哈萨克羊iNOS基因多态性与布鲁氏菌病的相关性分析

doi:10.16431/j.cnki.1671-7236.2021.03.035

摘要/Abstract

摘要： 试验旨在探讨哈萨克羊诱导型一氧化氮合酶（iNOS）基因多态性与布鲁氏菌病的相关性。使用虎红平板凝集试验（RBPT）方法对231只哈萨克羊血清进行布鲁氏菌病血清学检测，参考GenBank中绵羊iNOS基因序列，针对其第6、7、8外显子及其邻近内含子片段设计引物，利用PCR-SSCP技术和DNA测序技术对231只哈萨克羊的iNOS基因进行多态性检测，分析其SNPs与哈萨克羊布鲁氏菌病易感性的相关性。结果表明，67只哈萨克羊为布鲁氏菌感染阳性，阳性检出率为29.00%。在哈萨克羊iNOS基因的外显子6和8片段上未检测到多态位点，在外显子7片段上检测出F7-T18054C和F7-C18084T 2个多态位点，在F7-T18054C多态位点上检测到3种基因型（TC、TT、CC），优势等位基因和基因型分别是C型和CT型，其等位基因频率和基因型频率分别是0.660和0.446。在F7-C18084T多态位点上检测到2种基因型（CT、CC），优势等位基因频率和基因型分别是C和CC型，其等位基因和基因型频率分别是0.946和0.892。F7-C18084T属于低度多态（PIC<0.25），F7-T18054C属于中度多态（0.25 < PIC < 0.5）。相关性分析表明，F7-T18054C和F7-C18084T多态位点与布鲁氏菌病易感性无显著相关性（P>0.05）。试验结果表明，哈萨克羊iNOS基因F7-T18054C和F7-C18084T多态位点与布鲁氏菌病易感性不存在相关性。

关键词: iNOS基因; 哈萨克羊; PCR-SSCP; SNPs; 布鲁氏菌病

Abstract: The purpose of this experiment was to explore the correlation between polymorphism of inducible nitric oxide synthase (iNOS) gene and brucellosis in Kazakh sheep.The serum of 231 Kazakh sheep were tested for serology by brucellosis using RBPT method.Refer to the sheep iNOS gene sequence in GenBank,design primers for its exons 6,7,8,PCR-SSCP and DNA sequencing technology were used to detect the polymorphism of iNOS gene in 231 Kazakh sheep,and analyze the correlation between their SNPs and the susceptibility to brucellosis.The detection results showed that 67 Kazakh sheep were positive for Brucella infection,and the positive detection rate was 29.00%.No polymorphic sites were detected on the exons 6 and 8 fragments of iNOS gene in Kazakh sheep,F7-T18054C and F7-C18084T were detected on the exon 7 fragment,and three genotypes (TC,TT and CC) were detected on the F7-T18054C polymorphic site,the dominant allele and genotype were C and CT genotype,and the allele frequency and genotype frequency were 0.660 and 0.446,respectively.Two genotypes (CT and CC) were detected on the F7-C18084T polymorphic site.The dominant allele and genotype were C and CC,the allele frequency and genotype frequency were 0.946 and 0.892,respectively.F7-C18084T belonged to low polymorphism (PIC<0.25),F7-T18054C belonged to moderate polymorphism (0.25 < PIC < 0.5).The correlation analysis results showed that the correlation between F7-T18054C and F7-C18084T polymorphic sites and brucellosis susceptibility was not significant (P>0.05).The results showed that F7-T18054C and F7-C18084T of iNOS in Kazakh sheep didn't correlate with the susceptibility to brucellosis.

Key words: iNOS gene; Kazakh sheep; PCR-SSCP; SNPs; brucellosis

中图分类号:

S855.1⁺2

张凡, 徐杰, 李刚, 周永顺, 何营, 韩凯, 陈福龙, 高剑峰. 哈萨克羊iNOS基因多态性与布鲁氏菌病的相关性分析[J]. 中国畜牧兽医, 2021, 48(3): 1102-1111.

ZHANG Fan, XU Jie, LI Gang, ZHOU Yongshun, HE Ying, HAN Kai, CHEN Fulong, GAO Jianfeng. Correlation Analysis of iNOS Gene Polymorphism and Brucellosis in Kazakh Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 1102-1111.

