产蛋初期和后期蛋鸡胫骨转录谱的构建及骨代谢相关基因分析

doi:10.11843/j.issn.0366-6964.2023.10.015

摘要/Abstract

摘要： 旨在通过分析产蛋初期和产蛋后期蛋鸡胫骨转录表达谱筛选与胫骨骨代谢相关的候选基因，以揭示蛋鸡骨代谢变化的遗传机制。本研究选择19周龄（产蛋初期）和79周龄（产蛋后期）的海兰灰蛋鸡各3只，构建胫骨转录组文库，利用转录组数据筛选差异表达基因并预测蛋白质互作关系；随机选择5个差异表达基因，利用实时荧光定量PCR验证其表达水平。构建的6个海兰灰蛋鸡胫骨cDNA文库中获得有效读数19 034 718~22 668 315条，Q30值至少为93.68%，比对率为83.78%~85.72%。筛选出产蛋初期和产蛋后期蛋鸡胫骨差异表达基因共1 211个，其中645个基因下调，566个基因上调。GO功能富集分析结果显示，差异表达基因主要富集在细胞分化和细胞发育等过程。KEGG分析发现，差异表达基因显著富集在11条通路（P<0.05），其中5条与骨代谢相关，包括MAPK信号通路、TGF-β信号通路、Toll样受体信号通路、糖胺聚糖生物合成-硫酸软骨素/硫酸皮肤素和糖胺聚糖降解。在上述5条通路中涉及59个差异表达基因，其中29个上调，30个下调。此外，对骨代谢相关通路中的59个差异表达基因进行蛋白互作分析，共鉴定出53个编码蛋白，筛选出5个hub基因（IL6、IL1B、FOS、TGFB2和BMP4）。从1 211个差异表达基因中随机选取5个差异表达基因进行实时荧光定量PCR验证，结果与转录组测序结果一致。本研究揭示了产蛋初期和产蛋后期蛋鸡胫骨组织中的基因表达差异，筛选到多个影响蛋鸡胫骨骨代谢的差异表达基因和信号通路，为研究调控蛋鸡胫骨组织骨代谢的分子机制提供理论依据。

关键词: 蛋鸡, 胫骨, 差异表达基因, 骨代谢

Abstract: The purpose of this study was to screen candidate genes related to tibial bone metabolism and reveal the genetic mechanism of bone metabolism changes in laying hens by analyzing the transcriptional expression profile of the tibia of laying hens at the early and late stages of laying. Three Hy-line Grey laying hens aged 19 weeks old (early laying) and 79 weeks old (late laying) were selected to construct a tibia transcriptome library, and transcriptome data were used to screen differentially expressed genes and predict protein interactions; Five differentially expressed genes were randomly selected and their expression levels was verified using real-time fluorescence quantitative PCR. The effective reads of 19 034 718-22 668 315 were obtained from the 6 cDNA libraries of the tibia of Hy-line Grey laying hens with Q30 value of at least 93.68% and comparison rate of 83.78%-85.72%. A total of 1 211 differentially expressed genes were screened from the tibia of laying hens at the early and late stages of laying, of which 645 differentially expressed genes were down regulated and 566 differentially expressed genes were up regulated. GO analysis results showed that most of the differential genes were enriched in GO terms related to cell differentiation and cell development. KEGG analysis results showed that the differentially expressed genes were significantly enriched in 11 KEGG pathways (P<0.05). There were 5 pathways related to bone metabolism, namely MAPK signaling pathway, TGF-β signaling pathway, Toll-like receptor signaling pathway, glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate and glycosaminoglycan degradation. Among the above 5 pathways, 59 differentially expressed genes were involved, of which 29 were up regulated and 30 were down regulated. In addition, protein interaction analysis was conducted on 59 differentially expressed genes related to bone metabolism pathways, identifying a total of 53 coding proteins and screening out 5 hub genes (IL6, IL1B, FOS, TGFB2 and BMP4). Five differentially expressed genes were randomly selected from 1 211 differentially expressed genes for real-time fluorescent quantitative PCR verification, and the results were consistent with the results of RNA-Seq sequencing. This study revealed the differential gene expression in the tibia tissue of laying hens during the early and late stages of egg laying, and screened multiple differentially expressed genes and signaling pathways that affect tibia bone metabolism in laying hens, providing a theoretical basis for studying the molecular mechanism of tibia tissue regulation of bone metabolism in laying hens.

