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1 HEREDITAS (Beijing) 2013 *, 35(*): 1 8 ISSN 研究报告 DOI: /SP.J 绵羊发情周期不同组织 Cry1 mrna 转录水平相对定量研究高磊 1,2, 甘尚权 1,2, 杨井泉 1,2, 杨剑波 1,2, 梁耀伟 1,2, 阿不都艾尼努拉洪 3, 沈敏 1., ; 2., ; 3., ; 1,2* 研究表明, 时钟基因 Cry1 在哺乳动物季节性繁殖内分泌过程中可能发挥了重要作用文章以多浪羊 ( 非季节性繁殖 ) 和中国美利奴羊 ( 季节性繁殖 ) 为研究对象, 采用实时荧光定量 PCR 技术对下丘脑 - 垂体 - 性腺轴主要组织 Cry1 在发情周期不同阶段的表达变化情况进行了跟踪检测结果表明 : 绵羊 Cry1 在所检测组织中均有表达, 其中松果体和甲状腺 Cry1 的表达水平较高 ; 不同绵羊品种 Cry1 在发情周期不同阶段的组织表达谱基本一致, 除下丘脑外, 卵巢子宫松果体垂体和甲状腺中 Cry1 的表达水平均是在发情前期达到峰值 ; 卵巢子宫垂体和松果体 Cry1 在发情前期和发情期的表达变化幅度存在显著的品种间差异研究结果表明 :Cry1 可能具有启动发情和季节性繁殖的重要作用 Cry1 基因 ; 发情周期 ; 下丘脑 - 垂体 - 性腺轴 ; 绵羊 Relative quantification of mrna transcription of Cry1 in different tissues of sheep in oestrous cycle by real-time quantitative PCR GAO Lei 1,2, GAN Shang-Quan 1,2, YANG Jin-Quan 1,2, YANG Jian-Bo 1,2, Liang Yao-Wei 1,2, Abdulla Aini Nula Hong 3, SHEN Min 1,2 1. Animal Husbandry and Veterinary Institute, Xinjiang Academy of Agricultural and Reclamation Science, Shihezi , China; 2. Key Laboratory of sheep Breeding and Development Technology of Xinjiang Production and Construction Crops, Shihezi , China; 3. Agriculture Third Division forty-four regiment Veterinary Station of Xinjiang Production and Construction Corps, Tumushuke city , China Abstract: Studies have shown that clock gene Cry1 may have important roles in the endocrine process of seasonal reproduction in mammals. In this study, Duolang sheep (non-seasonal reproduction sheep breed) and Chinese Merino (seasonal reproduction sheep breed) were used to determine the expression change of Cry1 in hypothalamus-pituitary-ovary 收稿日期 : ; 修回日期 : 基金项目 : 973 ( 2009CB125907), ( ) ( 2012BB044) 作者简介 :,,, Tel: ; w.n007@163.com 通讯作者 :,,, shenmin0993@sina.com 网络出版时间 : URL:

2 2 HEREDITAS (Beijing) axis in different stage of estrous cycle by quantitative real-time PCR. The results showed that the Cry1 mrna was expressed in all tested tissues, in which the expression levels in pineal gland and thyroid gland were higher than in other tissues. As far as different sheep breeds were concerned, the tissue expression profiles of Cry1 at different stage of estrous cycle were broadly similar. Besides hypothalamus, the expression levels of Cry1 in ovary, uterus, pineal gland, pituitary gland, and thyroid gland were all reached to peak in proestrus. The differences of expression change extent for Cry1 in vary, uterus, pineal gland, and pituitary gland in proestrus and oestrus were significant between different sheep breeds. The results suggested that Cry1 may play roles in switching on the estrus and seasonal reproduction. Keywords: Cry1 gene; estrous cycle; hypothalamus-pituitary-ovary axis; sheep, 24 h, 8 (Clock Bmal1 Timeless Cry1 Cry2 Per1 Per2 Per3), [1] (Cricetidae) (Mus musculus) 24 h [2] (Ovis aries), - -,, 7 (Clock Bmal1 Timeless Cry1 Cry2 Per1 Per2) (PT) (SCN), [3], 1(Cryptochrome 1, Cry 1),, Cry1 [4,5], ;, Cry1, [6,7] Hazleriqq [8], Soay,, Cry1, Cry1 Martha [9] Sheanm [10], Cry1,, - 24h, Cry1, Cry1, Cry1,, PCR, Cry1, ~4 ( ) ( ) 12, 8:00 [11], 1~2, (12~15 d) (0~1.5 d) (3~4 d) (7~10 d)

