Wnt3a基因多态性与崇仁麻鸡皮肤毛囊性状相关性研究

doi:10.11843/j.issn.0366-6964.2023.07.014

摘要/Abstract

摘要： 旨在基于基因多态性分析Wnt家族成员3a （wnt family member 3a，Wnt3a）基因遗传变异位点与崇仁麻鸡皮肤毛囊密度的相关性。本研究随机选取600只在相同环境和相同管理水平下饲养至上市日龄（120 d），健康且状况良好的崇仁麻鸡，其中原种母鸡200只，公鸡100只和B系母鸡200只，公鸡100只。屠宰后选取背部、胸部和大腿部皮肤相同的位置测量毛囊密度，通过皮肤组织切片HE染色的方法观察毛囊的形态结构并测量毛囊直径和皮肤厚度。采血提DNA用PCR扩增后直接测序的方法筛选Wnt3a基因遗传变异位点，并与鸡皮肤毛囊密度进行相关性分析。通过实时荧光定量PCR技术检测Wnt3a基因在不同皮肤部位的表达量。结果表明：1）原种和B系同性别同皮肤部位相比较，原种母鸡背部毛囊密度极显著高于B系（P<0.01），大腿部毛囊密度显著高于B系（P<0.05），背部和大腿部毛囊直径显著低于B系（P<0.05）。原种公鸡背部皮肤毛囊密度极显著高于B系（P<0.01），胸部毛囊密度极显著低于B系（P<0.01），背部和大腿部毛囊直径显著低于B系（P<0.05）。B系公鸡胸部皮肤厚度显著高于原种（P<0.05），B系公、母鸡大腿皮肤厚度均显著高于原种（P<0.05）。2） Wnt3a基因筛选出的3个SNPs位点与各部位皮肤毛囊密度进行相关性分析后发现，g.2555812 T>C和g.2555377 T>C位点的CC基因型个体背部皮肤毛囊密度显著高于其他基因型个体背部（P<0.05）。3）实时荧光定量PCR结果显示，Wnt3a基因在原种和B系鸡的3个皮肤部位的毛囊组织上都有比较高的表达量，且在原种背部的表达量显著高于在B系背部的表达量（P<0.05）。综上所述，崇仁麻鸡原种皮肤毛孔相较于B系更加细密，Wnt3a基因g.2555812 T>C位点的CC基因型和g.2555377 T>C位点的CC基因型是崇仁麻鸡皮肤毛囊的有利基因型，可选作鸡皮肤毛囊密度性状的分子标记。本研究探讨了作为毛囊生长发育的候选基因Wnt3a的遗传效应及SNPs与皮肤毛囊性状的相关性，以期为优质鸡胴体包装性状的分子标记筛选与标记辅助选择提供参考依据。

