猪骨形成蛋白15基因多态性的PCR-RFLP检测方法
技术领域
本发明属于猪的分子生物学技术领域,具体地说本发明涉及猪骨形成蛋白15基因多态性的PCR-RFLP检测技术。
技术背景
产仔数是养猪生产中最重要的经济性状之一,提高每头母猪断奶仔猪将以最小的额外投入,增加养猪生产者的经济回报(Rothschild,1996)。一般来说,产仔数的多少取决于品种(遗传),不同品种间产仔数(从2头到20头,平均9到10头)和胚胎存活率(15-40%)存在很大差异。产仔数表型标准差在2.5到3头之间,遗传力为0.10到0.15(Johnson等,Responses in ovulation rate,embryonal survival,and litter traits in swine to 14generations of selection to increase litter size.J Anim Sci.77(3):541-57)。中国梅山猪比欧洲和美国商品猪每窝多产4到5头仔猪,比大白猪多***7.1枚。梅山猪多产基因导入欧洲和美国猪种中已产生了明显的商业价值。据文献报道,只要将猪产仔数提高1~1.5头,英国养猪业每年可获得7亿英镑的额外利润,而整个欧盟每年获得的额外利润将至少是20亿英镑(Clutter,1995)。
猪产仔数是一个遗传力低的限性性状,采用传统选择方法,直接选择产仔数而获得遗传进展是非常困难的。法国用了30多年时间来改良这个性状,结果毫无进展;而丹麦用了50多年的时间才将每胎产仔数提高了1.0头。Johnson等(Ovulatory response,and plasmaconcentrations of luteinizing hormone and progesterone following administration of syntheticmammalian or chicken luteinizing hormone-releasing hormone relative to the first or secondovulation in the sequence of the domestic hen.Biol Reprod.1984,31(4):646-55.)、Bennett和Leymaster(Integration of ovulation rate,potential embryonic viability and uterine capacity into amodel oflitter size in swine.J Anim Sci.1989,67(5):1230-41.)提出根据***率、胚胎存活率或子宫容量制定选择指数比直接选择产仔数,预计可获得较大选择反应。Johnson等人(1999)报道连续11代根据提高***率和胚胎存活率指数进行选择,接下来3个世代选择产仔数。经过14代的综合选择,与对照组(随机选择)相比,初生时总产仔数和活仔数分别提高3头和1.4头。也有文献报道,采用超多产选择法(hyperprolificacy selection)、BLUP(Best LinearUnbiased Prediction,最佳线性无偏估计)和繁殖激素水平的间接选择,能提高窝产仔数,但进展不大。
分子标记辅助选择(marker assisted selection,MAS)技术的建立和发展,给产仔数的遗传改良提供了新的方法。在公畜和母畜发育早期,同时直接选择影响产仔数表达的基因,将提高选择的准确性和选择反应。为了实施MAS,必须鉴别影响重要经济性状的单个候选基因和数量性状基因座位(quantitative trait loci,QTL),并估计它们的遗传效应。
产仔数受母体***数和胚胎存活率直接影响,而***率又是一个复杂性状,受遗传和众多环境因子影响。目前许多研究直接集中卵泡生成调控,主要研究方向是鉴别卵巢产生的自分泌或旁分泌调控因子的生理功能。有些调控因子由***合成和分泌,并扮演控制卵泡生长和分化的形态发生作用。大量证据显示***派生因子调控卵巢功能。***派生因子有生长分化因子-9(growth differentiation factor-9,GDF9)、骨形态发生蛋白6(bonemorphogenetic protein 6,BMP6)和骨形态发生蛋白15(BMP15),它们都属于最大的胞外信号蛋白家族转移生长因子-β(transforming growth factorβ,TGFβ)超家族成员。利用敲除GDF9基因鼠证实颗粒细胞生长和分化,以及***减数***能力均需要GDF9直接参与,缺乏GDF9基因母鼠导致早期卵泡生成受阻,以致不能生育。