3 * : Cry1 mrna 3 3,, 1.2 GenBank Cry1 (NM_ ), Olig 6.0 GAPDH [12] RNA cdna Trizol(Invitrogen) RNA 0.7% RNA, (Implen) RNA M-MLV first strand cdna Synthesis Kit(Invitrogen) cdna 1.4 Cry1 GAPDH PCR 20 µl, cdna 1 µl, Premix Taq Version 2.0(Loading dye mix)(takara) 10 µl, (10 µmol/l) 0.8 µl, ddh 2 O 7.4 µl PCR 94 3 min; s, s, s, 30 ; min PCR 1%, pmd19-t, E.coli.DH5α PCR, GenBank Cry1 GAPDH 1.5 Cry1 PCR 标准曲线绘制 PCR Cry1 GAPDH,, 10,, PCR 实时定量 PCR 检测 cdna, Cry1 GAPDH PCR, GAPDH Cry 1, 3 PCR 20 µl, :cdna 1 µl, Mix [1a(FastStart Enzyme) 10 µl +1b(Reaction Mix SYBR Green I, 10 conc)] (Roche) 2 µl, MgCl 2 (25 mmol/l) 2.4 µl, (10 μmol/l) 1 µl, H 2 O 12.6 µl min; s, s, s, 45, 0.5, PCR 数据分析 Ct (Qr=2 ΔΔ Ct ), 3, Ct, Ct Ct, ΔΔCt, 2 -ΔΔCt, SPSS(18.0), RNA 0.7%, RNA, 28S 18S 5S, RNA OD 260 /OD ~2.0, RNA cdna cdna, Cry1 表 1 实时荧光定量 PCR 引物序列 (5 3 ) (bp) Cry1 GAPDH F: TTCGTCAGGAGGGTTGGATT R: TGGCACTGAAGCAAGAAGAC F: CTGACCTGCCGCCTGGAGAAA R: GTAGAAGAGTGAGTGTCGCTGTT

4 4 HEREDITAS (Beijing) GAPDH,,, GenBank Cry1 GAPDH, 99%,,, RT-PCR PCR 2.2 Cry1 GAPDH, RT-PCR Cry1 Cry1, Cry1, ( 1) 图 1 Cry1 在绵羊不同组织的半定量 RT-PCR 结果 2.3 Cry1 PCR Cry1, Cry1 ( ) PCR, Cry1 GAPDH (Amplification efficiency, E values) , PCR Cry1 在不同品种绵羊发情周期不同阶段的组织表达谱变化 GAPDH, Cry1 1, Cry1 2 Cry1 ( 2A), Cry1 (2.49)> (1.01) (1.00) (1.00)> (0.75)> (0.26) Cry1, (P<0.01); Cry1, (P<0.05) Cry1 (2.69)> (1.00)> (0.61)> (0.38) (0.37)> (0.25) Cry1, (P<0.01); Cry1, (P<0.05) ( 2B), Cry1 (1.07) (1.00) (0.84)> (0.70) (0.52)> (0.30) Cry1, (P<0.05) (P<0.05) (P<0.01) ; Cry1 (P<0.05) Cry1 (1.22) (1.00)> (0.38) (0.30) (0.29) (0.24) Cry1, (P<0.01) ( 2C), Cry1 (1.00) (0.95)> (0.48)> (0.26) (0.25) (0.22) Cry1, (P<0.05), (P<0.01) Cry1 (1.00) (0.79)> (0.25) (0.23) (0.22) (0.13) CRY1 (P<0.01) ( 2D), Cry1 (1.00) (0.80)> (0.26) (0.18) (0.17) (0.09) Cry1, (P<0.01) Cry1 (1.00)> (0.58)> (0.22) (0.17) (0.15) (0.10) Cry1 (P<0.05), (P<0.01); Cry1

5 * : Cry1 mrna 5 图 2 Cry1 在不同绵羊品种发情周期不同阶段的组织表达变化 A ; B ; C ; D (P>0.05), (P<0.05), (P>0.05); * ** (P<0.05) (P<0.01) (P<0.05) 不同绵羊品种不同组织 Cry1 基因在发情周期不同阶段的表达变化 Cry1, 3, 6, Cry1 Cry1,,, Cry1,,,, Cry1 3A, Cry (P<0.01) 2.69 (P<0.01) 1.02 ; Cry (P<0.01) 1.74 (P<0.01) 1.14 Cry1 (P<0.01), 3B, Cry (P<0.01) 3.13 (P<0.01) 1.54 ; Cry (P<0.01) Cry1 (P<0.01), 3C, Cry (P<0.01) ; Cry (P<0.01) 2.08 (P<0.01) 1.35 Cry1 (P<0.01),

6 6 HEREDITAS (Beijing) 图 3 不同组织 Cry1 在发情周期不同阶段的表达变化 A: ; B ; C ; D ; E ; F (P>0.05), (P<0.05), (P>0.05); * ** (P<0.05) (P<0.01) 3D, Cry (P<0.01) 9.32 (P<0.01) 4.76 (P<0.01); Cry (P<0.01) 2.43 (P<0.01) 2.23 (P<0.01) Cry1 (P<0.01), 3E, Cry (P<0.01) 6.52 (P<0.01) 4.27 (P<0.01); Cry (P<0.01) 5.99 (P<0.01) 3.99 (P<0.01) Cry1 3F, Cry (P<0.05) 1.58 (P<0.05) 1.17 ; Cry (P<0.01) 1.73 (P<0.01) 0.87 Cry Cry1, Cry1