Key words: laying hen, tibia, differentially expressed genes, bone metabolism

中图分类号:

S831.2

张寅梁, 岳巧娴, 黄晨轩, 陈辉, 王德贺, 周荣艳. 产蛋初期和后期蛋鸡胫骨转录谱的构建及骨代谢相关基因分析[J]. 畜牧兽医学报, 2023, 54(10): 4164-4173.

ZHANG Yinliang, YUE Qiaoxian, HUANG Chenxuan, CHEN Hui, WANG Dehe, ZHOU Rongyan. Construction of Tibia Transcript Profile of Laying Hens at the Early and Late Laying Stages and Analysis of Genes Related to Bone Metabolism[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4164-4173.

参考文献 32

[1]	WEBSTER A B.Welfare implications of avian osteoporosis[J].Poult Sci, 2004, 83(2):184-192.
[2]	ALFONSO-CARRILLO C, BENAVIDES-REYES C, DE LOS MOZOS J, et al.Relationship between bone quality, egg production and eggshell quality in laying hens at the end of an extended production cycle (105 weeks)[J].Animals (Basel), 2021, 11(3):623.
[3]	WEI H D, CHEN Y Q, NIAN H Y, et al.Abnormal bone metabolism may me a primary causative factor of keel bone fractures in laying hens[J].Animals (Basel), 2021, 11(11):3133.
[4]	王泽伟, 高伦平, 王优灵, 等.笼养蛋鸡骨质疏松症的病因分析及预防措施[J].中国畜牧兽医文摘, 2014, 30(12):121.WANG Z W, GAO L P, WANG Y L, et al.Etiological analysis and preventive measures of osteoporosis in caged laying hens[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine, 2014, 30(12):121.(in Chinese)
[5]	王萍.延产500枚鸡蛋面临的挑战与应对措施[J].家禽科学, 2022(5):34-38.WANG P.Challenges and countermeasures for delaying the production of 500 eggs[J].Poultry Science, 2022(5):34-38.(in Chinese)
[6]	PREISINGER R.Innovative layer genetics to handle global challenges in egg production[J].Br Poult Sci, 2018, 59(1):1-6.
[7]	WHITEHEAD C C.Overview of bone biology in the egg-laying hen[J].Poult Sci, 2004, 83(2):193-199.
[8]	章世元, 俞路, 王雅倩, 等.日粮钙磷水平对笼养蛋鸡骨代谢及骨超微结构的影响[J].核农学报, 2008, 22(5):732-738.ZHANG S Y, YU L, WANG Y Q, et al.Effects of calcium and phosphorus levels on the bone micro-structure and metabolism of layers[J].Journal of Nuclear Agricultural Sciences, 2008, 22(5):732-738.(in Chinese)
[9]	KHOSLA S, MONROE D G.Regulation of bone metabolism by sex steroids[J].Cold Spring Harb Perspect Med, 2018, 8(1):a031211.
[10]	LECKA-CZERNIK B.Diabetes, bone and glucose-lowering agents:basic biology[J].Diabetologia, 2017, 60(7):1163-1169.
[11]	SIBANDA T Z, FLAVEL R, KOLAKSHYAPATI M, et al.The association between range usage and tibial quality in commercial free-range laying hens[J].Br Poult Sci, 2020, 61(5):493-501.
[12]	张亚男, 王爽, 阮栋, 等.蛋壳形成的钙代谢机制及其影响因素[J].动物营养学报, 2021, 33(3):1201-1207.ZHANG Y N, WANG S, RUAN D, et al.Calcium metabolism mechanism of eggshell formation and its influencing factors[J].Chinese Journal of Animal Nutrition, 2021, 33(3):1201-1207.(in Chinese)
[13]	LI L, ZHANG X Y, ZHAO L H, et al.Phosphorus restriction in brooding stage has continuous effects on growth performance and early laying performance of layers[J].Animals (Basel), 2021, 11(12):3546.
[14]	SILVERSIDES F G, KORVER D R, BUDGELL K L.Effect of strain of layer and age at photostimulation on egg production, egg quality, and bone strength[J].Poult Sci, 2006, 85(7):1136-1144.
[15]	DE OLIVEIRA PEIXOTO J, SAVOLDI I R, IBELLI A M G, et al.Proximal femoral head transcriptome reveals novel candidate genes related to epiphysiolysis in broiler chickens[J].BMC Genomics, 2019, 20(1):1031.
[16]	RUBIN C J, LINDBERG J, FITZSIMMONS C, et al.Differential gene expression in femoral bone from red junglefowl and domestic chicken, differing for bone phenotypic traits[J].BMC Genomics, 2007, 8:208.
