京海黄鸡白介素8基因5′调控区单核苷酸多态性及其与柔嫩艾美耳球虫抗性指标的关联性

王晓慧; 于海亮; 邹文斌; 糜长浩; 戴国俊; 张涛; 张跟喜; 谢恺舟; 王金玉; 施会强

doi:10.19303/j.issn.1008-0384.2019.11.004

京海黄鸡白介素8基因5′调控区单核苷酸多态性及其与柔嫩艾美耳球虫抗性指标的关联性

doi: 10.19303/j.issn.1008-0384.2019.11.004

1.
扬州大学动物科学与技术学院，江苏　扬州　225009
2.
江苏京海禽业集团有限公司，江苏　海门　226100

基金项目: 江苏现代农业产业技术体系建设专项（JATS[2018]303）；“十二五”国家科技支撑计划项目（2014BAD13B02）；江苏省高校优势学科建设工程（PAPD）；国家现代农业产业技术体系建设专项（CARS-41-G23）

详细信息

作者简介:
王晓慧（1994−），女，硕士，研究方向：家禽生产及抗病育种（E-mail：1216688368@qq.com）

通讯作者:
戴国俊（1963−），男，博士，教授，研究方向：家禽生产及抗病育种（E-mail：daigj@yzu.edu.cn）

中图分类号: S 831.2
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出版历程
- 收稿日期: 2019-06-21
- 修回日期: 2019-10-26
- 刊出日期: 2019-11-01

Correlation between Single Nucleotide Polymorphism in 5' Regulation Region of IL-8 and Coccidiosis-Resistance of Jinghai Yellow Chicken

1.
College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu　225009, China
2.
Jiangsu Jinghai Poultry Group Co., Ltd., Haimen, Jiangsu　226100, China