1998年,Dube等人克隆了鼠和人BMP15基因,与GDF9基因非常类似,在***中惟一表达,缺乏7个半胱氨酸残基中第4个(The bone morphogenetic protein 15 gene isX-linked and expressed in oocytes.Mol Endocrinol.12(12):1809-17.)。而7个半胱氨酸残基是40多个TGFβ超家族成员的典型保守区。由于第4个半胱氨酸能形成链间二硫键骨架,因此,BMP15和GDF9也许可能是单体或者以非共价键连接的异源或同源二聚体,并推测在卵巢中具有互补作用。鼠和人BMP15编码区序列全长1176bp,含有2个外显子,分别被3.5kb和4.2kb内含子隔开;编码392个氨基酸前肽原(其中信号肽17个氨基酸,前肽250个氨基酸和成熟肽125个氨基酸)。鼠和人前肽原、前肽和成熟肽氨基酸同源性分别为63%、60%和77%,核苷酸同源性分别为76%、74%和81%。人BMP15基因被定位于X染色体P11.2,证实与鼠BMP15基因所在X染色体上位置为保守同线性区域。绵羊BMP15基因已被克隆(Galloway等,2000,Mutations in an oocyte-derived growth factor gene(BMP15)cause increased ovulationrate and infertility in a dosage-sensitive manner.Nat Genet.25(3):279-83.)。编码区全长1179bp,含有2个外显子,被5.4kb内含子隔开,编码393个氨基酸。编码区序列与鼠和人同源性分别为78.8%和82.9%。绵羊BMP15基因被定位在X染色体10cM区域,与FecXI(多产X基因)为同一区域。在78个共信息母羊减数***中未发现BMP15和FecXI的表型重组。在携带多产X基因突变Inverdale羊(FecXI)和Hanna羊(FecXH)中,BMP15成熟肽序列发生了突变。BMP15基因突变杂合子绵羊***率增加,导致较多双胞胎或三胞胎。而BMP15基因纯合子绵羊阻碍卵泡生成,不能生育,类似敲除GDF9基因鼠表型。因此,BMP15基因也是母畜生育所必需的,自然突变即能增加***率(突变杂合子携带者),也能导致不育表型(突变纯合子携带者),可以作为***率或产仔数的候选基因进行深入研究。
发明内容
本发明的目的之一在于克隆猪BMP15基因编码区序列,通过序列比较分析获得单核苷酸多态性;本发明的目的之二是基于发现的单核苷酸多态性,建立BMP15基因单核苷酸多态性的PCR-RFLP检测方法,为猪***率或产仔数的标记辅助选择提供有用的分子标记。
本发明通过以下技术方案实现:
猪骨形成蛋白15(bone morphogenetic protein 15,BMP15)基因,其编码区核苷酸序列如猪序列表SEQ ID No.1和序列表SEQ ID No.2所示。
扩增和测序编码区核苷酸序列的10种引物分别如序列表SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQ ID No.9、SEQ ID No.10、序列表SEQ IDNo.11、序列表SEQ IDNo.12所示。
序列表SEQ ID No.3:
F1-1:5’-CTGCCTTTCACTGTTTCCTG-3’
序列表SEQ ID No.4:
R1-1:5’-CTCATTTTTCTCCCCTCCAG-3’
序列表SEQ ID No.5:
F2-1:5’-ACATCACAGCTCACAGCAAC-3’
序列表SEQ ID No.6:
R2-1:5’-ACATGAAGCGGAGTCGTAGA-3’
序列表SEQ ID No.7:
F2-2:5’-GTAGCGTCTGCCACCTACAT-3’
序列表SEQ ID No.8:
R2-2:5’-CCGGAACTCAAGAATCTCAC-3’
序列表SEQ ID No.9:
F2-3:5’-AGAGCCACTGTGGTTTATCG-3’
序列表SEQ ID No.10:
R2-3:5’-GAAAGGATGGAGGGAACACT-3’
序列表SEQ ID No.11:
F2-4:5’-TAACCAGTGTTCCCTCCATC-3’
序列表SEQ ID No.12:
R2-4:5’-CCTGGGAAACCTGATCTAGC-3’
如上所述的基因序列,其单核苷酸多态性(single nucleotide polymorphism,SNP)的特征在于BMP15基因编码区的核苷酸390位。
一种克隆或\和测序猪BMP15基因编码区核苷酸序列的方法,按照以下步骤:猪血液基因组DNA提取、设计特异引物、聚合酶链式反应(PCR)扩增、PCR产物纯化和直接测序,通过序列比较分析获得如序列表SEQ ID No.1和序列表SEQ ID No.2所示的核苷酸序列。
其中所述的引物如序列表SEQ ID No.7和SEQ ID No.8,能扩增出含有多态性位点的基因片段。
