CN111560378B - 一种鞍带石斑鱼gsdf基因及其应用 - Google Patents
一种鞍带石斑鱼gsdf基因及其应用 Download PDFInfo
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- CN111560378B CN111560378B CN202010272964.7A CN202010272964A CN111560378B CN 111560378 B CN111560378 B CN 111560378B CN 202010272964 A CN202010272964 A CN 202010272964A CN 111560378 B CN111560378 B CN 111560378B
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Abstract
本发明公开了一种鞍带石斑鱼gsdf基因及其编码蛋白,以及gsdf基因的构建方法。还公开了上述种鞍带石斑鱼gsdf基因作为雄性sertoli细胞的标记基因方面的应用。并进一步公开了一种鞍带石斑鱼gsdf重组表达载体以及该重组表达载体在制备具有鞍带石斑鱼性转变效果的诱导剂中的应用。
Description
技术领域
本发明属于gsdf基因技术领域,具体涉及一种鞍带石斑鱼gsdf基因及其应用。
背景技术
GSDF(gonadal soma-derived factor)是TGF-β家族成员,由Sawatari于2007年在研究虹鳟的性别分化机制时发现。随后,研究人员在多种硬骨鱼类都克隆到了该基因。在除鱼类以外的哺乳类、两栖类和鸟类等脊椎动物中尚未发现GSDF基因的存在。在吕宋青鳉的研究中发现gsdf在性别决定前高表达于XY胚胎中,在XX雌性个体中转基因过表达gsdfY(位于Y染色体上的gsdf基因)导致其性逆转为XX雄性,故研究认为gsdf可能是其雄性性别决定候选基因;在日本青鳉最近的研究中也发现用基因技术敲除gsdf基因,导致其性腺发育为卵巢,在XY型罗非鱼中敲除gsdf基因,也得到相同的结果。检测gsdf基因在各个组织的表达分布,发现其主要在性腺中特异表达,在***中的表达高于卵巢。说明gsdf基因参与了性腺特别是***的发育过程。在虹鳟的相关研究中,采用重组gsdf对***细胞进行培养,发现gsdf基因能促进原始生殖细胞和A型***的增殖。越来越多的研究表明gsdf基因在鱼类性别决定、分化和雄性生殖细胞的增殖过程中发挥必不可少的作用。
鞍带石斑鱼(Epinephelus lanceolatus)因其味道鲜美、营养丰富而广受欢迎,是一种具有很高经济价值的食用鱼类。鞍带石斑鱼是典型的雌雄同体鱼类,生殖过程中要经历雌性过程后,再转变为雄性,生殖周期很长,获得雄性一般都需要6年以上,在进行人工繁殖时,雄性亲鱼非常稀缺。而且由于环境的破坏和过渡捕捞,在自然海域中捕捞到野生雄性亲鱼机会更加渺茫。因此,如何获得功能性的雄性亲鱼成为鞍带石斑鱼人工繁育工作能否顺利进行的关键。所以对于研究其生殖机制是很有必要的。
现有技术中尚未有诱导鞍带石斑鱼性逆转的成熟方法,但有相关的研究,相关的研究主要是采用雄激素投喂或埋植处理来促进鞍带石斑鱼的性逆转。使用激素的方法有明显的不足之处:投喂处理时,使用的激素量较大,投喂周期长,造成的水体环境污染严重和诱导性逆转效率低等问题;雄激素埋植时,需要开展外科手术,操作麻烦,并且手术导致外伤而且病原感染,死亡率高。
发明内容
本发明的目的在于提供一种鞍带石斑鱼gsdf基因及其编码蛋白,以及gsdf基因的构建方法,其构建了一种新的基因,该基因可以作为雄性sertoli细胞的标记基因,具有鞍带石斑鱼性转变效果,无需投喂激素处理和雄激素埋植。
本发明的目的还在于提供上述种鞍带石斑鱼gsdf基因或所述gsdf基因的编码蛋白作为雄性sertoli细胞的标记基因方面的应用。
本发明的最后一个目的在于提供一种鞍带石斑鱼gsdf重组表达载体以及上述鞍带石斑鱼gsdf基因、编码蛋白以及含有鞍带石斑鱼gsdf基因的重组表达载体在制备具有鞍带石斑鱼性转变效果的诱导剂中的应用。
本发明的上述第一个目的可以通过以下技术方案来实现:一种鞍带石斑鱼gsdf基因,其核苷酸序列如SEQ ID NO:1所示。
上述鞍带石斑鱼gsdf基因的编码蛋白,其氨基酸序列如SEQ ID NO:2所示。
上述的鞍带石斑鱼gsdf基因的构建方法,包括以下步骤:
(1)从鞍带石斑鱼中提取总RNA;