摘要

摘要: 目的研究白介素8（IL-8）基因5′调控区单核苷酸突变对鸡柔嫩艾美耳球虫（E. tenella）抗性指标的影响。方法本试验利用DNA直接测序技术，检测京海黄鸡IL-8基因5′调控区单核苷酸多态性（SNPs），并对5′调控区的SNPs突变前后的转录因子进行预测，然后分析SNPs与柔嫩艾美耳球虫抗性指标的关联性。结果测序结果表明：IL-8基因5′调控区共检测到3个突变位点（T-550C、G-398T和T-360C），均形成了3种基因型，杂合度在0.436~0.471，PIC值在0.25~0.50，均属于中度多态，且3个突变位点均处于哈代-温伯格平衡状态。生物信息学分析表明：3个突变位点均改变了其原有的转录因子结合位点。关联分析显示：T-550C突变位点的TC型个体的IL-8表达量与TT型差异显著，TC型GSH-PX、CAT、IL-2、IL-6、IFN-γ指标均高于其他2种基因型，但差异不显著。G-398T突变位点的TT型个体SOD活性和GT型个体CAT活性均显著高于GG型；TT型个体NO含量与GG型差异极显著，与GT型差异显著；TT型IL-2表达量显著高于GT型。T-360C突变位点的TT型和TC型个体SOD活性与CC型差异极显著或差异显著；TT型和TC型个体的NO含量均显著高于CC型；TT型个体的IL-2、IL-8表达量均显著高于CC型。结论 G-398T和T-360C突变位点的TT型个体球虫抗性优于其他基因型个体，T-550C突变位点的杂合型个体球虫抗性优于纯合型个体，表明IL-8基因5′调控区的突变对球虫抗性指标有显著的调控作用，可作为球虫病抗性鸡新品种或新品系的育种参考依据。
- 京海黄鸡 /
- 柔嫩艾美耳球虫 /
- 白介素8基因 /
- 5′调控区SNPs /
- 抗性指标
Abstract: Objective Correlation between the single nucleotide polymorphism (SNPs) in the 5' regulation region of IL-8 and the disease resistance to Eimeria tenella of chickens was studied. Method Direct DNA sequencing was performed to determine SNPs in IL-8 gene of Jinghai yellow chicken. Predicted transcription factors before and after mutation were analyzed to correlate between SNPs and coccidiosis-resistance of the birds. Result There were 3 SNPs at the mutation sites of T-550C, G-398T and T-360C detected in the target region that formed CT-, TT- and GG-genotypes with heterozygous degrees between 0.436 and 0.471 and PIC between 0.25 and 0.5. In a Hardy-Weinberg equilibrium state, SNPs were moderately polymorphic. A bioinformatics analysis confirmed that the original transcription factor binding sites in the genes had all been altered after the mutation. The expression of IL-8 of TC-genotype mutated at T-550C was significantly higher than that of TT-genotype. The coccidiosis-resistance indicators including GSH-PX, CAT, IL-2, IL-6 and IFN-γ in TC-genotype were higher than that in the other two genotypes, though not significantly. In genotypes mutated at G-398T, the SOD activity of TT-genotype and the CAT activity of GT-genotype were significantly higher than those of GG-genotype; the NO content of TT-genotype extremely significantly different from that of GG-genotype and significantly different from that of GT-genotype; and, the IL-2 expression of TT-genotype significantly higher than that of GT-genotype. Whereas, in the genotypes mutated at T-360C, the SOD activities of TT- and TC-genotypes were extremely significantly different or significantly different from that of CC-genotype; the NO contents of TT- and TC-genotypes significantly higher than that of the CC genotype; and, the IL-2 and IL-8 expressions of the TT genotype significantly higher than those of CC-genotypes. Conclusion It appeared that the TT-genotype with mutated G-398T or T-360C site would be more resistant to the parasitic attack by E. tenella than the other genotypes, and that hybrids of the genotypes carrying T-550C mutation would be more resistant than the homozygous counterparts. Thus, the significant regulation function of the polymorphism in the 5' regulation region of IL-8 gene could conceivably be targeted for breeding coccidiosis-resistant chickens.
- Jinghai yellow chicken /
- Eimeria tenella /
- Interleukin-8 gene /
- 5' regulatory region SNP /
- disease-resistance indicators

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图 1 IL-8基因位点位点峰图及序列比对

注：A：T-550C位点峰图比对；B：G-398T位点峰图比对；C：T-360C位点峰图比对；D：T-550C位点序列比对图；E：G-398T位点序列比对图；F：T-360C位点序列比对图

Figure 1. Peak and sequence alignments of IL-8

Note： A: T-550C peak alignment; B: G-398T peak alignment; C: T-360C peak alignment; D: T-550C sequence alignment; E: G-398T sequence alignment; F: T-360C sequence alignment.

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表 1 引物序列信息

Table 1. Information on primer sequence

引物 Primer	引物序列（5′→3′）Primers sequence(5′→3′)	退火温度 Annealing temperature/℃	长度 Length/bp
P1	F: TTCCATTCGCATAAGTCATC	51	638
	R: AAAGTTGATTTGGGGATACC
P2	F: TGTAATTGGGAATTCAAGGGGGA	58	708
	R: CCCATTTGGTGTGTGATAAGATGA
P3	F: AGTCCACAGACCACAAAGCA	58	693
	R: TCGCAATATAAGTTTCTGATGGCTT
P4	F: AAACCAGCAACACAAAGTC	60	574
	R: CATCTCAGCAAGTGCCAAG

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表 2 IL-8基因5′调控区单核苷酸突变位点信息

Table 2. Information on SNPs in 5′ regulation region of IL-8

序号 Site number	染色体位置 Chromosome position	序列号 Serial number	单核苷酸多态性 SNP
1	51282560	rs740065165	T-550C
2	51282712	rs731947764	G-398T
3	51282750	rs16409254	T-360C
注：A：T-550C位点峰图比对；B：G-398T位点峰图比对；C：T-360C位点峰图比对；D：T-550C位点序列比对图；E：G-398T位点序列比对图；F：T-360C位点序列比对图 Note： A: T-550C peak alignment; B: G-398T peak alignment; C: T-360C peak alignment; D: T-550C sequence alignment; E: G-398T sequence alignment; F: T-360C sequence alignment.

