CN1587409A - 一种斜带石斑鱼SRY盒相关基因Sox及其应用 - Google Patents
一种斜带石斑鱼SRY盒相关基因Sox及其应用 Download PDFInfo
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Abstract
一种斜带石斑鱼SRY盒相关基因Sox及其应用。本发明从斜带石斑鱼性腺中发现了基因Sox,该基因在斜带石斑鱼的性腺发育和性逆转中有重要的应用。
Description
技术领域
本发明涉及基因工程领域,具体地说是一种斜带石斑鱼SRY盒相关基因Sox及其应用。
背景技术
斜带石斑鱼(Epinephelus coioides),属鲈形目、鮨科、石斑鱼亚科,石斑鱼属。其肉质鲜美,营养丰富,为餐桌中的上等佳肴。由于其生长快、市场价格高而稳定,是目前我国沿海地区重要的海水养殖经济鱼类。斜带石斑鱼是一种雌性先熟的雌雄同体鱼类,开始为雌性,在体长为250~400mm时发生性逆转,雄性需6龄左右才成熟,在人工繁殖时常会遇到难以同时得到功能性雄性和雌性亲鱼的问题,给种苗生产造成很大困难。因此,如何使斜带石斑鱼提早性转变,尽早获得功能性的雄鱼显得非常重要。目前,在诱导斜带石斑鱼提前性逆转的研究中,主要还是应用性类固醇激素,如甲基睾酮(MT)。但MT是一种禁药,不易购买,而且使用后可能会造成环境激素污染。
动物性腺分化发育受多种内在和外在因素调节,归根结底,它是受到基因表达的调节。有关哺乳动物的研究表明:SRY基因是雄性决定基因,它编码一个转录调节因子,含有一个与DNA结合的HMG区域。随后的研究发现动物体内存在一大类这样的转录调节因子,它们的HMG区域与SRY的HMG区域有高度的同源性,这一类转录因子被命名为SRY盒相关基因,即Sox基因。目前,根据HMG区域同源性的高低,Sox基因分为10大类(包括SRY),即A,B,C,D,E,F,G,H,I,J类。不同的Sox有不同的功能,它们参与动物体多种生命活动的调节,如神经系统的发育,软骨和血的生成,性腺的分化发育等。在鱼类中,目前还没有找到一种决定性别分化的SRY盒基因。如能找到与性腺分化发育相关的基因,则可以通过多种方法,如转基因技术、基因敲除技术等,调控该基因的表达,从而达到控制性腺分化发育的目的。
发明内容
本发明的目的在于提供一种与斜带石斑鱼性腺分化发育相关的功能基因。
本发明的另一个目的提供该基因在斜带石斑鱼性腺发育和性逆转中的应用。
本发明从4龄斜带石斑鱼性腺(III期早期卵巢)中提取总RNA,然后用SMART cDNA文库构建试剂盒(Invitrogen)构建了斜带石斑鱼卵巢cDNA文库。根据Sox基因的保守序列,设计了一对简并引物,以斜带石斑鱼卵巢反转录产物为模板,进行PCR扩增,获得一条220bp的扩增片段,测序结果表明它是一种Sox基因的片段。然后,分别进行3’和5’RACE,获得了该Sox基因的全长cDNA序列。
根据该Sox基因的核苷酸序列设计了特异性引物,应用RT-PCR方法分析了Sox基因表达的组织分布和处于不同发育阶段的斜带石斑鱼性腺Sox基因的表达水平,结果表明:斜带石斑鱼Sox基因表达水平在性腺比在其他组织高,另外,Sox基因在IV期早期的卵巢表达量比II期卵巢高,而当性腺处于间性初期时表达量开始下降,在雄性时表达量进一步下降。这些结果显示了该Sox基因参与了斜带石斑鱼性腺发育和性逆转的调节。因此,可以应用该基因的产品或调控该基因的表达来控制斜带石斑鱼的性腺发育。
目前,在诱导斜带石斑鱼提前性逆转的研究中,主要还是应用性类固醇激素,如甲基睾酮(MT)。但MT是一种禁药,不易购买,而且使用后可能会造成环境激素污染。而应用该Sox基因的产品或调控该Sox基因的表达来控制斜带石斑鱼的性腺发育,具有高特异性和敏感性,以及无环境激素污染等优点。
附图说明
图1为用于构建卵巢cDNA文库的卵巢组织切片图;
图2为卵巢总RNA电泳图;
图3为克隆斜带石斑鱼Sox基因初始cDNA片段的PCR扩增产物电泳图;
图4为斜带石斑鱼Sox基因5’RACE产物电泳图;
图5为斜带石斑鱼Sox基因3’RACE产物电泳图;
图6为一步法RT-PCR分析斜带石斑鱼Sox基因表达的组织分布图;
图7为两步法RT-PCR分析斜带石斑鱼Sox基因在不同发育状况的性腺中的表达图。
