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陨司徒
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1 ( ) Journal of Hunan Agricultural University (Natural Sciences), Apr. 2014, 40(2): DOI: /j.cnki.jhau hspa9 基因 5 侧翼区 SNPs 对矮脚黄羽肉鸡耐热性的影响 1,2 1,2 1,2 1,2 1,2* ( ( ) ) 摘要 hspa9 5 PCR PCR RFLP 5 (T3(H/L) CD3 + T CD4 + T ) hspa9 5 3 (SNP) (C. 85C>A C. 485G>A C. 568G>A) C. 85C>A AA AC CC C. 485G>A GG GA AA C. 568G>A GG GA AA C. 485G>A (P 0.01) GG GA C. 485G>A T3 (P 0.05) GA T3 AA C. 568G>A T3 (P 0.05) GG T3 GA AA C. 568G>A CD4 + T (P 0.01) GG CD4 + T AA C. 485G>A C. 568G>A 关键词 hspa9 中图分类号 S831.2 Q75 文献标志码 :A 文章编号 : (2014) Effect of SNPs of hspa9 gene 5 flanking region with thermal tolerance traits in yellow feather dwarf broiler ZHANG Wen-wu 1,2, GAN Jian-kang 1,2, ZHANG Xi-quan 1,2, ZHANG De-xiang 1,2, LUO Qing-bin 1,2* (1.College of Animal Science, South China Agricultural University, Guangzhou , China; 2.Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture(Guangzhou), Guangzhou , China) Abstract: To observe the role of hspa9 of domestic chicken in tolerance of heat stress, hspa9 gene 5 'flanking region of yellow feather dwarf broiler pure line was amplified by PCR and sequenced, and then analyzed by PCR RFLP. The relationships between the mutation sites detected in hspa9 gene and the five heat resistant traits (triiodothyronine (T3), corticosterone, heterophil/lymphocyte ratio (H/L), CD3 + and CD4 + T cell) were analyzed. The results indicated that three SNP sites (C. 85C>A, C. 485G>A and C. 568G>A) were found. According to the C. 85C>A specific enzyme sites, hspa9 gene is divided into AA, AC and CC genotypes; according to the C. 485G>A specific enzyme sites, this gene is divided into GG, GA and AA genotypes; according to the C. 568G>A specific enzyme sites, this gene is divided into GG, GA and AA genotypes. Under high temperature, C. 485G>A site was highly significantly associated with corticosterone (P 0.01), corticosterone value in individual of GG genotype was highly significantly higher than that of AA genotype. At room temperature, C. 485G>A site was significantly associated with T3 (P 0.05), the content of T3 in individual of GA genotype was significantly higher than that of AA genotype; C. 568G>A site was significantly associated with T3 (P 0.05), the content of T3 in individual of GG genotype was significantly higher than that of AA T3 genotype; C. 568G>A site was significantly associated with CD4 + T cell (P 0.01), the percentage of CD4 + T cell in individual of GG genotype was significantly higher than that of AA genotype. Both C.+258A>G and C.+276C>G sites play an 收稿日期 : 基金项目 ( ) 作者简介 (1985 ) @qq.com *

2 184 ( ) important role in thermal stress resistance, indicating their potential in marker assisted selection of chicken. Key words: yellow feather dwarf broiler; hspa9 gene; thermal stress; thermal stress resistance (heat shock protein, HSP) ( ) [1] [2] HSP70 [3] hspa9 hsp70 hspa9 [4] [5] hspa9 apoptinhepg2 [6] hspa9 [7] mrna hspa9 hspa9 [8] hspa9 hspa9 PCR RFLPhspA9 1 材料与方法 1.1 供试鸡 333 (N301 ) 1.2 外周血采集 35 (20 ) 15 (32 ) 1.5 ml 1.5 ml 1.5 ml 1.3 鸡基因组 DNA 的提取 30 μl [9] DNA DNA 300 μl ddh 2 O 耐热性状相关指标的测定 (triiodothyronine T3) [10] Alfred [11] Campo Davila [12] 458 H/L 458 CD3 + T CD4 + T 1.5 hspa9 基因的 PCR RFLP 分析 1) PCR GenBank HspA9 cdna Primier5.0 hspa95 F1(5 GCACCACG CTGTTCCATCA 3 ) R1(5 AGGGCGATTGCAG CGAGCT 3 ) 10 DNA PCR PCR (Gen Star ) PCR (20 μl) Mix 10 μl 20 μmol/l 0.2 μl 10 U/μL Taq 0.15 μl DNA 1.5 μl (35 DNA) ddh 2 O 7.95 μl PCR 94 3 min 32 (94 30 s s s) 72 5 min, 16 PCR 1.2% 2) PCR RFLP PCR 666 DNA PCR RFLP PCR 10 μl Mix 5 μl 20 μmol/l 0.1 μl 10 U/μL Taq 0.1 μl DNA 1 μl ddh 2 O 3.7 μl PCR PCR 10 μl ddh 2 O 2.1 μl (TaKaRa ) 0.3 μl Buffer 0.6 μl PCR 7 μl 37 2% 1.6 关联分析 SAS 9.0 (General Linear Model GLM)