[17]	YUE Q X, CHEN Y, CHEN H, et al.Transcriptome profile reveals novel candidate genes associated with bone strength in end-of-lay hens[J/OL].Animal Biotechnology, 2022, doi:10.1080/10495398.2022.2134884.
[18]	WANG C C, SHEN J, YING J, et al.FoxO1 is a crucial mediator of TGF-β/TAK1 signaling and protects against osteoarthritis by maintaining articular cartilage homeostasis[J].Proc Natl Acad Sci U S A, 2020, 117(48):30488-30497.
[19]	WANG Q, TAN Q Y, XU W, et al.Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice[J].Osteoarthritis Cartilage, 2017, 25(11):1868-1879.
[20]	RICE S J, ROBERTS J B, TSELEPI M, et al.Genetic and epigenetic fine-tuning of TGFB1 expression within the human osteoarthritic joint[J].Arthritis Rheumatol, 2021, 73(10):1866-1877.
[21]	ELSAFADI M, MANIKANDAN M, ALMALKI S, et al.TGFβ1-induced differentiation of human bone marrow-derived MSCs is mediated by changes to the actin cytoskeleton[J].Stem Cells Int, 2018, 2018:6913594.
[22]	OFITERU A M, BECHERU D F, GHARBIA S, et al.Qualifying osteogenic potency assay metrics for human multipotent stromal cells:TGF-β2 a telling eligible biomarker[J].Cells, 2020, 9(12):2559.
[23]	FU X T, QIE J, FU Q C, et al.miR-20a-5p/TGFBR2 axis affects pro-inflammatory macrophages and aggravates liver fibrosis[J].Front Oncol, 2020, 10:107.
[24]	马敏, 史雪莹, 刘黎莎, 等.硫酸软骨素药理学活性研究进展[J].食品与药品, 2017, 19(6):448-453.MA M, SHI X Y, LIU L S, et al.Research advances in pharmacological activities of chondroitin sulfate[J].Food and Drug, 2017, 19(6):448-453.(in Chinese)
[25]	NISHIDA Y.Pathophysiology of hyaluronan accumulation/depolymerization in osteoarthritic joints[J].Am J Pathol, 2021, 191(11):1963-1965.
[26]	HADLEY J A, HORVAT-GORDON M, KIM W K, et al.Bone sialoprotein keratan sulfate proteoglycan (BSP-KSPG) and FGF-23 are important physiological components of medullary bone[J].Comp Biochem Physiol A Mol Integr Physiol, 2016, 194:1-7.
[27]	YOSHIMOTO T, KITTAKA M, DOAN A A P, et al.Osteocytes directly regulate osteolysis via MYD88 signaling in bacterial bone infection[J].Nat Commun, 2022, 13(1):6648.
[28]	龙雨.依降钙素注射液对老年骨质疏松患者血清中IL-6、性激素水平的影响及其临床价值[J].中国老年学杂志, 2022, 42(8):1912-1914.LONG Y.Effect of calcitonin injection on serum IL-6 and sex hormone levels in elderly osteoporosis patients and its clinical value[J].Chinese Journal of Gerontology, 2022, 42(8):1912-1914.(in Chinese)
[29]	吴迎爽.吡格列酮对去卵巢大鼠IL-1β、IL-6、TNF-a的影响及与骨代谢关系的实验研究[D].太原:山西医科大学, 2012.WU Y S.Influence of pioglitazone on IL-1β, IL-6, TNF-α in ovaries rats and experimental study on the relation of them with bone metabolism[D].Taiyuan:Shanxi Medical University, 2012.(in Chinese)
[30]	OFITERU A M, BECHERU D F, GHARBIA S, et al.Qualifying osteogenic potency assay metrics for human multipotent stromal cells:TGF-β2 a telling eligible biomarker[J].Cells, 2020, 9(12):2559.
[31]	汪玉海, 王志斌, 张小钰, 等.脂肪干细胞对绝经后骨质疏松大鼠c-Fos、c-Jun表达的影响[J].宁夏医学杂志, 2020, 42(10):865-868.WANG Y H, WANG Z B, ZHANG X Y, et al.Effects of c-Fos and c-Jun expression on adipose-derived stem cells in postmenopausal osteoporosis rats[J].Ningxia Medical Journal, 2020, 42(10):865-868.(in Chinese)
[32]	YE Y E, JIANG Z W, PAN Y Q, et al.Role and mechanism of BMP4 in bone, craniofacial, and tooth development[J].Arch Oral Biol, 2022, 140:105465.