关键词: 崇仁麻鸡, 毛囊性状, Wnt3a基因, SNP位点, 荧光定量

Abstract: The aim of this study was to analyze the correlation between genetic variant loci of Wnt family member 3a (Wnt3a) gene and skin hair follicle density in Chongren partridge chicken based on gene polymorphism.Six hundred Chongren partridge chickens, 200 protospecies hens and 100 roosters and 200 B strain hens and 100 roosters, reared under the same environment and the same management level until date of listing (120 d), were randomly selected in good health and condition. After slaughter, hair follicle density was measured at the same locations on the skin of the back, chest and thighs, the morphological structure of hair follicles was observed by HE staining of skin tissue sections, and the diameter of hair follicles and skin thickness were measured. The genetic variant loci of Wnt3a gene were screened by PCR amplification followed by direct sequencing, and their correlation with the hair follicle density of chicken skin was analyzed. The expression of Wnt3a gene in different skin sites was detected by real-time fluorescence quantitative PCR technique. The results showed that:1) Comparing the same sex and skin area of protospecies and B strain, the hair follicle density on the back of protospecies hens was extremely significantly higher than that of B strain (P<0.01), the hair follicle density on the thighs was significantly higher than that of B strain (P<0.05), and the hair follicle diameter on the back and thighs was significantly lower than that of B strain (P<0.05). The density of skin follicles on the back of the protospecies cock was highly significant higher than that of B strain (P<0.01), the density of hair follicles on the chest was highly significant lower than that of B strain (P<0.01), and the diameter of hair follicles on the back and thighs was significantly lower than that of B strain (P<0.05).The thickness of the skin on the chest of males in B strain was signifi-cantly higher than that of protospecies (P<0.05), and the thickness of the skin on the thighs of both males and females in B strain was significantly higher than that of protospecies (P<0.05). 2) Three SNPs loci were screened for the Wnt3a gene and their correlation with the skin follicle density at each site was analyzed. g.2555812 T>C and g.2555377 T>C loci were found to have significantly higher skin follicle density on the back of individuals with CC genotype than that of individuals with other genotypes (P<0.05). 3) Real-time fluorescence quantitative PCR results showed that the Wnt3a gene in hair follicle tissues of all 3 skin sites of the protospecies and the B strain had relatively high expression, and the expression in the dorsal part of the protospecies was significantly higher than that in the dorsal part of the B strain (P<0.05). In conclusion, the skin pores of protospecies of Chongren partridge chicken were finer compared to the B strain, and the CC genotype of the Wnt3a gene at the g.2555812 T>C locus and the CC genotype at the g.2555377 T>C locus were favorable genotypes for the skin follicles of Chongren partridge chicken and could be used as molecular markers for the chicken skin follicle density trait. In this study, we investigated the genetic effects of Wnt3a as a candidate gene for hair follicle growth and develop-ment and the correlation between SNPs and skin hair follicle traits, in order to provide a reference basis for molecular marker screening and marker-assisted selection of quality chicken carcass packaging traits.

Key words: Chongren partridge chicken, hair follicle traits, Wnt3a gene, SNP loci, fluorescence quantification

中图分类号:

S831.2

陈春, 康昭风, 魏岳, 黎观红, 武艳平, 谢金防. Wnt3a基因多态性与崇仁麻鸡皮肤毛囊性状相关性研究[J]. 畜牧兽医学报, 2023, 54(7): 2810-2823.

CHEN Chun, KANG Zhaofeng, WEI Yue, LI Guanhong, WU Yanping, XIE Jinfang. Correlation of Wnt3a Gene Polymorphism with Skin Follicle Traits in Chongren Partridge Chicken[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2810-2823.