所述的方法还包括建立了BMP15基因多态性的PCR-限制性片段长度多态性(RFLP)检测方法,所述的限制性内切酶是Spe I。
所述的方法,PCR反应是在含有50ng基因组DNA、15mmol/L Tri s-HCl、50mmol/L KCl(pH8.0)、2mmol/L MgCl2,200μmol/L dNTPs、10μmol/L引物、4%二甲亚砜和0.5U TaqDNA聚合酶的25μl反应体系中进行,反应条件是95℃ 5min、35循环(94℃ 45S、58℃ 45S和72℃ 1min 30S)、72℃延长5min。17μl PCR产物,加入0.5μl(10U/μl)的限制性内切酶Spe I酶37℃消化过夜,消化产物用1.5%琼脂糖凝胶电泳。
其中所述的多态性是226bp和277bp限制性片段长度多态性。
所述的猪BMP15基因序列,其单核苷酸多态性(single nucleotide polymorphism,SNP)的特征在于BMP15基因编码区的核苷酸390位。
比较序列表SEQ ID No.1和SEQ ID No.2 DNA序列(如图1所示),发现BMP15基因编码区核苷酸390位T→A的SNP位点。所述的用于猪BMP15基因SNP分型的检测技术是PCR-RFLP。PCR扩增的引物如序列表SEQ ID No.7和SEQ ID No.8所示。PCR反应是在含有50ng基因组DNA、15mmol/L Tris-HCl、50mmol/L KCl(pH8.0)、2mmol/L MgCl2,200μmol/LdNTPs、10μmol/L引物、4%二甲亚砜和0.5U Taq DNA聚合酶的25μl反应体系中进行,反应条件是95℃ 5min、35循环(94℃ 45S、58℃ 45S和72℃ 1min 30S)、72℃延长5min。17μl PCR产物,加入0.5μl(10U/μl)的限制性内切酶Spe I酶37℃消化过夜,消化产物用1.5%琼脂糖凝胶电泳。所述的多态性是226bp和277bp限制性片段长度多态性。
序列表及其说明:
1、序列表SEQ ID No.1:是来源于欧洲血统的“大白猪”克隆的核苷酸序列;
2、序列表SEQ ID No.2:是来源于中国地方猪种血统“梅山猪”克隆的核苷酸序列;
3、序列表SEQ ID No.3:是实施BMP15基因克隆所用特异引物的核苷酸序列;
4、序列表SEQ ID No.4:是实施BMP15基因克隆所用特异引物核苷酸序列;
5、序列表SEQ ID No.5:是实施BMP15基因克隆所用特异引物核苷酸序列;
6、序列表SEQ ID No.6:是实施BMP15基因克隆所用特异引物核苷酸序列;
7、序列表SEQ ID No.7:是实施BMP15基因克隆和PCR-RFLP分析所用特异引物核苷酸序列;
8、序列表SEQ ID No.8:是实施BMP15基因克隆和PCR-RFLP分析所用特异引物核苷酸序列;
9、序列表SEQ ID No.9:是实施BMP15基因克隆所用特异引物核苷酸序列;
10、序列表SEQID No.10:是实施BMP15基因克隆所用特异引物核苷酸序列;
11、序列表SEQ ID No.11:是实施BMP15基因克隆所用特异引物核苷酸序列;
12、序列表SEQ ID No.12:是实施BMP15基因克隆所用特异引物核苷酸序列;
附图及其说明:
图1:大白猪和梅山猪BMP15基因编码区核苷酸序列比较
本发明具有以下效果:
本发明的基因标记和SNP的PCR-RFLP检测技术可用于不同猪品种的BMP15基因多态性分析,以及研究基因与猪繁殖性能的关系。
具体实施方式
实施例1BMP15基因编码区DNA的测序
选择中国地方品种梅山猪(***血统)和外来品种大白猪(欧洲猪血统)作为典型试验材料,根据猪和人BMP15基因DNA序列设计5对引物扩增含有BMP15基因外显子1和外显子2区域。
F1-1:5’-CTGCCTTTCACTGTTTCCTG-3’
R1-1:5’-CTCATTTTTCTCCCCTCCAG-3’
F2-1:5’-ACATCACAGCTCACAGCAAC-3’
R2-1:5’-ACATGAAGCGGAGTCGTAGA-3’
F2-2:5’-GTAGCGTCTGCCACCTACAT-3’
R2-2:5’-CCGGAACTCAAGAATCTCAC-3’
F2-3:5’-AGAGCCACTGTGGTTTATCG-3’
R2-3:5’-GAAAGGATGGAGGGAACACT-3’
F2-4:5’-TAACCAGTGTTCCCTCCATC-3’
R2-4:5’-CCTGGGAAACCTGATCTAGC-3’