参考文献

[1] SANDOUGHDARAN S,SADEGHIPOUR H,SADEGHIPOUR H R.Effect of acute lithium administration on penile erection:Involvement of nitric oxide system[J].International Journal of Reproductive Biomedicine,2016,14(2):109-116.
[2] UMAR S,LAARSE A.Nitric oxide and nitric oxide synthase isoforms in the normal,hypertrophic,and failing heart[J].Molecular & Cellular Biochemistry,2010,333(1-2):191-201.
[3] BALLIGAND J L,FERON O,DESSY C.eNOS activation by physical forces:From short-term regulation of contraction to chronic remodeling of cardiovascular tissues[J].Physiological Reviews,2009,89(2):481-534.
[4] KUMAR A,SINGH K P,BALI P,et al.iNOS polymorphism modulates iNOS/NO expression via impaired antioxidant and ROS content in P. vivax and P. falciparum infection[J].Redox Biology,2018,15:192-206.
[5] JOHANNESEN J,ERIKSEN V,ANDERSEN M L M,et al.The role of an iNOS polymorphism at the post-diagnosis diabetes development in children with type 1 diabetes[J].Journal of Endocrinology and Diabetes Research,2016,2(2):18-27.
[6] NEGI V S,MARIASELlVAM C M,MISRA D P,et al.Polymorphisms in the promoter region of iNOS predispose to rheumatoid arthritis in South Indian Tamils[J].International Journal of Immunogenetics,2017,44(3):114-121.
[7] NAGAREDDY P R,MCNEILL J H,MACLEOD K M.Chronic inhibition of inducible nitric oxide synthase ameliorates cardiovascular abnormalities in streptozo-tocin diabetic rats[J].European Journal of Pharmaco-logy,2009,611(1-3):53-59.
[8] TUPITSYNA T V,BONDARENKO E A,BOTSINA A Y.The role of polymorphic variants of gene of inducible NO-synthase NOS2 in brain infarction in patients with acute ischemic stroke[J].Molekuliarnaia Genetika Mikrobiologiia I Virusologiia,2010,3:3-7.
[9] ZHAI Z,WANG Z,WANG L,et al.Relationship between inducible NOS single-nucleotide polymor-phisms and hypertension in Han Chinese[J].Herz,2018,43(5):461-465.
[10] 王光辉,陈楠,王虎霞,等.iNOS在不同乳腺病理组织中的表达水平及其与肿瘤临床预后的相关性[J].现代肿瘤医学,2018,26(1):52-56. WANG G H,CHEN N,WANG H X,Expression and clinical significance of iNOS in breast cancer[J].Journal of Modern Oncology,2018,26(1):52-56.(in Chinese)
[11] JOHANNESEN J,PIE A,POCIOT F,et al.Linkage of the human inducible nitric oxide synthase gene to type 1 diabetes[J].The Journal of Clinical Endocrino-logy and Metabolism,2001,86(6):2792-2796.
[12] 朱瑾英,张慧娟,姜晓艳,等.诱导型一氧化氮合酶基因多态性与2型糖尿病关系研究[J].现代生物医学进展,2011,22:141-142. ZHU J Y,ZHANG H J,JIANG X Y,et al.Correlation between polymorphism of iNOS gene and type 2 diabetes[J].Advances in Modern Biomedicine,2011,22:141-142.(in Chinese)
[13] NEGI V S,MARIASELVAM C M,MISRA D P,et al.Polymorphisms in the promoter region of iNOS predispose to rheumatoid arthritis in South Indian Tamils[J].International Journal of Immuno-genetics,2017,44(3):114-121.
[14] BLANC-GRUYELLE A L,LEMAIRE X,GUAGUERE A,et al.An atypical presentation of human brucellosis[J].Medecineet Maladies Infectieuses,2017,47(2):164.
[15] VOLLMAR P,ZANGE S,ZOELLER L,et al.Brucellosis overview and current significance[J].Deutsche Medizinische Wochenschrift,2016,141(14):1014-1416.