3 40 2 hspa9 5 SNPs 185 hspa9 y ij =μ+g i +e ij y ij μ g i e ij i j 2 结果与分析 2.1 hspa9 基因 5 侧翼区的 PCR 结果 PCR hspa9 5 3 SNP C. 85C>A C. 485G>A C. 568G>A C. 85C>A 5 85 C A Hae II ( GGCGCC) C. 485G>A G A Sml I ( CTTGAG) C. 568G>A G A Ban II ( GAGCTC) 2.2 hspa9 基因 3 个 SNP 的分型 C. 85C>A F2(5 ATGGGGGTGCTCGGAAAAC 3 ) R2(5 AGGG CGATTGCAGCGAGCT 3 ) DNA PCR bp Hae II PCR ( 1) hspa9 CC AC AA CC hspa9 Hae II bp AC hspa bp AA hspa bp C. 485G>A C. 568G>A F1 R1 DNA PCR Sml I Fig.1 1 AC 2 CC 3 AA M DL 2000 DNA marker 图 1 hspa9 基因 C. 85C>A 位点特异酶切结果 Digestion of hspa9 gene by Hae II specific for C. 85C>A site Ban II C. 485G>A ( 2) hspa9 GG GA AA GG hspa9 Sml I bp GA hspa bp AA hspa bp C. 568G>A ( 3) hspa9 GG GA AA GG hspa9 Ban II bp GA hspa bp AA hspa bp Fig.2 Fig.3 M 1 GG 2 AA 3 GA DL 2000 DNA marker 图 2 hspa9 基因 C. 485G>A 位点特异酶切结果 Digestion of hspa9 gene by Sml I specific for C. 485G>A site M 1 AA 2 GA 3 GG DL 2000 DNA marker 图 3 hspa9 基因 C. 568G>A 位点特异酶切结果 Digestion of hspa9 gene by Ban II specific for C. 568G>A site 2.3 hspa9 基因多态位点与耐热性状的相关性 hspa C. 485G>A (P 0.01) GG GA (P 0.01) GG AA GA AA (P>0.05) C. 85C>A C. 568G>A ( 1)

4 186 ( ) 表 1 热应激状态下 3 个 SNP 与耐热性状的关联分析 Table 1 Association analysis between 3 SNPs and thermal tolerance traits at high temperature CD3 + T CD4 + T H/L / T3 / C. 85C>A CC (1.51±0.08)(207) (40.70±0.73)(207) (0.42±0.01)(181) (4.17±0.65)(187) (18.21±2.77)(193) AA (1.71±0.41)(7) (44.60±3.97)(7) (0.36±0.07)(6) (7.94±3.65)(6) (25.80±14.56)(7) CA (1.41±0.15)(52) (41.16±1.46)(52) (0.39±0.02)(44) (6.96±1.23)(49) (28.84±5.56)(48) C. 485G>A GG (1.54±0.19)(45) (40.58±1.82)(33) (0.43±0.03)(28) (9.40±1.52)(33)A (24.18±4.59)(34) AA (1.41±0.16)(55) (42.36±1.59)(43) (0.38±0.02)(36) (5.82±1.32)(44)AB (20.48±4.03)(44) GA (1.47±0.08)(197) (40.82±0.74)(197) (0.41±0.01)(175) (3.64±0.66)(174)B (17.69±2.02)(176) C. 568G>A GG (1.54±0.16)(47) (41.25±1.51)(47) (0.42±0.02)(41) (4.50±1.33)(45) (22.14±5.68)(46) AA (1.39±0.10)(117) (40.97±0.96)(117) (0.39±0.02)(102) (4.77±0.87)(105) (24.06±3.67)(110) GA (1.57±0.11)(103) (40.37±1.02)(103) (0.42±0.02)(93) (4.42±0.93)(91) (16.50±3.97)(94) χ 2 Hardy Weinberg χ 2 15 hspa9 C. 485G>A T3 (P 0.05) GA T3 AA (P 0.05) GG AA GG GA (P>0.05) C. 568G>A T3 (P 0.05) GG T3 AA GA (P 0.05) GA AA (P>0.05) C. 568G>A CD4 + T (P 0.01) GG CD4 + T AA (P 0.01) GG GA GA AA (P>0.05) C. 85C>A ( 2) 表 2 常温状态下 3 个 SNP 与耐热性状的关联分析 Table 2 Association analysis between 3 SNPs and thermal tolerance traits at room temperature CD3 + T CD4 + T H/L / T3 / C. 85C>A CC (3.41±0.15)(161) (35.01±0.78)(161) (0.56±0.03)(82) (7.53±0.74)(146) (9.37±0.64)(286) AA (4.12±0.78)(6) (38.97±4.05)(6) (0.64±0.14)(4) (6.18±4.01)(5) (11.48±3.41)(10) CA (3.08±0.30)(42) (33.60±1.53)(42) (0.55±0.06)(18) (6.77±1.45)(38) (7.99±1.23)(72) C. 485G>A GG (3.13±0.38)(24) (35.00±2.10)(24) (0.46±0.09)(8) (9.12±1.82)(24) (11.38±2.03)(27)ab AA (3.18±0.31)(35) (33.03±1.74)(35) (0.47±0.07)(15) (7.45±1.57)(32) (7.81±0.79)(177)a GA (3.41±0.14)(162) (34.99±0.81)(162) (0.58±0.03)(89) (7.18±0.74)(144) (10.03±0.80)(175)b C. 568G>A GG (3.69±0.28)(42) (38.23±1.60)(75)A (0.53±0.05)(27) (6.67±1.16)(36) (12.82±1.68)(40)a AA (3.17±0.19)(99) (32.74±1.04)(99)B (0.60±0.04)(42) (6.70±0.75)(87) (9.01±0.70)(230)b GA (3.39±0.21)(75) (35.15±1.19)(75)AB (0.57±0.04)(41) (7.28±0.83)(71) (8.10±1.05)(102)b χ 2 3 讨论 T3 Koluman [13] T3 T3 [14] T3 T3 Maak [15] T3 T3 T3 hspa9 hspa9 C. 485G>A GA hspa9 C. 568G>A GG [12] [16 17] Zulkifli [18] hspa9 hspa9 C. 485G>A GG CD4 + T