聚合酶链式反应(PCR)是在含有50ng基因组DNA、15mmol/L Tris-HCl、50mmol/L KCl(pH8.0)、2mmol/L MgCl2,200μmol/L dNTPs、10μmol/L引物、4%二甲亚砜和0.5U TaqDNA聚合酶的25μl反应体系中进行,反应条件是95℃ 5min、35循环(94℃ 45S、58℃ 45S和72℃ 1min 30S)、72℃延长5min,4℃保存。扩增的PCR产物被纯化后用正向引物和反向引物直接进行序列测定。DNA序列用Blast软件(http://www.ncbi.nlm.nih.gov/)进行排列和比较:大白猪和梅山猪BMP15基因编码区DNA序列见序列表SEQ ID No.1和序列表SEQ ID No.2所示。
实施例2 BMP15基因SNP的PCR-RFLP检测技术
用Blast软件比较大白猪和梅山猪BMP15基因编码区DNA序列,在核苷酸390位发现了1个碱基改变(T→A),即SNP(见附图1):梅山猪是A,而大白猪是T,并且导致了限制性内切酶Spe I(A↓CTAGT)酶切位点的改变。据此,建立了基于限制性内切酶Spe I的PCR-RFLP检测方法检测SNP。PCR反应在含有50ng基因组DNA、15mmol/L Tris-HCl、50mmol/L KCl(pH8.0)、2mmol/L MgCl2,200μmol/L dNTPs、引物F2-2和R2-210μmol/L、4%二甲亚砜和0.5U Taq DNA聚合酶的25μl反应体系中进行,反应条件是95℃ 5min、35循环(94℃ 60S、58℃ 45S和72℃ 90S)、72℃延长10min。PCR产物用1.5%琼脂糖凝胶电泳检测:等位基因A(BMP15基因编码区核苷酸390位为A)限制性片段长度为226bp和277bp,等位基因B(BMP15基因编码区核苷酸390位为T)限制性片段长度为503bp;杂合基因型AB限制性片段长度为503bp、277bp和226bp。对***地方品种二花脸、梅山猪、采用中国血统的湖北省通城猪培育品种中国瘦肉猪新品系(DIV)与欧洲血统的外来品种大白猪、长白猪和杜洛克猪BMP15基因多态性进行了检测分析,其基因型和基因频率见表1所示。从表1结果可以看出,不同品种BMP15基因型不同,外来品种猪均为BB型,二花脸猪均为AA型,梅山猪中出现AA型和AB型,而培育品种DIV只有BB型。下一步将扩大样本含量,研究BMP15基因型与***率或产仔数的关系。
表1不同品种猪的BMP15基因型和基因频率检测结果
品种 |
样品数 |
基因型频率(%) |
基因频率(%) |
AA |
AB |
BB |
A |
B |
二花脸梅山猪DIV大白猪长白猪杜洛克猪 |
371247371213 |
10033.3---- |
-66.7---- |
--100100100100 |
10066.7---- |
33.3100100100100 |
SEQUENCE LISTING
<110>华中农业大学
<120>猪骨形成蛋白15基因多态性的PCR-RFLP检测方法
<130>
<141>2003-04-17
<160>14
<170>PatentIn version 3.1
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<211>1185
<212>DNA
<213>猪(Sus scrofa)
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atc ctc ctg att gag gca aat ggg agt atc ttg tac aag gag tat gag 1152
Ile Leu Leu Ile Glu Ala Asn Gly Ser Ile Leu Tyr Lys Glu Tyr Glu
370 375 380
gat atg att gcc cag ccc tgt aca tgc aga tga 1185
Asp Met Ile Ala Gln Pro Cys Thr Cys Arg
385 390
<210>2
<211>394
<212>PRT
<213>猪(Sus scrofa)
<400>2
Met Val Leu Leu Ser Ile Ile Arg Thr Leu Leu Leu Trp Gly Leu Val
1 5 10 15
Leu Phe Met Glu His Arg Val Gln Met Thr Gln Val Gly Gln Pro Ser
20 25 30
Val Ala Leu Leu Pro Glu Ala Cys Thr Leu Pro Leu Ile Arg Glu Leu
35 40 45
Leu Glu Glu Ala Pro Gly Lys Gln Gln Arg Lys Pro Gln Val Leu Gly
50 55 60
His Pro Leu Arg Tyr Met Leu Glu Leu Tyr Gln Arg Ser Ala Asp Ala
65 70 75 80
