绵羊KAP11.1基因克隆、原核表达及皮肤毛囊表达研究

doi:10.16431/j.cnki.1671-7236.2022.03.009

Abstract

Abstract: 【Objective】 The aim of this study was to clone and prokaryotic express the keratin associated protein 11.1(KAP11.1) gene of hair follicles, compare the expression of KAP11.1 gene in skin follicles of different sheep breeds, and explore the expression difference of KAP11.1 gene in local sheep breeds in Southern Xinjiang and its effect on wool quality.【Method】 The skin follicles on the body side of plain-type Hetian sheep, mountain-type Hetian sheep and Karakul sheep were used as the research materials, and the sequence of KAP11.1 gene in sheep (accession No.:HQ595347.1) in GenBank was used as the reference to design primers.PCR amplification of KAP11.1 gene was carried out to construct pMD19-T-KAP11.1 clone plasmid, and pET-28a(+)-KAP11.1 prokaryotic recombinant expression plasmid was constructed after double-enzyme digestion identification.Sequence analysis was carried out after PCR and double-enzyme digestion identification.The recombinant plasmid was transformed into E.coli BL21(DE3).SDS-PAGE and Western blotting were used for detection.The expression of KAP11.1 gene in skin follicles of different sheep was detected by Real-time quantitative PCR.【Result】 The CDS sequence of KAP11.1 gene in 3 sheep breeds was 480 bp, encoding 159 amino acids, which was an unstable hydrophobic protein.The similarity alignment results showed that compared with the reference gene, the two types of Hetian sheep was 99.79%, and mutations occurred at 423 bp, from C to T.The sequence homology of Karakul sheep was 99.38%, from G to T at 69 bp, from C to T at 93 bp, and from C to T at 423 bp.Phylogenetic tree analysis showed that 3 sheep breeds had the closest genetic relationship with Capra hircus, and the farthest genetic relationship with Bos indicus.The secondary structure of KAP11.1 was mainly composed of random coil.The prokaryotic recombinant expression plasmid pET-28a(+)-KAP11.1 was successfully constructed, and KAP11.1 protein (19 ku) was purified.The expression of KAP11.1 gene in skin follicles of mountain-type Hetian sheep and Karakul sheep were significantly higher than that of plain-type Hetian sheep (P<0.05), and there was no significant difference between mountain-type Hetian sheep and Karakul sheep (P>0.05).【Conclusion】 The 480 bp CDS sequence of KAP11.1 gene in sheep was cloned, the prokaryotic recombinant expression plasmid pET-28a(+)-KAP11.1 was successfully constructed, 19 ku KAP11.1 protein was obtained, and KAP11.1 gene was expressed in skin follicles of 3 sheep breeds.

Key words: Hetian sheep; Karakul sheep; KAP11.1 gene; cloning; prokaryotic expression; skin follicles

CLC Number:

GONG Shujuan, SHI Ruijun, WU Tinghui, LI Shuwei. Cloning, Prokaryotic Expression and Skin Follicles Expression of KAP11.1 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 876-886.