具体实施方式
实施例1 Sox基因的获得
(1)取样:4龄斜带石斑鱼购自广东省大亚湾水产养殖中心海水养殖基地。解剖鱼体,取出性腺,一部分性腺样品冻于液氮,另一部分性腺样品用Bouin’s固定液固定,石蜡切片,组织切片观察发现性腺为III期早期卵巢,如图1所示。
(2)RNA提取:用TrizolReagent提取性腺总RNA,在1%的琼脂糖凝胶上电泳,检查RNA质量。电泳显示:18S和28S核糖体RNA条带清晰,表明所提取的卵巢RNA质量较高。如图2所示,图中已标出18S和28S核糖体RNA的位置。
(3)cDNA文库构建:取1μg性腺总RNA,应用SMARTTTM cDNA文库构建试剂盒(Invitrogen),构建了斜带石斑鱼卵巢cDNA文库。文库扩增后的滴度为1.2×1010pfu/ml,文库的重组率达到99%,表明cDNA文库质量较高。
(4)Sox基因的克隆:以性腺反转录产物为模板,应用简并引物F1(5’cac at(ca)aa(ag)cg(ag)cc(cga)atg aa(tc)gc 3’)和R1(5’gc ttt ggttt(ct)ttc(tc)t(gc)gg(tc)c 3’)进行PCR,获得一个220bp的扩增片段,如图3所示,把该片段克隆到T-载体pUCm-T,测序分析表明该片段为一种Sox基因cDNA片段。以性腺cDNA文库为模板,引物5’LDAMP(5’CTC GGG AAG CGC GCC ATT GTG TTG GT 3’)和基因特异性引物Qo-SOXRl(5’ACC GCT TCC CGA GCC GTT TAG AG3’)进行5’RACE,获得Sox cDNA的5’端序列,如图4所示,箭头所示400bp左右的扩增片段;以性腺反转录产物为模板(Promega试剂盒),基因特异性引物QO-SOX-F3(5’ACA CCG AGA AGA TCC CGTTCA TCC 3’)和3’接头引物BRL2(5’GGC CAC GCG GCG ACTAGT AC3’)进行3’RACE,获得Sox cDNA的3’端序列,如图5所示,箭头所示2.0kb左右的扩增片段;然后,拼接3’RACE和5’RACE的测序结果,得出斜带石斑鱼的Sox全长cDNA核苷酸序列,并推导出其编码的氨基酸序列,如序列表所示。
实施例2 Sox基因的应用
(1)斜带石斑鱼Sox基因表达的组织分布:从斜带石斑鱼不同组织中提取总RNA,用RNase-free的DNaseI去除其中可能存在的基因组DNA的污染,然后取0.5ug RNA,应用一步法反转录PCR(One-stepRT-PCR)分析试剂盒(Invitrogen)分析Sox基因在不同组织的表达,所用引物为QOSOXFl(5’GAT CAT GGT TCA ACA CAC GGA AC3’)和QOSOXRl(5’ACC GCT TCC CGA GCC GTT TAG AG3’),PCR循环数为39,扩增产物大小为220bp。一步法RT-PCR结果表明斜带石斑鱼Sox基因表达水平在性腺比在其他组织高,如图6所示,M,100bp DNA分子量标准;1为阴性对照(模板为水);2为4龄斜带石斑鱼性腺(III期早期卵巢);3-10分别为2龄斜带石斑鱼性腺(II期卵巢)、垂体、延脑、下丘脑、后脑、中脑、前脑、和嗅球。
(2)斜带石斑鱼Sox基因在处于不同发育时期的性腺中的表达:选取处于不同发育阶段(雌性、间性和雄性)的性腺,提取总RNA,然后取5ug RNA,用DNaseI处理去除基因组DNA污染后,用Thermoscript RT-PCR系统(Invitrogen)进行RT-PCR分析,研究Sox基因表达状况,PCR循环数为28。RT-PCR分析结果表明:IV期早期的卵巢表达量比II期卵巢高,另外,性腺处于间性初期时表达量开始下降,在雄性时表达量进一步下降,表示该基因参与了调控斜带石斑鱼性腺发育和性逆转,如图7所示,M,100bp DNA分子量标准;1,II期卵巢;2,IV期早期卵巢;3,间性早期性腺;4,雄性精巢;5,阴性对照(模板为水)。
在所检测的二龄斜带石斑鱼各组织中,性腺表达Sox基因的水平最高。在斜带石斑鱼处于间性期,该Sox基因表达量下降,而在雄性阶段,表达量进一步下降,表明该Sox基因在斜带石斑鱼性腺发育和性逆转过程中起重要的调节作用。因此,可以通过调节该Sox基因的表达来控制斜带石斑鱼的性腺发育和性逆转,人工诱导斜带石斑鱼提前性逆转。