参考文献 45

[1]	周勇, 李知新, 鲁宏伟, 等.我国H5和H7N9亚型高致病性禽流感的监测及疫情暴发分析[J].畜牧兽医学报, 2022, 53(9):3093-3106.ZHOU Y, LI Z X, LU H W, et al.Surveillance and outbreak analysis of H5 and H7N9 subtypes of highly pathogenic avian influenza in China[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(9):3093-3106.(in Chinese)
[2]	ZENG H, DE REU K, GABRIËL S, et al.Salmonella prevalence and persistence in industrialized poultry slaughterhouses[J]. Poult Sci, 2021, 100(4):100991.
[3]	刘国信.转变消费观念推广"冰鲜鸡"是大势所趋[J].养禽与禽病防治, 2016(11):34-36.LIU G X.Change the concept of consumption, the promotion of "chilled chicken" is the trend[J].Poultry Husbandry and Disease Control, 2016(11):34-36.(in Chinese)
[4]	WANG X S, TREDGET E E, WU Y J.Dynamic signals for hair follicle development and regeneration[J].Stem Cells Dev, 2012, 21(1):7-18.
[5]	LEE J, RABBANI C C, GAO H Y, et al.Hair-bearing human skin generated entirely from pluripotent stem cells[J].Nature, 2020, 582(7812):399-404.
[6]	LI G, TANG X L, ZHANG S P, et al.SIRT7 activates quiescent hair follicle stem cells to ensure hair growth in mice[J].EMBO J, 2020, 39(18):e104365.
[7]	XIE W Y, CHEN M J, JIANG S G, et al.Investigation of feather follicle morphogenesis and the expression of the Wnt/β-catenin signaling pathway in Yellow-feathered broiler chick embryos[J].Br Poult Sci, 2020, 61(5):557-565.
[8]	WU P, JIANG T X, LEI M X, et al.Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling[J].Development, 2021, 148(18):dev198671.
[9]	GEYFMAN M, PLIKUS M V, TREFFEISEN E, et al.Resting no more:re-defining telogen, the maintenance stage of the hair growth cycle[J].Biol Rev Camb Philos Soc, 2015, 90(4):1179-1196.
[10]	STENN K S, PAUS R.Controls of hair follicle cycling[J].Physiol Rev, 2001, 81(1):449-494.
[11]	JI S F, ZHU Z Y, SUN X Y, et al.Functional hair follicle regeneration:an updated review[J].Signal Transduct Target Ther, 2021, 6(1):66.
[12]	HARDY M H.The secret life of the hair follicle[J].Trends Genet, 1992, 8(2):55-61.
[13]	SHANG F Z, WANG Y, MA R, et al.Screening of microRNA and mRNA related to secondary hair follicle morphogenesis and development and functional analysis in cashmere goats[J].Funct Integr Genomics, 2022, 22(5):835-848.
[14]	KIRIKOSHI H, SEKIHARA H, KATOH M.Expression of WNT14 and WNT14B mRNAs in human cancer, up-regulation of WNT14 by IFNγ and up-regulation of WNT14B by β-estradiol[J].Int J Oncol, 2001, 19(6):1221-1225.
[15]	GUO H Y, YANG K, DENG F, et al.Wnt3a inhibits proliferation but promotes melanogenesis of melan-a cells[J].Int J Mol Med, 2012, 30(3):636-642.
[16]	GUO H Y, XING Y Z, LIU Y X, et al.Wnt/β-catenin signaling pathway activates melanocyte stem cells in vitro and in vivo[J].J Dermatol Sci, 2016, 83(1):45-51.
[17]	TANG M S, WANG T, ZHANG X F.A review of SNP heritability estimation methods[J].Brief Bioinform, 2022, 23(3):bbac067.
[18]	束婧婷, 姬改革, 屠云洁, 等.鸡Wnt3a基因组织表达差异与SNPs对鸡毛囊密度性状的遗传效应[J].中国畜牧兽医, 2021, 48(5):1672-1680.SHU J T, JI G G, TU Y J, et al.Tissue expression analysis of Wnt3a gene and genetic effects of SNPs on feather follicle density in chicken[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(5):1672-1680.(in Chinese)
[19]	吴静文.鸡皮肤黄度的遗传规律及相关分子标记的鉴定[D].广州:华南农业大学, 2020.WU J W.Characterization of chicken skin yellowness and ldentification of related molecular markers[D].Guangzhou:South China Agricultural University, 2020.(in Chinese)
[20]	YANG X, XIAN Y R, LI Z H, et al.G0S2 gene polymorphism and its relationship with carcass traits in chicken[J].Animals (Basel), 2022, 12(7):916.
[21]	SUN G R, LI M, LI H, et al.Molecular cloning and SNP association analysis of chicken PMCH gene[J].Mol Biol Rep, 2013, 40(8):5049-5055.
[22]	赵振华, 黎寿丰, 黄华云, 等.优质肉鸡胴体外观性状遗传力及相关性分析[J].四川农业大学学报, 2015, 33(1):89-92.ZHAO Z H, LI S F, HUANG H Y, et al.Estimates of heritability and correlations for the carcass appearance performance of high-quailty broiler chickens[J].Journal of Sichuan Agricultural University, 2015, 33(1):89-92.(in Chinese)