References

[1] GONG H, ZHOU H, FORREST R, et al.Wool keratin-associated protein genes in sheep——A review[J].Genes, 2016, 7(6):24.
[2] 晏华春, 马晓燕, 依明·苏来曼, 等.和田羊KRT31基因遗传多态性相关分析[J].中国草食动物科学, 2015, 35(5):8-10. YAN H C, MA X Y, YIMING S L M, et al.Polymorphism of KRT31 gene and its correlation with the wool fineness in Hetian sheep[J].China Herbivore Science, 2015, 35(5):8-10.(in Chinese)
[3] 郭欣芸.羊毛纤维品质对地毯性能的影响[D].乌鲁木齐:新疆大学, 2017. GUO X Y.The influence of wool fiber quality on carpet perfermance[D].Urumqi:Xinjiang University, 2017.(in Chinese)
[4] 李树伟, 任述强, 杨飞, 等.新疆4种绵羊羊毛氨基酸含量测定[J].中国畜牧兽医, 2011, 38(11):32-37. LI S W, REN S Q, YANG F, et al.Wool amino acid assaying of local variety sheep in Xinjiang[J].China Animal Husbandry & Veterinary Medicine, 2011, 38(11):32-37.(in Chinese)
[5] 李莲瑞, 陈根元, 王晓斌, 等.新疆卡拉库尔羊的研究现状及前景[J].中国草食动物, 2008, 2:63-64. LI L R, CHEN G Y, WANG X B, et al.Research status and prospect of Xinjiang Karakul sheep[J].China Herbivore Science, 2008, 2:63-64.(in Chinese)
[6] POPESCU C, HOCKER H.Hair——The most sophisticated biological composite material[J].Chemical Society Reviews, 2007, 36(8):1282-1291.
[7] GONG H, ZHOU H, HICKFORD J G.Polymorphism of the ovine keratin-associated protein 1-4 gene (KRTAP1-4)[J].Molecular Biology Reports, 2010, 37(7):3377-3380.
[8] STRNAD P, USACHOV V, DEBES C, et al.Unique amino acid signatures that are evolutionarily conserved distinguish simple-type, epidermal and hair keratins[J].Journal of Cell Science, 2011, 124(Pt 24):4221-4232.
[9] 李志刚, 尹阔, 李树伟.山区型和田羊毛囊HGT蛋白KAP6.1、KAP7与KAP8的基因克隆分析[J].畜牧与兽医, 2016, 48(12):42-46. LI Z G, YIN K, LI S W.Gene cloning analysis for follicle high glycine/tyrosine proteins of KAP6.1, KAP7 and KAP8 in the mountain-type Hetain sheep[J].Animal Husbandry & Veterinary Medicine, 2016, 48(12):42-46.(in Chinese)
[10] 余路菲, 赵军, 李树伟.山区型和田羊毛囊KAP1.1基因表达载体构建及生物信息学分析[J].塔里木大学学报, 2020, 32(3):21-28. YU L F, ZHAO J, LI S W.Construction and bioinformatics analysis of KAP1.1 gene expression vector in wool follicles of mountain-type Hetian sheep[J].Journal of Tarim University, 2020, 32(3):21-28.(in Chinese)
[11] 武振辉, 张亮, 李树伟.山区型和田羊毛囊KIF2.9基因的克隆与生物信息学分析[J].黑龙江畜牧兽医, 2020, 3:6-11. WU Z H, ZHANG L, LI S W.Cloning and bioinformatics analysis of hair follicle KIF2.9 gene of mountain-type Hetian sheep[J].Heilongjiang Animal Science and Veterinary Medicine, 2020, 3:6-11.(in Chinese)
[12] 金梅, 王瑞, 王婧, 等.HGT KAPs基因家族在辽宁绒山羊皮肤毛囊中的表达研究[J].中国畜牧杂志, 2017, 53(2):40-46. JIN M, WANG R, WANG J, et al.The expression of glycine/tyrosin-rich keratin-associated proteins genes in hair follicle of Liaoning cashmere goat[J].Chinese Journal of Animal Science, 2017, 53(2):40-46.(in Chinese)
[13] SULAYMAN A, TURSUN M, SULAIMAN Y, et al.Association analysis of polymorphisms in six keratin genes with wool traits in sheep[J].Asian-Australasian Journal of Animal Sciences, 2018, 31(6):775-783.
[14] SHANAZ S, GANAI T A S, AYAZ A, et al.Genetic polymorphism of keratin intermediate filament-1 gene in Changthangi goats and its effect on fibre quality[J].Indian Journal of Small Ruminants, 2018, 24(2):209.
[15] ROGERS M A, LANGBEIN L, WINTER H, et al.Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein (KAP) genes on chromosome 21q22.1.[J].The Journal of Biological Chemistry, 2002, 277(50):48993-49002.
[16] RECHICHE O, PLOWMAN J E, HARLAND D P, et al.Expression and purification of high sulfur and high glycine-tyrosine keratin-associated proteins (KAPs) for biochemical and biophysical characterization[J].Protein Expression and Purification, 2018, 146:34-44.
[17] 朱建勋, 王继卿, 胡江, 等.五个绵羊群体KAP11-1基因核苷酸序列变异分析[J].华北农学报, 2016, 31(5):101-107. ZHU J X, WANG J Q, HU J, et al.Nucleotide sequence variations of KAP11-1 gene in 5 Chinese sheep populations[J].Acta Agriculturae Boreali-Sinica, 2016, 31(5):101-107.(in Chinese)
[18] BARRY C P, JANE A, ANTONIETTA N.Characterization of a gene encoding a cysteine-rich keratin associated protein synthesized late in rabbit hair follicle differentiation[J].Differentiation, 1995, 58(3):227-232.
[19] JIN M, CAO Q, WANG R, et al.Molecular characterization and expression pattern of a novel keratin-associated protein 11.1 gene in the Liaoning cashmere goat (Capra hircus).[J].Asian-Australasian Journal of Animal Sciences, 2017, 30(3):328-337.
[20] 唐晓惠.藏绵羊毛囊不同发育时期miRNAs表达变化研究[D].武汉:华中农业大学, 2012. TANG X H.The study of expression of miRNAs in wool follicle during different development phases[D].Wuhan:Huazhong Agricultural University, 2012.(in Chinese)
[21] 苏波.绵羊毛囊皮质层特异表达基因KAP11.1上游调控序列研究[D].武汉:华中农业大学, 2017. SU B.characterization of upstream sequence regulating the wool follicle cortex specific expression of sheep KAP11.1 gene[D].Wuhan:Huazhong Agricultural University, 2017.(in Chinese)
[22] ARORA R, BHATIA S, SEHRAWAT A, et al.Genetic polymorphism of type 1 intermediate filament wool keratin gene in native Indian sheep breeds[J].Biochemical Genetics, 2008, 46(9-10):549-556.
[23] 李托, 李陇平.绒山羊主要经济性状相关基因研究进展[J].安徽农业科学, 2020, 48(4):15-17. LI T, LI L P.Research advances on related genes to main economic traits in cashmere goat[J].Journal of Anhui Agricultural Sciences, 2020, 48(4):15-17.(in Chinese)
[24] 车陇杰, 王继卿, ZHOU H T, 等.山羊KRTAP26-1基因鉴定及其遗传特征研究[J].基因组学与应用生物学, 2018, 37(2):710-717. CHE L J, WANG J Q, ZHOU H T, et al.Identification of caprine KRTAP26-1 gene and its genetic characteristics study[J].Genomics and Applied Biology, 2018, 37(2):710-717.(in Chinese)
[25] HUA G, HUITONG Z, JIQING W, et al.Characterisation of an ovine keratin associated protein (KAP) gene, which would produce a protein rich in glycine and tyrosine, but lacking in Cysteine[J].Genes, 2019, 10(11):848.
[26] AN X, LI T, CHEN N, et al.PGAM1 regulates the glycolytic metabolism of SCs in Tibetan sheep and its influence on the development of SCs[J].Gene, 2021, 804:145897.
[27] 李淑娟, 张超, 张彦妮, 等.毛果杨HDAC基因家族序列及其表达分析[J].西北农林科技大学学报(自然科学版), 2015, 43(3):63-76. LI S J, ZHANG C, ZHANG Y N, et al.Sequence and gene expression of histone deacetylases (HDAC) gene family of Populus trichocarpa[J].Journal of Northwest A&F University (Natural Science Edition), 2015, 43(3):63-76.(in Chinese)
[28] 张亭亭.KAP26.1、KAP11.1基因在辽宁新品系绒山羊皮肤中的表达及分析[D].大连:辽宁师范大学, 2013. ZHANG T T.Analysis the expression of KAP26.1, KAP11.1 gene in Liaoning new breed of cashmere goat skin[D].Dalian:Liaoning Normal University, 2013.(in Chinese)
[29] 梁玫岩.新疆南部地方品种绵羊KAP13.3和KRT17基因的表达分析[D].阿拉尔:塔里木大学, 2021. LIANG M Y.Expression analysis of KAP13.3 and KRT17 genes in Southern Xinjiang landrace sheep[D].Alar:Tarim University, 2021.(in Chinese)
[30] 王丽.平原型与山区型和田羊皮肤毛囊形态学观察及其KAPs、KIFs角蛋白基因的转录水平差异分析[D].阿拉尔:塔里木大学, 2018. WANG L.Wool follicle morphological analysis and their KAPs and KIFs gene signifiance difference on transcription level between plain-type and mountain-type Hetian sheep[D].Alar:Tarim University, 2018.(in Chinese)
[31] 宫淑娟, 铁鑫, 李树伟.舍饲条件下南疆地方品种绵羊的被毛品质研究[J].黑龙江畜牧兽医, 2021, 19:10-14. GONG S J, TIE X, LI S W.Study on the fur quality of Southern Xinjiang local breed sheep under stall-feeding condition[J].Heilongjiang Animal Science and Veterinary Medicine, 2021, 19:10-14.(in Chinese)