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ttgttttgcg gatcagaaac tgatccggtc tatgcagact gacctctaat gacctcagat 1745
atcaggacgg acacctccgt ccttccagct ccacctggac tcctcttgga ctcacctgca 1805
cccgttttgg actcctcctg taggactgtt ttatgtgact cagtttttta tttgctgtct 1865
gatgttaagc tccgcctcct tcgggaggaa acaggaaacc ttattttgat aagaacaaac 1925
SOXST2~1.TXT
atacctcatc ttttttaagg agtaaagata ttttcaggac tatttttgta cagtcggatt 1985
ttcaggcact tgttgtgaat ataataagta atctgagtta tgaacatgtg accgacctgc 2045
tgttgtttta aacgtgttca gacaggaagt cagaccttta ttgtgaaagg gaggaagttg 2105
tatatgttgt gaaaaatagt taaagttgga ttttctgtta catgttttat tttcggcacg 2165
tttgtttttc tcatcatttt ggtgcaatat aaagaaaagt gaaaaattaa aggaccaaaa 2225
gataaaaaaa aaaaaaaaa aaaaaaaa 2253
<210>2
<211>389
<212>PRT
<213>斜带石斑鱼(Epinephelus coioides)
<400>2
Met Val Gln His Thr Glu His Gly Gly Pro Arg Ile His Ser Gly Ser
1 5 10 15
Arg Glu Ala Ala Asp Thr Glu Asp Ser Glu Leu Leu Ala Ala Ser Val
20 25 30
Cys Gly Pro Val Pro Gln Lys Ala Asp Trp Cys Lys Thr Ala Ser Gly
35 40 45
His Ile Lys Arg Pro Met Asn Ala Phe Met Val Trp Ser Lys Ile Glu
50 55 60
Arg Arg Lys Ile Met Glu Gln Ser Pro Asp Met His Asn Ala Glu Ile
65 70 75 80
Ser Lys Arg Leu Gly Lys Arg Trp Lys Met Leu Lys Asp Thr Glu Lys
85 90 95
Ile Pro Phe Ile Arg Glu Ala Glu Arg Leu Arg Leu Lys His Met Ala
100 105 110
Asp Tyr Pro Asp Tyr Lys Tyr Arg Pro Lys Lys Lys Pro Lys Thr Asp
115 120 125
Gly Cys Ala Phe Pro Thr Gly Lys Leu Ala Ala Gln Ser Pro Glu Lys
130 135 140
Thr Ser Val Arg Pro Val Tyr Lys Lys Asn Leu Ser Lys Leu Lys pro
145 150 155 160
Ser Lys Pro Val Thr Gly Ser Gly Arg Ala Ile Ser Leu Leu Pro Gln
165 170 175
His Gln His Pro Asp His Arg Tyr Arg Tyr Val Leu Thr Thr Ser Phe
180 185 190
Lys Pro Asn Lys Phe Gly Lys Arg Asp Phe Thr Asp Asp Glu Glu Glu
