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1 HEREDITAS (Beijing) , 31(1): ISSN 研究报告 DOI: /SP.J 猪 PRLR 和 RBP4 基因多态性与产仔性能的关系孙延晓, 曾勇庆, 唐辉, 樊新忠, 陈其美, 李华, 钱源, 宋一萍, 采用 PCR-RFLP 方法, 对莱芜黑猪鲁莱黑猪里岔黑猪鲁烟白猪新沂蒙黑猪 5 个山东地方 / 培育猪种和大约克夏长白杜洛克 3 个引进猪种共 8 个猪种 323 头繁殖母猪进行 PRLR 和 RBP4 基因的多态性检测, 并采用最小二乘法分析其对产仔数影响的遗传效应结果表明 : 两个基因位点在 8 个猪种的测定群体中均存在多态性, 但山东地方 / 培育猪种与引进猪种间在基因型频率上存在较大差异 PRLR 和 RBP4 基因对产仔数性状有显著影响 (P<0.05), AA 均为优良基因型对于 PRLR 基因, 山东地方 / 培育猪种内 AA 基因型母猪的总产仔数和活产仔数比 BB 基因型母猪平均多产 1.03 头和 0.89 头, 引进猪种中 AA 基因型母猪比 BB 基因型母猪平均多产分别为 1.26 头和 1.11 头对于 RBP4 基因, 山东地方 / 培育猪种内 AA 基因型母猪的总产仔数和活产仔数比 BB 基因型母猪平均多产 0.59 头和 0.51 头, 引进猪种中 AA 基因型母猪比 BB 基因型母猪平均多产分别为 0.72 头和 0.64 头猪 ; PRLR 基因 ; RBP4 基因 ; 多态性 ; 产仔数 Relationship of genetic polymorphism of PRLR and RBP4 genes with litter size traits in pig SUN Yan-Xiao, ZENG Yong-Qing, TANG Hui, FAN Xin-Zhong, CHEN Qi-Mei, LI Hua, QIAN Yuan, SONG Yi-Ping College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai an , China Abstract: The polymorphism of prolactin receptor (PRLR) and retinol-binding protein 4 (RBP4) genes was detected by PCR-RFLP method, and the effects of PRLR and RBP4 genes on litter size traits in pig were analyzed by the least square analysis. Data of total number born (TNB) and number born alive (NBA) from 323 sows, including five Shandong local pig breeds and three foreign pig breeds, were collected. The results showed that the polymorphic sites of both PRLR and RBP4 genes were found in all populations tested. The genotype distribution, however, revealed great differences between Shandong local pig breeds and foreign pig breeds. The effects of genotypes on TNB and NBA were significant (P<0.05). The homozygote AA was the most prolific genotype. For PRLR gene, AA genotypic sows of Shandong local pig breeds and foreign pig breeds produced 1.03 TNB, 0.89 NBA, and 1.26 TNB, 1.11 NBA more than BB genotypic sows, respectively. For RBP4 gene, AA genotypic sows of Shandong local pig breeds and foreign pig breeds produced 0.59 TNB, 0.51 NBA and 0.72 TNB, 0.64 NBA more than BB genotypic sows, respectively. Keywords: pig; PRLR gene; RBP4 gene; polymorphism; litter size 收稿日期 : ; 修回日期 : 基金项目 : ( ), ( 2007LZ013) ( J07WF08) 作者简介 : (1981 ),,, [email protected] 通讯作者 : (1964 ),,,, Tel: ; [email protected]

2 64 HEREDITAS (Beijing) ,, [1],,,, (Prolactin, PRL),,,,,, (Prolactin receptor, PRLR) [2, 3] 1998, Vincent [4], PRLR (Retinol-binding proteins, RBPs) A A, A [5], RBP4, RBP4,, [6, 7],,,, 5 3, PCR-RFLP PRLR RBP4,, : 36 48, ; 30, ; 30, ; 54, 3 : , ( 1 276, 2 7, ),,,,, 基因组 DNA 提取 - DNA, 0.8%, DNA, 25 ng/μl, 引物设计 [8, 9] PRLR : F: 5 -CGTGGCTCCGTTTGAAGAACC-3 R: 5 -C TGAAAGGAGTGCATAAAGCC-3 ; RBP4 F: 5 -GAGCAAGATGGAATGGGTT-3 R: 5 - CTCGGTGTCTGTAAAGGTG PCR 扩增 (1)PRLR : 20 μl, : 10 Taq buffer 2.0 μl; 2.5 mmol/l dntp Mixture 1.6 μl; 20 μmol/l 0.5 μl; 2.5 U/μL Taq DNA 0.4 μl; DNA 1.0 μl; 14.0 μl PCR : 94 4 min; s; s; s, 32, 72 7 min 1.5%, UVP (2)RBP4 : 25 μl, : 10 Taq buffer 2.5 μl; 2.5 mmol/l dntp Mixture 2.0 μl; 20 μmol/l 0.5 μl; 2.5 U/μL Taq DNA 0.4 μl; DNA 1.0 μl; 18.1 μl PCR : 95 5 min; s; s; s, 35, 72 7 min 1.5%, UVP