5 40 2 hspa9 5 SNPs 187 CD4 + T [19 20] CD4 + T [21] CD4 + T [22] CD4 + T hspa9 C. 568G>A GG hspa9 C. 485G>A C. 568G>A SNP 参考文献 : [1] Gray C C Amrani M Yacoub M H Heat stress proteins and myocardial protection Experimental model or potential clinical tool[j] The International Journal of Biochemistry and Cell Biology (5) [2] Kaldis A Atkinson B G Heikkila J J Molecular chaperone function of the Rana catesbeiana small heat shock protein hsp30[j] Comparative Biochemistry and Physiology (2) [3] Hartl F U Molecular chaperones in cellular protein folding[j] Nature (6583) [4] Schlieben P Meyer A Weise C et al Differences in the proteome of high grade versus low-grade canine cutaneous mast cell tumours[j] Veterinary Journal (2) [5] Pawar H Kashyap M K Sahaspabuddhe N A et al. Quantitative tissue proteomics of esophageal squamous cell carcinoma for novel biomarker discovery[j] Cancer Biology and Therapy (6) [6] Peng C Yang P Cui Y et al HspA9 overexpression inhibits apoptin induced apoptosis in the HepG2 cell line[j] Oncology Reports (6) [7] Zhu Y Ren C Wan X et al Gene expression of Hsp70 Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis[j] Toxicology and Industrial Health (8) [8] Chen T H Kambal A Krysiak K et al Knockdown of HspA9 a del(5q31.2) gene results in a decrease in hematopoietic progenitors in mice[j] Blood (5) [9] F R R E [M] 1998 [10] MGG [J] (1) 77 [11] Alfred M L Casimir J Atlas of Avian Hematology[M]. Washington United States Department of Agriculture 1961 [12] Campo J L Davila S G Estimation of heritability for heterophil Lymphocyte ratio in chickens by restricted maximum likelihood effects of age sex and crossing[j]. Poultry Science (10) [13] Koluman N Daskiran I Effects of ventilation of the sheep house on heat stress growth and thyroid hormones of lambs[j] Tropical Animal Health and Production (6) [14] [J] (6) [15] Makk S Melesse A Schmidt R et al Effect of long term heat exposure on peripheral concentrations of heat shock protein 70 (Hsp70) and hormones in laying hens with different genotypes[j] British Poultry Science (1) [16] Post J Rebel J M Ter Huurne A A Physiological effects of elevated plasma corticosterone concentrations in broiler chickens An alternative means by which to assess the physiological effects of stress[j] Poultry Science (8) [17] Star L Nieuwland M G Kemp B et al Effect of single or combined climatic and hygienic stress on natural and specific hum oral immune competence in four layer lines[j] Poultry Science (9) [18] Zulkifli I Al Aqil A Omar A R et al Crating and heat stress influence blood parameters and heat shock protein 70 expression in broiler chickens showing short or long tonic immobility reactions[j] Poultry Science (3) [19] Trout J M Mashaly M M The effects of adrenocorticotropic hormone and heat stress on the distribution of lymphocyte populations in immature male chickens[j]. Poultry Science (11) [20] Hammami M M Bouchama A Shail E et al. Lymphocyte subsets and adhesion molecules expression in heatstroke and heat stress[j] Journal of Applied Physiology (5) [21] CD4 + CD8 + T [J] (6) 5 7 [22] Chen Z Y Gan J K Xiao X et al The association of SNPs in Hsp90β gene 5 flanking region with thermo tolerance traits and tissue mrna expression in two chicken breeds[j] Molecular Biology Reports (9) 责任编辑 : 罗维英文编辑 : 罗维

5-25袁宏钧.indd

5-25袁宏钧.indd 第 24 卷第 5 期 2015 年 5 月碘 131 doi:10.3978/j.issn.1005-6947.2015.05.005 http://dx.doi.org/10.3978/j.issn.1005-6947.2015.05.005 Chinese Journal of General Surgery, 2015, 24(5):643 647. Chinese Journal of