195 200 205
Glu Asp Asp Asp Asp Glu Asp Asp Asp Glu Gly Asp Ser Glu Glu Glu
210 215 220
SOXST2~1.TXT
Arg Gly Phe Lys Leu Glu Glu Glu Asp Glu Pro Ser Arg Tyr Ser Leu
225 230 235 240
Gly Leu Ser Ser Ala Ala Ala Glu Pro Glu Gly Thr Gly Met Tyr Glu
245 250 255
Ser Ser Gly Arg Leu Phe Tyr Ser Phe Lys Asn Ile Thr Arg Gln Gly
260 265 270
Thr Gly Thr Gly Thr Gly Ser Tyr Pro Ala Ser Leu Ser Pro Ala Ser
275 280 285
Ser Ser Arg Ser Ser Ser Ser Cys Cys Gly Glu Asp Leu Asp Asp Leu
290 295 300
Pro Ala Glu Gly Thr Asp Phe Thr Leu Ser Leu Leu Ala Gly Leu His
305 310 315 320
Ala Ser Ser Glu Pro Trp Ser Ser Ser Ser Ser Ser Ala Pro Gly Asn
325 330 335
Leu Gly Leu Ser Leu Val Asp Leu Asp Leu Asp Ser Cys Gly Ser Glu
340 345 350
Arg Thr Leu Gly Ser His Phe Asp Phe Pro Asp His Cys Thr Pro Glu
355 360 365
Leu Ser Glu Met Ile Ala Gly Asn Trp Leu Glu Val Asn Phe Ser Asp
370 375 380
Leu Val Phe Thr Tyr
385
Claims (4)
1、一种斜带石斑鱼SRY盒相关基因Sox,其核苷酸序列如序列表所示。
2、根据权利要求1所述的斜带石斑鱼SRY盒相关基因Sox,其特征在于所述基因Sox的核苷酸序列所编码的氨基酸序列如序列表所示。
3、根据权利要求1所述的斜带石斑鱼SRY盒相关基因Sox,其特征在于所述基因Sox在石斑鱼性腺发育和性逆转中的应用。
4、根据权利要求1所述的斜带石斑鱼SRY盒相关基因Sox,其特征在于所述基因Sox在斜带石斑鱼性腺发育和性逆转中的应用。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100421551C (zh) * | 2006-12-26 | 2008-10-01 | 中国水产科学研究院黄海水产研究所 | 半滑舌鳎性逆转的诱导方法 |
CN103740701A (zh) * | 2014-01-07 | 2014-04-23 | 四川农业大学 | 一种克隆猪Sox6基因编码区全序列的方法 |
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2004
- 2004-08-24 CN CN 200410051198 patent/CN1587409A/zh active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100421551C (zh) * | 2006-12-26 | 2008-10-01 | 中国水产科学研究院黄海水产研究所 | 半滑舌鳎性逆转的诱导方法 |
CN103740701A (zh) * | 2014-01-07 | 2014-04-23 | 四川农业大学 | 一种克隆猪Sox6基因编码区全序列的方法 |
CN103740701B (zh) * | 2014-01-07 | 2016-04-06 | 四川农业大学 | 一种克隆猪Sox6基因编码区全序列的方法 |
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