Arg Gly His Pro Arg Glu Asn Arg Thr Ile Gly Ala Thr Met Val Arg
85 90 95
Leu Val Arg Pro Leu Val Asn Gly Ala Arg Pro Leu Arg Gly Pro Trp
100 105 110
His Ile Gln Thr Leu Asp Phe Pro Leu Arg Pro Asn Arg Val Ala Tyr
115 120 125
Gln Leu Val Arg Ala Thr Val Val Tyr Arg His Gln Leu His Leu Ala
130 135 140
Pro Phe His Leu Ser Cys His Val Glu Pro Trp Ile Gln Lys Ser Thr
145 150 155 160
Thr Ser His Phe Pro Ser Ser Gly Arg Gly Ser Leu Lys Pro Ser Leu
165 170 175
Leu Pro Gln Ala Trp Thr Glu Met Asp Val Thr Gln His Val Gly Gln
180 185 190
Lys Leu Trp Asn His Lys Gly Arg Arg Val Leu Arg Leu Arg Phe Met
195 200 205
Cys Gln Gln Gln Asn Gly Ser Glu Ile Leu Glu Phe Arg Gly Arg Gly
210 215 220
Ile Ser Ser Leu Asp Thr Ala Phe Leu Leu Leu Tyr Phe Asn Asp Thr
225 230 235 240
Arg Ser Val Gln Lys Ala Lys Leu Leu Pro Arg Gly Leu Glu Glu Phe
245 250 255
Met Ala Arg Asp Pro Ser Leu Leu Leu Arg Lys Ala Arg Gln Ala Gly
260 265 270
Ser Ile Ala Ser Glu Val Leu Gly Pro Ser Arg Glu His Asp Gly Pro
275 280 285
Glu Ser Asn Gln Cys Ser Leu His Pro Phe Gln Val Ser Phe His Gln
290 295 300
Leu Gly Trp Asp His Trp Ile Ile Ala Pro His Phe Tyr Thr Pro Asn
305 310 315 320
Tyr Cys Lys Gly Val Cys Pro Arg Val Leu His Tyr Gly Leu Asn Ser
325 330 335
Pro Asn His Ala Ile Ile Gln Asn Leu Val Asn Glu Leu Val Asp Gln
340 345 350
Ser Val Pro Gln Pro Ser Cys Val Pro Tyr Lys Tyr Val Pro Ile Ser
355 360 365
Ile Leu Leu Ile Glu Ala Asn Gly Ser Ile Leu Tyr Lys Glu Tyr Glu
370 375 380
Asp Met Ile Ala Gln Pro Cys Thr Cys Arg
385 390
<210>3
<211>1185
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>CDS
<222>(1)..(1185)
<223>
<400>3
atg gtc ctc ctc agc atc att aga acc ctt ctt ctt tgg gga ctg gtg 48
Met Val Leu Leu Ser Ile Ile Arg Thr Leu Leu Leu Trp Gly Leu Val
1 5 10 15
ctt ttt atg gaa cac agg gtc caa atg acc cag gta ggg caa ccc tct 96
Leu Phe Met Glu His Arg Val Gln Met Thr Gln Val Gly Gln Pro Ser
20 25 30
gtg gcc ctc ctg cct gag gcc tgt acc ttg ccc ctg att agg gag ctg 144
Val Ala Leu Leu Pro Glu Ala Cys Thr Leu Pro Leu Ile Arg Glu Leu
35 40 45
cta gag gaa gcc cct ggc aaa cag cag agg aag cca cag gtc ctg ggg 192