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表 3 IL-8基因5′调控区SNPs突变前后转录因子变化预测结果

Table 3. Predicted transcription factors before and after mutation of IL-8 in 5' regulation region

突变位点 Mutation site	碱基 Base	转录因子 Transcription factor	转录因子结合位点碱基序列 Transcription factor binding site base sequence	转录因子位置 Transcription factor position
−550 bp	T	Oct-1	GTTGCATTTG	−551~−542 bp
	C
−398 bp	G	C/EBPalp	GAAATAAATA	−398~−389 bp
	T	Pit-1a	TAAATAAATA	−398~−389 bp
		C/EBPalp	ACATAAATAA	−401~−392 bp
−360 bp	T	NF-1	AGCCAGTTAT	−362~−353 bp
	C
注：图中下划线标注为突变碱基。 Note: The underline in the figure is the mutant base.

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表 4 IL-8基因5′调控区多态性

Table 4. SNPs in 5′ regulation region of IL-8

突变位点 Mutation site	基因型 Genotype	数量 Number	基因型频率 Genotype frequency	等位基因 Allelic gene	等位基因频率 Allele frequency	χ²值 χ² value	P值 P value	杂合度 H	有效等位基因数 Ne	多态信息含量 PIC
T-550C	TT	40	0.435	T	0.679	1.390	0.500	0.436	1.772	0.341
	CC	7	0.076	C	0.321
	TC	45	0.489
G-398T	GG	13	0.141	G	0.380	0.023	0.990	0.471	1.892	0.360
	TT	35	0.381	T	0.720
	GT	44	0.478
T-360C	TT	40	0.435	T	0.674	0.725	0.699	0.440	1.784	0.343
	CC	8	0.087	C	0.326
	TC	44	0.478
注：PIC＞0.50为高度多态；0.25＜PIC＜0.50为中度多态；PIC＜0.25为低度多态^[15]。 Note: PIC＞0.50 is highly polymorphic; 0.25＜PIC＜0.50 is moderately polymorphic; PIC＜0.25 means low polymorphism^[15].

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表 5 IL-8基因T-550C突变位点各基因型与鸡球虫抗性指标关联分析

Table 5. Correlation between genotypes mutated at T-550C in IL-8 and coccidiosis-resistance indicators

指标 Indices	基因型 Genotype
指标 Indices	TT（40）	TC（45）	CC（7）
超氧化物歧化酶（SOD）/（U·mL⁻¹）	750.63±88.37	720.62±78.91	723.95±67.57
丙二醛（MDA）/（nmol·mL⁻¹）	3.66±0.40	3.74±1.26	4.16±1.03
谷胱甘肽过氧化物酶（GSH-PX）/（U·mL⁻¹）	151.77±16.94	152.22±17.53	150.83±22.52
过氧化氢酶（CAT）/（U·mL⁻¹）	0.87±0.17	1.01±0.16	0.95±0.19
一氧化氮（NO）/（μmol·L⁻¹）	52.35±5.25 A	49.62±3.79 AB	32.12±3.69 B
白介素1-β（IL-1β）/（ng·L⁻¹）	20.54±2.00	19.75±3.04	20.22±1.19
白介素2（IL-2）/（ng·L⁻¹）	14.78±2.66	15.82±2.04	15.18±2.69
白介素6（IL-6）/（ng·L⁻¹）	69.93±3.55	72.40±8.64	67.81±4.29
白介素8（IL-8）/（ng·L⁻¹）	90.87±7.63 b	119.11±10.42 a	109.06±11.76 ab
干扰素-γ（IFN-γ）/（ng·mL⁻¹）	189.47±15.25	201.21±13.67	195.58±16.06
注：同行数据后不同大写字母表示差异极显著（P＜0.01），不同小写字母表示差异显著（P＜0.05），相同字母或无字母表示差异不显著（P＞0.05），表6～7同。 Note: Different uppercase letters on a same row indicate significant differences (P<0.01); different lowercase letters, significant differences (P<0.05); and, same or no letter, no significant differences (P>0.05). Same for Tables 6–7.