3 1 : PRLR RBP RFLP 分析 (1)PRLR : 20 μl, 10 U/μL Alu 0.4 μl, 10 L buffer 2.0 μl, PCR 10 μl, 7.6 μl, 37 5 h, 3.0%, (2)RBP4 : 20 μl, 10 U/μL Msp 1.0 μl, 10 T buffer 2.0 μl, 0.1% BSA 2.0 μl, PCR 8.0 μl, 7.0 μl, 37 8 h, 1.5%, 统计分析 2, PopGen 32, SAS 8.1 GLM(General linear model),, : Y = / ; : Pi=[2(ii)+(ij 1 )+(ij 2 )+ +(ij n )]/2N : Pi i ; j 1, j 2,, j n i 1 n ; : n 2 ( Oi Ei 1/ 2) χ = Ei: E, Oi:, n: PCR PRLR 基因 PCR 扩增结果和 PCR-RFLP 分析 PCR 1.5%, 163 bp ( 1A) PCR-RFLP PCR Alu, 3 : 85/59/19 bp 104/85/59/19 bp 104/59 bp, AA AB BB ( 1B) RBP4 基因 PCR 扩增结果和 PCR-RFLP 分析 PCR 1.5%, 480 bp ( 2A) PCR-RFLP PCR Msp, 3 : 190/154/136 bp 190/154/136/125/75 bp 154/136/125/75 bp, AA AB BB ( 2B) i i 2 图 1 PRLR 基因 PCR 扩增 (A) 及 PCR-RFLP 检测 (B) 结果 : BB ; 2~4 7: AB ; 10: AA ; M: 20 bp DNA ladder 图 2 RBP4 基因 PCR 扩增 (A) 及 PCR-RFLP 检测 (B) 结果 1 2 5: AB ; 3 6: AA ; 4 7: BB ; M: Marker I

4 66 HEREDITAS (Beijing) PRLR RBP4 1, PRLR B( 0.5 ), AB>BB>AA; A, AA BB (P<0.05), A χ 2, - (P>0.05) 2, RBP4 3 A( 0.6 ), AA BB (P<0.05) /, AB>BB>AA, B, AA BB (P<0.05), A χ 2, - (P>0.05) 2.3 PRLR RBP4 / 3 PRLR (P<0.05); RBP4 / (P>0.05), / (P<0.05) /, AA, /, PRLR AA BB , RBP4 AA BB , PRLR AA BB , RBP4 AA BB 表 1 不同猪种 PRLR 基因型分布和等位基因频率 * AA AB BB A B Laiwu (3) (21) (12) Licha (7) (27) (20) Lulai (5) (25) (18) Yimeng (12) (11) (7) Luyan (10) (12) (8) Landrace (8) (24) (19) Yorkshire (9) (27) (13) Duroc (16) (8) (1) *: χ (1) 3.84, χ (1) 6.63 Laiwu: ; Licha: ; Lulai: ; Yimeng: ; Luyan: ; Landrace:, Yorkshire: ; Duroc: 表 2 不同猪种 RBP4 基因型分布和等位基因频率 AA AB BB A B Laiwu (5) (22) (9) Licha (7) (29) (12) Lulai (11) (28) (15) Yimeng (10) (15) (5) Luyan (9) (16) (5) Landrace (24) (21) (6) Yorkshire (18) (24) (7) Duroc (19) (5) (1)

5 1 : PRLR RBP4 67 表 3 山东地方 / 培育猪种和引进猪种总产仔数和活产仔数的最小二乘分析 TNB NBA TNB NBA / PRLR AA 11.23±0.41 a 10.32±0.53 a 11.78±0.37 a 11.14±0.29 a AB 10.69±0.35 b 9.89±0.19 a 11.44±0.25 a 10.87±0.39 a BB 10.02±0.58 c 9.28±0.61 b 10.93±0.51 b 10.40±0.62 b RBP4 AA 11.13±0.31 a 10.31±0.41 a 11.67± ±0.27 AB 10.81±0.29 ab 10.02±0.42 ab 11.46± ±0.21 BB 10.38±0.21 b 9.64±0.45 b 11.24± ±0.45 PRLR AA 11.27±0.41 a 10.13±0.56 a 12.05±0.25 a 11.16±0.31 a AB 10.66±0.27 b 9.56±0.35 b 11.58±0.43 a 10.79±0.57 a BB 9.79±0.19 c 8.81±0.31 c 11.02±0.37 b 10.27±0.29 b RBP4 AA 11.18±0.21 a 10.24±0.39 a 11.72±0.32 a 10.85±0.27 a AB 10.79±0.43 ab 9.93±0.34 ab 11.51±0.21 ab 11.66±0.53 ab BB 10.31±0.18 b 9.45±0.42 b 11.15±0.41 b 10.37±0.52 b (P<0.05) TNB: ; NBA: 3 PRLR,,, [8, 10] [11] PRLR 10 Alu,, AA>AB>BB Rothschild [12] PRLR / / 6 PIC,, PRLR 6 4, AA BB 1 Terman [13] PRLR, PRLR (P<0.01),, AA AB BB, [14],, AA BB (P<0.01), AA BB (P<0.01), /, PRLR AA BB (P<0.05), AA BB (P<0.05), RBP4 [15, 16], RBP4,, [6, 7] Rothschild [17] , RBP4 AA BB 0.50 ( ) 0.26 ( ) Drogemuller [8] AA 1~10 BB 0.35, /, RBP4 AA BB , AA BB (P<0.05), PRLR RBP4,, PRLR RBP4, PRLR RBP4 ;,