Leu Glu Glu Ala Pro Gly Lys Gln Gln Arg Lys Pro Gln Val Leu Gly
50 55 60
cat ccc ttg cga tat atg ctg gag ttg tac cag cgt tca gcc gac gca 240
His Pro Leu Arg Tyr Met Leu Glu Leu Tyr Gln Arg Ser Ala Asp Ala
65 70 75 80
cgt ggg cac cct agg gag aac cgc acc att ggg gcc acc atg gtg agg 288
Arg Gly His Pro Arg Glu Asn Arg Thr Ile Gly Ala Thr Met Val Arg
85 90 95
ctg gtg agg cca ttg gtt aat gga gca agg cct ctc aga ggg ccc tgg 336
Leu Val Arg Pro Leu Val Asn Gly Ala Arg Pro Leu Arg Gly Pro Trp
100 105 110
cat ata cag acc ttg gac ttt cct ctg aga cca aac cgg gta gcc tac 384
His Ile Gln Thr Leu Asp Phe Pro Leu Arg Pro Asn Arg Val Ala Tyr
115 120 125
caa cta gtc aga gcc act gtg gtt tat cgc cat caa ctt cac cta gct 432
Gln Leu Val Arg Ala Thr Val Val Tyr Arg His Gln Leu His Leu Ala
130 135 140
ccc ttc cac ctc tcc tgc cat gtg gag ccc tgg atc cag aaa agc aca 480
Pro Phe His Leu Ser Cys His Val Glu Pro Trp Ile Gln Lys Ser Thr
145 150 155 160
acc agt cac ttt cct tcc tca gga aga ggc tcc tta aag cct tcc ctg 528
Thr Ser His Phe Pro Ser Ser Gly Arg Gly Ser Leu Lys Pro Ser Leu
165 170 175
ctg ccc caa gct tgg acg gag atg gat gtc acg caa cat gtt gga caa 576
Leu Pro Gln Ala Trp Thr Glu Met Asp Val Thr Gln His Val Gly Gln
180 185 190
aag ctc tgg aat cac aag ggg cgc agg gtt cta cga ctc cgc ttc atg 624
Lys Leu Trp Asn His Lys Gly Arg Arg Val Leu Arg Leu Arg Phe Met
195 200 205
tgt cag cag caa aat ggt agt gag att ctt gag ttc cgg ggg cgt ggc 672
Cys Gln Gln Gln Asn Gly Ser Glu Ile Leu Glu Phe Arg Gly Arg Gly
210 215 220
att tca tcc ctg gac act gcc ttc ttg tta ctc tat ttc aat gac act 720
Ile Ser Ser Leu Asp Thr Ala Phe Leu Leu Leu Tyr Phe Asn Asp Thr
225 230 235 240
cgg agt gtt cag aag gcc aaa ctt ctt ccc aga ggc ctg gaa gag ttt 768
Arg Ser Val Gln Lys Ala Lys Leu Leu Pro Arg Gly Leu Glu Glu Phe
245 250 255
atg gca aga gac cct tct ctt ctt ttg cgg aag gcc cgg caa gca ggc 816
Met Ala Arg Asp Pro Ser Leu Leu Leu Arg Lys Ala Arg Gln Ala Gly
260 265 270
agc atc gca tct gag gtt ctt ggc ccc tcc agg gag cac gat ggg cct 864
Ser Ile Ala Ser Glu Val Leu Gly Pro Ser Arg Glu His Asp Gly Pro