7 * : Cry1 mrna 7, (SCN),, [4,13,14], Rataiczak [15] Cry1, Cry1 RT-PCR, Cry1 Cry1 mrna ( ),, Cry1, 3.2 Cry1 - - Cry1, - -, Cry1, Cry1, Cry1, [16,17], Cry1, T3 T4, T4, T3 [18,19] Cry1,,, 3.3 Cry1,,,,, Cry1 PCR,, Cry1, ( )Cry1 ( ),, Cry1 参考文献 (References): [1] King DP, Takahashi JS. Molecular genetics of circadian rhythms in mammals. Annu Rev Neurosci, 2000, 23(1): [2] Ralph MR, Menaker M. A mutation of the circadian system in golden hamsters. Science, 1988, 241(4870): [3] Lincoln G, Messager S, Andersson H, Hazlerigg D. Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer. Proc Natl Acad Sci USA, 2002, 99(21): [4] Miyamoto Y, Sancar A. Vitamin B 2 -based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals. Proc Natl Acad Sci USA, 1998, 95(11): [5] Lincoln GA, Andersson H, Loudon A. Clock genes in calendar cells as the basis of annual timekeeping in mammals-a unifying hypothesis. J Endocrinol, 2003, 179(1): [6] van der Horst GT, Muijtjens M, Kobayashi K, Takano R, Kanno S, Takao M, de Wit J, Verkerk A, Eker AP, van Leenen D, Buijs R, Bootsma D, Hoeijmakers JH, Yasui A. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature, 1999, 398(6728): [7] Park K, Kang HM. Cloning and circadian expression of rat Cry1. Mol Cells, 2004, 18(2): [8] Hazlerigg DG, Andersson H, Johnston JD, Lincoln G. Molecular characterization of the long-day response in the Soay sheep, a seasonal mammal. Curr Biol, 2004, 14(4): [9] Vitaterna MH, Selby CP, Todo T, Niwa H, Thompson C,

8 8 HEREDITAS (Beijing) Fruechte EM, Hitomi K, Thresher RJ, Ishikawa T, Miyazaki J, Takahashi JS, Sancar A. Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc Natl Acad Sci USA, 1999, 96(21): [10] Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng BH, Kume K, Lee CC, van der Horst GTJ, Hastings MH, Reppert SM. Interacting molecular loops in the mammalian circadian clock. Science, 2000, 288(5468): [11],,,.., 2012, (21): [12] Mulsant P, Lecerf F, Fabre S, Schibler L, Monget P, Lanneluc I, Pisselet C, Riquet J, Monniaux D, Callebaut I, Cribiu E, Thimonier J, Teyssier J, Bodin L, Cognié Y, Chitour N, Elsen JM. Mutation in bone morphogenetic protein receptor-ib is associated with increased ovulation rate in Booroola Mérino ewes. Proc Natl Acad Sci USA, 2001, 98(9): [13] van der Spek PJ, Kobayashi K, Bootsma D, Takao M, Eker APM, Yasui A. Cloning, tissue expression, and mapping of a human photolyase homolog with similarity to plant blue-light receptors. Genomics, 1996, 37(2): [14] Kobayashi K, Kanno SI, Takao M, Yasui A, Smit B, van der Horst GTJ. Characterization of photolyase/blue-light receptor homologs in mouse and human cells. Nucleic Acids Res, 1998, 26(22): [15] Rataiczak CK, Herzoq ED, Muqlia LJ. Clock gene expression in gravid uterus and extra-embryonic tissues during late gestation in the mouse. Reprod Fertil Dey, 2010, 22(5): [16] Lincoln GA, Andersson H, Hazlerigg D. Clock genes and the long-term regulation of prolactin secretion: evidence for a photoperiod/circannual timer in the pars tuberalis. J Neuroendocrinol, 2003, 15(4): [17] Johnston JD, Tournier BB, Andersson H, Masson-Pévet M, Lincoln GA, Hazlerigg DG. Multiple effects of melatonin on rhythmic clock gene expression in the mammalian pars tuberalis. Endocrinology, 2006, 147(2): [18] Barrett P, Ebling FJP, Schuhler S, Wilson D, Ross AW, Warner A, Jethwa P, Boelen A, Visser TJ, Ozanne DM, Archer ZA, Mercer JG, Mrgan PJ. Hypothalamic thyroid hormone catabolism acts as a gatekeeper for the seasonal control of body weight and reproduction. Endocrinology, 2007, 148(8): [19],.., 2011, 33(7):

Microsoft Word 高磊.doc

Microsoft Word 高磊.doc HEREDITAS (Beijing) 2013 1, 35(1): 85 92 ISSN 0253-9772 www.chinagene.cn 研究报告 DOI: 10.3724/SP.J.1005.2013.00085 绵羊发情周期不同组织 Cry1 mrna 转录水平相对定量研究高磊 1,2, 甘尚权 1,2, 杨井泉 1,2, 杨剑波 1,2, 梁耀伟 1,2, 阿不都艾尼努拉洪 3,