6 68 HEREDITAS (Beijing) , ESR FSHβ,, 参考文献 (References): [1] Terqui M. Mechanisms Involved in the High Prolificacy of the Meishan Breed. In: Proceedings of the Chinese Pig Symposium. International Academic Press, 1990, [2] Ben-Jonathan N, Mershon JL, Allen DL, Steinmetz R. Extrapituitary prolactin-distribution, regulation, functions and clinical aspects. Endocr Rev, 1996, 17(6): [DOI] [3] Bignon C, Binart N, Ormandy C, Schuler LA, Kelly PA. Long and short forms of the ovine prolactin receptor cdna cloning and genomic analysis reveal that the two forms arise by different alternative splicing mechanisms in ruminants and in rodents. J Mol Endocrinol, 1997, 19(2): [DOI] [4] Vincent AL, Evans G, Short TH. The prolactin receptor gene is associated with increased litter size in pigs. In Proceedings of the 6th World Congress on genetics Applied to Livestock Production. Armidale, Australia, 1998, 27: [5],,.., 2004, 26(2): [6] Harney JP, Ott TL, Geisert RD. Retinol-binding protein gene expression in cyclic and pregnant endometrium of pigs, sheep, and cattle. Biol Reprod, 1993, 49(5): [DOI] [7] Blowe CD, Boytte KE, Ashwell MS, Eisen EJ, Robison OW and Cassady JP. Characterization of a line of pigs previously selected for increased litter size for RBP4 and follistatin. J Anim Breed Genet, 2006, 123(6): [DOI] [8] Drogemuller C, Hamann H, Distl O. Candidate gene markers for litter size in different German pig lines. J Anim Sci, 2001, 79(10): [9] Linville RC, Pomp D, Johnson RK, Rothschild MF. Candidate gene analysis for loci affecting litter size and ovulation rate in swine. J Anim Sci, 2001, 79(1): [10] Putnova L, Knol A, Drorak J. A new Hpa II PCR-RFLP within the porcine prolactin receptor (PRLR) gene and study of its effect on litter size and number of teats. J Anim Breed Genet, 2002, 119(1): [DOI] [11],,,. (PRLR) Alu., 2004, 24(3): [12] Rothschild ME, Vincent AL, Tuggle CK, Evan SG, Short TM, Southwood OI, Wales R, Platsow GS. A mutation in the prolactin receptor gene is associated with increased litter size in pigs. Anim Genet, 1998, 29(Supp1.1): [13] Terman A. Effect of the polymorphism of prolactin receptor (PRLR) and leptin (LEP) genes on litter size in Polish pigs. J Anim Breed Genet, 2005, 122(6): [DOI] [14],,,,,. 5., 2006, 28(6): [15] MacKenzie SH, Roberts MP, Liu KH. Bovine endometrial retinol-binding protein secretion, messager ribonucleic acid expression, and cellular localization during the estrous cycle and early pregnancy. Biol Reprod, 1997, 57(6): [DOI] [16] Dore JJ, Roberts MP, Godkin JD. Early gestational expression of retinal binding protein mrna by the ovine conceptus and endometrium. Mol Reprod Dev, 1994, 38(1): [DOI] [17] Rothschild MF, Messer L, Day A, Wales R, Short T, Southwood O, Plastow G. Investigation of the retinol-binding protein 4 (RBP4) gene as a candidate gene for increased litter size in pigs. Mamm Genome, 2000, 11(1): [DOI]

第二部分

第二部分中国农通报 0,7(04):478-48 Chinese Agricultural Science Bulletin 基于网络平台的生物统计改革及实践效果分析袁文, 彭惠茹, 张洪亮, 刘文欣, 华金平, 苏胜宝, 孔繁玲 ( 中国农大科技术发展研究院, 北京 0093; 中国农大农与生物