275 280 285
gaa agt aac cag tgt tcc ctc cat cct ttc caa gtc agc ttc cac caa 912
Glu Ser Asn Gln Cys Ser Leu His Pro Phe Gln Val Ser Phe His Gln
290 295 300
ctg ggt tgg gat cat tgg atc att gct ccc cat ttc tat acc cca aac 960
Leu Gly Trp Asp His Trp Ile Ile Ala Pro His Phe Tyr Thr Pro Asn
305 310 315 320
tac tgt aag ggg gtc tgc cct cgg gta cta cac tat ggt ctc aat tcc 1008
Tyr Cys Lys Gly Val Cys Pro Arg Val Leu His Tyr Gly Leu Asn Ser
325 330 335
ccc aat cat gcc atc atc cag aac ctt gtc aat gag ctg gtg gac cag 1056
Pro Asn His Ala Ile Ile Gln Asn Leu Val Asn Glu Leu Val Asp Gln
340 345 350
agt gtc cct cag ccc tcc tgt gtc cct tat aag tat gtg cct att agc 1104
Ser Val Pro Gln Pro Ser Cys Val Pro Tyr Lys Tyr Val Pro Ile Ser
355 360 365
atc ctc ctg att gag gca aat ggg agt atc ttg tac aag gag tat gag 1152
Ile Leu Leu Ile Glu Ala Asn Gly Ser Ile Leu Tyr Lys Glu Tyr Glu
370 375 380
gat atg att gcc cag ccc tgt aca tgc aga tga 1185
Asp Met Ile Ala Gln Pro Cys Thr Cys Arg
385 390
<210>4
<211>394
<212>PRT
<213>猪(Sus scrofa)
<400>4
Met Val Leu Leu Ser Ile Ile Arg Thr Leu Leu Leu Trp Gly Leu Val
1 5 10 15
Leu Phe Met Glu His Arg Val Gln Met Thr Gln Val Gly Gln Pro Ser
20 25 30
Val Ala Leu Leu Pro Glu Ala Cys Thr Leu Pro Leu Ile Arg Glu Leu
35 40 45
Leu Glu Glu Ala Pro Gly Lys Gln Gln Arg Lys Pro Gln Val Leu Gly
50 55 60
His Pro Leu Arg Tyr Met Leu Glu Leu Tyr Gln Arg Ser Ala Asp Ala
65 70 75 80
Arg Gly His Pro Arg Glu Asn Arg Thr Ile Gly Ala Thr Met Val Arg
85 90 95
Leu Val Arg Pro Leu Val Asn Gly Ala Arg Pro Leu Arg Gly Pro Trp
100 105 110
His Ile Gln Thr Leu Asp Phe Pro Leu Arg Pro Asn Arg Val Ala Tyr
115 120 125
Gln Leu Val Arg Ala Thr Val Val Tyr Arg His Gln Leu His Leu Ala
130 135 140
Pro Phe His Leu Ser Cys His Val Glu Pro Trp Ile Gln Lys Ser Thr
145 150 155 160
Thr Ser His Phe Pro Ser Ser Gly Arg Gly Ser Leu Lys Pro Ser Leu
165 170 175
Leu Pro Gln Ala Trp Thr Glu Met Asp Val Thr Gln His Val Gly Gln
180 185 190
Lys Leu Trp Asn His Lys Gly Arg Arg Val Leu Arg Leu Arg Phe Met
195 200 205
Cys Gln Gln Gln Asn Gly Ser Glu Ile Leu Glu Phe Arg Gly Arg Gly