Cloning, Prokaryotic Expression and Skin Follicles Expression of KAP11.1 Gene in Sheep

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[3]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[4]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[5]	FAN Bingfeng, LI Wen, LIU Lixiang, ZHAO Xiangyuan, SHAO Jing, LIN Lefeng, SUN Huimin, ZHANG Xulin, XU Baozeng. Cloning and Bioinformatics Analysis of AANAT Gene in Mink [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1307-1318.
[6]	LIU Hongrun, ZHU Siran, FENG Lingli, ZHANG Kun, YAN Gang, WANG Yubin, ZHANG Shuai, JIANG Shan, XU Di, LAN Ganqiu, LIANG Jing. Cloning,Bioinformatics Analysis and Tissue Expression Localization of CDO1 Gene in Large White Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1352-1363.
[7]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[8]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[9]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[10]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[11]	ZHANG Yongjie, SUI Zhiyuan, ZHANG Zhishuai, WANG Chenguang, LI Xiaojun, XING Feng. Cloning and Bioinformatics Analysis of NPTX1 Gene and Its Tissues Expression at Puberty in Dolang Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1048-1058.
[12]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[13]	WEI Yirong, ZHENG Zihua, ZHANG Sanbao, SONG Ying, JIANG Haiyu, SUN Wenyue, CHENG Pengfei, LIU Yufan, ZOU Jianwei, HUANG Yanna, PAN Yan, JIANG Qinyang. Cloning,Bioinformatics Analysis and Tissue Expression of UCP1 Gene in Nubian Goats [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 440-450.
[14]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[15]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.