[16] 贾怀妙,赵效国,师茂林,等.2005-2016年第八师石河子市人间布鲁氏菌病流行病学特征分析及对策探讨[J].疾病监测与控制.2017,11(9):714-716. JIA H M,ZHAO X G,SHI M L,et al.Epidemiological characteristics of human brucellosis in Shihezi city,8th division,2005-2016[J].Disease Surveillance and Control,2017,11(9):714-716.(in Chinese)
[17] 随海田,安允萍,克依赛尔·阿布都克尤木,等.新疆喀什地区2005-2016年布鲁氏菌病流行病学特征分析[J].中国人兽共患病学报,2018,34(1):18-22. SUI H T,AN Y P,KEYISAIER A,et al.Brucellosis epidemiological characteristics in Kashgar,Xinjiang,China,2005-2016[J].Chinese Journal of Zoonoses,2018,34(1):18-22.(in Chinese)
[18] 徐杰,李刚,张凡,等.哈萨克羊iNOS基因多态性与布鲁菌易感性的关系[J].西北农林科技大学学报(自然科学版), 2021, 49(1):17-24. XU J,LI G,ZHANG F,et al.Relationship between iNOS gene polymorphism and Brucella susceptibility in Kazak sheep[J].Journal of Northwest A&F University (Natural Science Edition),2021,49(1):17-24.(in Chinese)
[19] 冯华.改良试管凝集试验和虎红平板凝集试验检测羊布鲁氏菌病结果比较[J].中国草食动物科学,2017,37(4):69-70. FENG H.Comparison of the results of improved tube agglutination test and tiger red plate agglutination test for brucellosis in sheep[J].China Herbivore Science,2017,37(4):69-70.(in Chinese)
[20] 王会敏.中国美利奴羊TLR4、TLR2基因多态性及其与布鲁氏菌病的相关性分析[D].石河子:石河子大学,2017. WANG H M.Analysis of correlation between the polymorphisms of TLR4 and TLR2 genes with brucellosis in Chinese Merino sheep[D].Shihezi:Shihezi University,2017.(in Chinese)
[21] 魏丽君.四个绵羊品种MHC-DRB3基因外显子2的多态性分析[J].中国畜牧兽医,2007,34(7):63-66. WEI L J.Polymorphisms of exon 2 of MHC-DRB3 gene in four sheep breeds[J].China Animal Husbandry & Veterinary Medicine,2007,34(7):63-66.(in Chinese)
[22] 陈月娥,苟亚峰,周路,等.中国美利奴羊MHC-DRB1基因exon 2 SNPs及其与布鲁氏菌病易感性相关性[J].中国畜牧杂志,2014,50(1):15-20. CHEN Y E,GOU Y F,ZHOU L,et al.The SNPs of Chinese Merino sheep MHC-DRB1 gene exon 2 and its association with brucellosis susceptibility[J].Chinese Journal of Animal Husbandry,2014,50(1):15-20.(in Chinese)
[23] SHIN H J,PARK Y H,KIM S U,et al.Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase[J].Journal of Biological Chemistry,2011,286(34):29872-29881.
[24] 韩京军,张艳炜,伍德强,等.诱导型一氧化氮合酶基因多态性与冠心病的关联性研究[J].中国心血管杂志,2018,23(1):31-35. HAN J J,ZHANG Y W,WU D Q,et al.Correlation between inducible nitric oxide synthase (iNOS) gene polymorphisms and coronary heart disesse in Chinese[J].Chinese Journal of Cardiovascular Medicine,2018,23(1):31-35.(in Chinese)
[25] 王元元,严国,罗成,等.哈萨克羊DRB1基因外显子3多态性及生物信息学分析[J].中国畜牧兽医,2018,45(3):721-729. WANG Y Y,YAN G,LUO C,et al.Polymorphism and bioinformatics analysis of DRB1 gene exon 3 in Kazakh sheep[J].China Animal Husbandry & Veterinary Medicine,2018,45(3):721-729.(in Chinese)
[26] 邢翀.RNA二级结构预测算法的研究[D].长春:吉林大学,2012. XING C.Research on RNA secondary structure prediction algorithms[D].Changchun:Jilin University,2012.(in Chinese)
[27] 毛颖颖,汪洪波,刘海平.iNOS基因rs10459953/rs1060822多态性与低氧训练效果的关联性研究[J].山东体育学院学报,2014,30(1):85-89. MAO Y Y,WANG H B,LIU H P.Association between polymorphisms rs10459953/rs1060822 gene and variation in response to hypoxia training[J].Journal of Shandong Institute of Physical Education and Sports,2014,30(1):85-89.(in Chinese)