210 215 220
Ile Ser Ser Leu Asp Thr Ala Phe Leu Leu Leu Tyr Phe Asn Asp Thr
225 230 235 240
Arg Ser Val Gln Lys Ala Lys Leu Leu Pro Arg Gly Leu Glu Glu Phe
245 250 255
Met Ala Arg Asp Pro Ser Leu Leu Leu Arg Lys Ala Arg Gln Ala Gly
260 265 270
Ser Ile Ala Ser Glu Val Leu Gly Pro Ser Arg Glu His Asp Gly Pro
275 280 285
Glu Ser Asn Gln Cys Ser Leu His Pro Phe Gln Val Ser Phe His Gln
290 295 300
Leu Gly Trp Asp His Trp Ile Ile Ala Pro His Phe Tyr Thr Pro Asn
305 310 315 320
Tyr Cys Lys Gly Val Cys Pro Arg Val Leu His Tyr Gly Leu Asn Ser
325 330 335
Pro Asn His Ala Ile Ile Gln Asn Leu Val Asn Glu Leu Val Asp Gln
340 345 350
Ser Val Pro Gln Pro Ser Cys Val Pro Tyr Lys Tyr Val Pro Ile Ser
355 360 365
Ile Leu Leu Ile Glu Ala Asn Gly Ser Ile Leu Tyr Lys Glu Tyr Glu
370 375 380
Asp Met Ile Ala Gln Pro Cys Thr Cys Arg
385 390
<210>5
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>Intron
<222>(1)..(20)
<223>
<400>5
ctgcctttca ctgtttcctg 20
<210>6
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>6
ctc att ttt ctc ccc tcc ag 20
Leu Ile Phe Leu Pro Ser
1 5
<210>7
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>Intron
<222>(1)..(20)
<223>
<400>7
acatcacagc tcacagcaac 20
<210>8
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>8
aca tga agc gga gtc gta ga 20
Thr Ser Gly Val Val
1 5
<210>9
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>Intron
<222>(1)..(20)
<223>
<400>9
gtagcgtctg ccacctacat 20
<210>10
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>10
ccg gaa ctc aag aat ctc ac 20
Pro Glu Leu Lys Asn Leu
1 5
<210>11
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>11
aga gcc act gtg gtt tat cg 20
Arg Ala Thr Val Val Tyr
1 5
<210>12
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>12
gaa agg atg gag gga aca ct 20
Glu Arg Met Glu Gly Thr
1 5
<210>13
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>exon
<222>(1)..(20)
<223>
<400>13
taa cca gtg ttc cct cca tc 20
Pro Val Phe Pro Pro
1 5
<210>14
<211>20
<212>DNA
<213>猪(Sus scrofa)
<220>
<221>Intron
<222>(1)..(20)
<223>
<400>14
cctgggaaac ctgatctagc 20