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表 6 IL-8基因G-398T突变位点各基因型与鸡球虫抗性指标关联分析

Table 6. Correlation between genotypes mutated at G-398T in IL-8 and coccidiosis-resistance indicators

指标 Indices	基因型 Genotype
指标 Indices	GG（13）	GT（44）	TT（35）
超氧化物歧化酶（SOD）/（U·mL⁻¹）	631.43±50.73 b	667.21±27.41 ab	724.97±87.91 a
丙二醛（MDA）/（nmol·mL⁻¹）	3.66±0.40	3.63±0.39	3.19±0.73
谷胱甘肽过氧化物酶（GSH-PX）/（U·mL⁻¹）	164.04±21.41	155.20±20.63	158.58±19.50
过氧化氢酶（CAT）/（U·mL⁻¹）	0.84±0.10 b	1.05±0.15 a	0.91±0.18
一氧化氮（NO）/（μmol·L⁻¹）	24.18±4.26 Bab	32.67±9.88 ABb	45.89±11.63 Aa
白介素1-β（IL-1β）/（ng·L⁻¹）	20.45±1.70	22.51±1.30	23.17±1.65
白介素2（IL-2）/（ng·L⁻¹）	15.17±2.54 ab	13.66±1.38 b	16.51±3.10 a
白介素6（IL-6）/（ng·L⁻¹）	65.66±6.05	71.01±7.81	68.40±7.30
白介素8（IL-8）/（ng·L⁻¹）	94.31±9.86	104.24±7.70	105.89±13.30
干扰素-γ（IFN-γ）/（ng·mL⁻¹）	183.25±17.99	179.94±15.83	175.99±17.26

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表 7 IL-8基因T-360C突变位点各基因型与鸡球虫抗性指标关联分析

Table 7. Correlation between genotypes mutated at T-360C in IL-8 and coccidiosis-resistance indicators

指标 Indices	基因型 Genotype
指标 Indices	TT（40）	TC（44）	CC（8）
超氧化物歧化酶（SOD）/（U·mL⁻¹）	781.29±76.94 Aab	706.31±72.85 ABa	652.43±44.92 Bb
丙二醛（MDA）/（nmol·mL⁻¹）	4.01±1.20	3.91±0.49	4.23±0.79
谷胱甘肽过氧化物酶（GSH-PX）/（U·mL⁻¹）	124.34±11.73	117.17±13.46	116.68±14.53
过氧化氢酶（CAT）/（U·mL⁻¹）	0.77±0.16	0.86±0.30	0.74±0.14
一氧化氮（NO）/（μmol·L⁻¹）	46.91±11.24 a	45.50±9.28 a	35.03±8.11 b
白介素1-β（IL-1β）/（ng·L⁻¹）	20.11±1.03	20.65±1.84	21.55±2.73
白介素2（IL-2）/（ng·L⁻¹）	15.82±2.04 a	13.82±1.84 ab	12.46±1.55 b
白介素6（IL-6）/（ng·L⁻¹）	62.74±10.21	60.80±9.47	57.92±9.38
白介素8（IL-8）/（ng·L⁻¹）	103.19±14.94 a	91.30±13.82 ab	83.15±13.01 b
干扰素-γ（IFN-γ）/（ng·mL⁻¹）	163.28±13.49 b	168.75±15.29 b	193.31±16.32 a

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