxia adaptation in Xueyu White chickens[J].Acta Veterinaria et Zootechnica Sinica,2023,54(10):4154-4163.(in Chinese)
[18] ROZENBOIM I,PIESTUN Y,MOBARKEY N,et al.Monochromatic light stimuli during embryogenesis enhance embryo development and posthatch growth[J].Poult Sci,2004,83(8):1413-1419.
[19] ZHU H X,SHAO X B,CHEN Z,et al.Induction of out-of-season egg laying by artificial photoperiod in Yangzhou geese and the associated endocrine and molecular regulation mechanisms[J].Anim Reprod Sci,2017,180:127-136.
[20] LI M Y,LIANG C,ZHAO X H,et al.Reproductive performance of Zi-goose promoted by red color illumination[J].Front Vet Sci,2022,9:879478.
[21] MARTINSEN B J.Reference guide to the stages of chick heart embryology[J].Dev Dyn,2005,233(4):1217-1237.
[22] ZHOU P Z,ZHANG Y,SETHI I,et al.GATA4 regulates developing endocardium through interaction with ETS1[J].Circ Res,2022,131(11):e152-e168.
[23] AFOUDA B A.Towards understanding the gene-specific roles of GATA Factors in heart development:Does GATA4 lead the way?[J].Int J Mol Sci,2022,23(9):5255.
[24] KUO C T,MORRISEY E E,ANANDAPPA R,et al.GATA4 transcription factor is required for ventral morphogenesis and heart tube formation[J].Genes Dev,1997,11(8):1048-1060.
[25] MOLKENTIN J D,LIN Q,DUNCAN S A,et al.Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis[J].Genes Dev,1997,11(8):1061-1072.
[26] GARG V,KATHIRIYA I S,BARNES R,et al.GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5[J].Nature,2003,424(6947):443-447.
[27] MOSKOWITZ I P,WANG J,PETERSON M A,et al.Transcription factor genes Smad4 and Gata4 cooperatively regulate cardiac valve development[J].Proc Natl Acad Sci U S A,2011,108(10):4006-4011.
[28] LI R G,XU Y J,WANG J,et al.GATA4 loss-of-function mutation and the congenitally bicuspid aortic valve[J].Am J Cardiol,2018,121(4):469-474.
[29] 张全波,陆长青,冉黎,等.TGF-β1/Smad4信号通路在SD大鼠胚胎心脏发育中的作用及意义[J].实用医学杂志,2010,26(17):3105-3108.
ZHANG Q B,LU C Q,RAN L,et al.Effect of TGF-β1/Smad4 signaling pathway on the development of heart of SD rat embryo[J].The Journal of Practical Medicine,2010,26(17):3105-3108.(in Chinese)
[30] QI X,YANG G,YANG L L,et al.Essential role of Smad4 in maintaining cardiomyocyte proliferation during murine embryonic heart development[J].Dev Biol,2007,311(1):136-146.
[31] NIE X G,DENG C X,WANG Q,et al.Disruption of Smad4 in neural crest cells leads to mid-gestation death with pharyngeal arch,craniofacial and cardiac defects[J].Dev Biol,2008,316(2):417-430.
[32] HALMOS G,SZABO Z,JUHASZ E,et al.Signaling mechanism of growth hormone-releasing hormone receptor[J].Vitam Horm,2023,123:1-26.
[33] HARVEY S,JOHNSON C D,SANDERS E J.Extra-pituitary growth hormone in peripheral tissues of early chick embryos[J].J Endocrinol,2000,166(3):489-502.
[34] 李青竹,李润航,郑艳秋,等.鹅多个组织GHR和IGF-Ⅰ基因表达的发育性变化[J].中国兽医学报,2015,35(5):739-744.
LI Q Z,LI R H,ZHENG Y Q,et al.Developmental changes of GHR and IGF-Ⅰ gene expression in multiple tissues of geese[J].Chinese Journal of Veterinary Science,2015,35(5):739-744.(in Chinese)
[35] KVHN E R,VLEURICK L,EDERY M,et al.Internalization of the chicken growth hormone receptor complex and its effect on biological functions[J].Comp Biochem Physiol Part B:Biochem Mol Biol,2002,132(1):299-308.
[36] DISHON L,AVITAL-COHEN N,ZAGURI S,et al.The effect of selected in ovo green light photostimulation periods on post-hatch broiler growth and somatotropic axis activity[J].Poult Sci,2021,100(8):101229.