[23]	SCHNEIDER M R, SCHMIDT-ULLRICH R, PAUS R.The hair follicle as a dynamic miniorgan[J].Curr Biol, 2009, 19(3):R132-R142.
[24]	YU M K, WU P, WIDELITZ R B, et al.The morphogenesis of feathers[J].Nature, 2002, 420(6913):308-312.
[25]	姬改革, 束婧婷, 单艳菊, 等.鸡皮肤毛囊性状研究进展[J].中国家禽, 2019, 41(10):46-49.JI G G, SHU J T, SHAN Y J, et al.Research progress of skin hair follicle traits in chicken[J].China Poultry, 2019, 41(10):46-49.(in Chinese)
[26]	RISHIKAYSH P, DEV K, DIAZ D, et al.Signaling involved in hair follicle morphogenesis and development[J].Int J Mol Sci, 2014, 15(1):1647-1670.
[27]	SONG Y P, LIU C, ZHOU Y X, et al.Regulation of feather follicle development and Msx2 gene SNP degradation in Hungarian white goose[J].BMC Genomics, 2022, 23(1):821.
[28]	JENNI L, GANZ K, MILANESI P, et al.Determinants and constraints of feather growth[J].PLoS One, 2020, 15(4):e0231925.
[29]	RIM E Y, CLEVERS H, NUSSE R.The Wnt pathway:from signaling mechanisms to synthetic modulators[J].Annu Rev Biochem, 2022, 91:571-598.
[30]	CHATTERJEE A, PAUL S, BISHT B, et al.Advances in targeting the WNT/β-catenin signaling pathway in cancer[J].Drug Discov Today, 2022, 27(1):82-101.
[31]	SICK S, REINKER S, TIMMER J, et al.WNT and DKK determine hair follicle spacing through a reaction-diffusion mechanism[J].Science, 2006, 314(5804):1447-1450.
[32]	李瑶, 孙远, 鲍忠省, 等.Wnt5a基因在鹅皮肤中的定量定位表达特性[J].吉林农业大学学报, 2014, 36(4):475-478, 493.LI Y, SUN Y, BAO Z S, et al.Characteristics of quantitative and positioning expression of Wnt5a Gene in Goose Skin[J].Journal of Jilin Agricultural University, 2014, 36(4):475-478, 493.(in Chinese)
[33]	XING Y Z, MA X G, GUO H Y, et al.Wnt5a Suppresses β-catenin Signaling during Hair Follicle Regeneration[J].Int J Med Sci, 2016, 13(8):603-610.
[34]	KANDYBA E, KOBIELAK K.Wnt7b is an important intrinsic regulator of hair follicle stem cell homeostasis and hair follicle cycling[J].Stem Cells, 2014, 32(4):886-901.
[35]	TAO Y F, ZHOU X L, LIU Z W, et al.Expression patterns of three JAK-STAT pathway genes in feather follicle development during chicken embryogenesis[J].Gene Expr Patterns, 2020, 35:119078.
[36]	XIE W Y, CHEN M J, JIANG S G, et al.The Wnt/β-catenin signaling pathway is involved in regulating feather growth of embryonic chicks[J].Poult Sci, 2020, 99(5):2315-2323.
[37]	HU X W, ZHANG X K, LIU Z W, et al.Exploration of key regulators driving primary feather follicle induction in goose skin[J].Gene, 2020, 731:144338.
[38]	ASADOLLAHPOUR NANAEI H, KHARRATI-KOOPAEE H, ESMAILIZADEH A.Genetic diversity and signatures of selection for heat tolerance and immune response in Iranian native chickens[J].BMC Genomics, 2022, 23(1):224.
[39]	ZHUANG Z X, CHEN S E, CHEN C F, et al.Genomic regions and pathways associated with thermotolerance in layer-type strain Taiwan indigenous chickens[J].J Therm Biol, 2020, 88:102486.
[40]	GILLEN S L, WALDRON J A, BUSHELL M.Codon optimality in cancer[J].Oncogene, 2021, 40(45):6309-6320.
[41]	BAILEY S F, MORALES L A A, KASSEN R.Effects of synonymous mutations beyond codon bias:the evidence for adaptive synonymous substitutions from microbial evolution experiments[J].Genome Biol Evol, 2021, 13(9):evab141.
[42]	PARVATHY S T, UDAYASURIYAN V, BHADANA V.Codon usage bias[J].Mol Biol Rep, 2022, 49(1):539-565.
[43]	CALLENS M, PRADIER L, FINNEGAN M, et al.Read between the Lines:diversity of nontranslational selection pressures on local codon usage[J].Genome Biol Evol, 2021, 13(9):evab097.
[44]	PANARO M A, CALVELLO R, MINIERO D V, et al.Imaging intron evolution[J].Methods Protoc, 2022, 5(4):53.
[45]	刘继强, 郝晓东, 武丽娜, 等.全基因组SNP分型技术在畜禽遗传育种研究中的应用[J].畜牧兽医学报, 2022, 53(12):4123-4137.LIU J Q, HAO X D, WU L N, et al.Application of whole genome SNP genotyping technology in livestock and poultry genetics and breeding[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(12):4123-4137.(in Chinese)