(2)以步骤(1)所提取的总RNA为模板,以RNA Oligo dT为引物,其核苷酸序列如SEQ ID NO:7所示,逆转录合成cDNA;
(3)以步骤(2)所合成的cDNA为模板,以SEQ ID NO:3、SEQ ID NO:4所示的序列为上、下游引物,通过PCR扩增,得到鞍带石斑鱼gsdf基因。
本发明的上述第二个目的可以通过以下技术方案来实现:上述鞍带石斑鱼gsdf基因或所述gsdf基因的编码蛋白作为雄性sertoli细胞的标记基因方面的应用。
本发明的上述第三个目的可以通过以下技术方案来实现:一种鞍带石斑鱼gsdf重组表达载体,包括上述鞍带石斑鱼gsdf基因。
作为本发明一种优选的实施方式,所述重组表达载体为pcDNA4.0-gsdf。
上述鞍带石斑鱼gsdf重组表达载体的制备方法,包括以下步骤:
(1)从鞍带石斑鱼中提取总RNA;
(2)以步骤(1)所提取的总RNA为模板,以RNA Oligo dT为引物,其核苷酸序列如SEQ ID NO:7所示,逆转录合成cDNA;
(3)以步骤(2)所合成的cDNA为模板,以SEQ ID NO:5、SEQ ID NO:6所示的序列为上、下游引物,通过PCR扩增,获得具有酶切位点的鞍带石斑鱼gsdf基因,与KpnI和XhoI酶切的pcdan4.0载体连接,得到鞍带石斑鱼gsdf重组表达载体pcDNA4.0-gsdf。
上述鞍带石斑鱼gsdf基因、上述gsdf基因的编码蛋白或所述重组表达载体在制备具有鞍带石斑鱼性转变效果的诱导剂中的应用。
与现有技术相比,本发明具有如下优点:本发明构建了一种鞍带石斑鱼gsdf基因,该基因可以作为雄性sertoli细胞的标记基因,具有鞍带石斑鱼性转变效果,本发明可以采用体内注射的微创方式,将鞍带石斑鱼内源性的调控因子gsdf基因实现体内重组表达,操作简便,安全健康及效率高,显示出比现有技术更高的优势。
附图说明
图1是实施例1中鞍带石斑鱼gsdf cDNA全长克隆电泳图,其中M:DNA 2kb分子量标准;
图2是实施例2中鞍带石斑鱼gsdf基因在***中的表达定位,白色箭头指示的为Steroid细胞;
图3是实施例3中鞍带石斑鱼gsdf基因重组质粒在体注射斜带石斑鱼的性腺组织学切片观察结果,其中A:对照组;B:gsdf基因重组质粒注射组。
具体实施方式
下面结合附图和实施例对本发明做进一步说明,但不以任何形式限制本发明。
实施例1鞍带石斑鱼gsdf基因、其编码的蛋白
本实施例中的鞍带石斑鱼gsdf基因,其通过引物扩增基因片段和RACE全长扩增的方法得到鞍带石斑鱼gsdf基因开放读码框。
具体的,本实施例提供的鞍带石斑鱼gsdf基因的构建方法,包括以下步骤:
(1)从鞍带石斑鱼中提取总RNA;
(2)以步骤(1)所提取的总RNA为模板,以RNA Oligo dT为引物,其核苷酸序列如SEQ ID NO:7所示(tttttttttttttttttt),逆转录合成cDNA;
(3)以步骤(2)所合成的cDNA为模板,以SEQ ID NO:3(具体为atgtcctttacgttcatcgt)、SEQ ID NO:4(具体为ttactctgtgccgggctgct)所示的序列为上、下游引物,构建PCR扩增反应体系(表1),反应程序为:94℃预变性4min,94℃变性30s,58℃退火30s,72℃延伸1min,35个循环,72℃延伸1min。
表1 PCR扩增反应体系
扩增片段大小为633bp,电泳分离DNA片段,切胶回收目的产物(图1),将纯化后的目的产物连接至pGEM-T载体,转化DH5α大肠杆菌,挑选阳性克隆,送专业的测序公司进行序列测序,获得gsdf基因的cDNA序列片段即鞍带石斑鱼gsdf基因的开放读码框(鞍带石斑鱼gsdf基因,引物设计直接从起始密码开始到终止密码,克隆出来的序列直接是开放阅读框,克隆出来的cDNA序列和开放阅读框一样长,直接翻译过来就是氨基酸序列)
获得的鞍带石斑鱼gsdf基因,其核苷酸序列如SEQ ID NO:1所示。
具体为:
atgtcctttacgttcatcgtcatgatgatgcttctgggctcttcagtggtgattgcatttgtcttgcagccatccagggaagaacctgcagcctctgctaactctccagtttaccatcacaggtgtgagtcattgcagtccatgaggaagggtctcctcagggctctcaacttgcaggctgagccacagctgcctgttggtgggctggacggtgtcagagagcagtggaggaagaccttcaggaccctcactcacagagctaaggacactgcagttccagcagtgtctgtgtcacctgatgctgggaatggaacgagcctggagtgctgctccatggcttctgagatctttatgaaagatctgggatgggacaactgggtgatccatcctgccagccttaccatcgttcactgtgcaatctgtaaccctgatctgaacactgtgcaatgtccatcatcccatgccaacgtccaagaagctgactcacaggtgtcctgttgtcagcccacctcccaggaaatggtgcctgttctctacgtggatgaattcagcaccctggtcatttcctccgtgcagctgacccgcagctgtggctgtgggcctggcaccctccagcagcccggcacagagtaa
对应的氨基酸序列如SEQ ID NO:2所示:
MSFTFIVMMMLLGSSVVIAFVLQPSREEPAASANSPVYHHRCESLQSMRKGLLRALNLQAEPQLPVGGLDGVREQWRKTFRTLTHRAKDTAVPAVSVSPDAGNGTSLECCSMASEIFMKDLGWDNWVIHPASLTIVHCAICNPDLNTVQCPSSHANVQEADSQVSCCQPTSQEMVPVLYVDEFSTLVISSVQLTRSCGCGPGTLQQPGTE 210。
该鞍带石斑鱼gsdf基因编码的蛋白,其编码210个氨基酸,分子量为22.8千道尔顿。
实施例2鞍带石斑鱼gsdf基因***组织原位杂交
(1)质粒提取
将连入pGEM-T载体的gsdf基因的ORF区片段,进行无内毒素质粒提取。
(2)片段线性化
按照表2的酶切体系,将质粒分别进行SphI和PstI的单酶切,并将片段线性化后进行纯化;
表2酶切反应体系
(3)体外转录及RNA纯化
参考DIG RNALabelingKit(SP6/T7)(Roche,美国)说明书进行体外转录及RNA纯化。
①在冰上配置体外转录反应体系(表3),在PCR仪上37℃反应2h;
表3 PCR反应溶液
②加入2μLDNaseI,在PCR仪上37℃反应15min后加入2μL EDTA终止反应。
③向上述体系中加入75μL预冷的无水乙醇和2.5μL LiCl,-80℃放置30min;
④取出,转移到1.5mL离心管中,4℃,12000rpm,离心15min,弃上清;
⑤加入50μL预冷的DEPC水配置的70%乙醇,4℃,12000rpm,离心5min,清洗RNA;
⑥弃上清,待乙醇挥发2min后加入50μL DEPC水和1μLRNaseinhibitor;
⑦测浓度和电泳检测,剩余探针分装并放入-80℃冰箱备用。
(4)原位杂交
第一天:杂交(所有试剂均使用DEPC水配置)
①室温用1×PBS浸泡,10min×2;
②室温用DEPC水浸泡,5min;
③室温条件用0.2M HCl浸泡,10min;
0.2M HCl:40mL1×PBS+200μL 40%HCl
④室温用1×PBS浸泡,5min×2;
⑤37℃用1μg/mL蛋白酶K浸泡15min;
1μg/mL蛋白酶K:50mL1×PBS+2.5μL蛋白酶K(20μg/mL)
⑥室温用1×PBS浸泡,5min×2,;
⑦室温用2×SSC浸泡,5min×2,配置杂交液(表4);
表4探针杂交液:
注:先将1、2、3配好,60℃水浴促溶,之后再加入4、5、6。
⑧切片放入湿盒,每片加入200μL探针杂交液,加入探针,放入分子杂交箱,55℃过夜(12-16h)。
第二天:洗片、显色(所有试剂均可以不使用DEPC水配置)
①室温用2×SSC浸泡,30min;
②60℃用2×SSC浸泡,30min×2;
②60℃用0.1×SSC浸泡,30min×2;
③室温用配置的Buffer1缓冲液(表5)浸泡5min;
表5 Buffer 1缓冲液
④将切片放入湿盒,室温用Blocking Solution溶液(表6)敷育30min;
表6 Blocking Solution溶液
⑥室温用Anti-DIG敷育2h(也可4℃过夜);
Anti-DIG:DIG-AP 2μL+1mL Buffer 1。
⑦转移到新的染缸,室温用Buffer 1清洗10min×2;
⑧室温用Buffer 2缓冲液(表7)清洗5min;
表7 Buffer 2缓冲液
⑨室温显色,200μL每张片,尽量到显微镜下显色观察;
⑩显色完成后,放入PBS中停止显色;加入DAPI显色。
原位杂交结果:
用鞍带石斑鱼***的冰冻切片,采取原位杂交的方法检测了gsdfmRNA在鞍带石斑鱼斑鱼性腺中的定位。如图2所示:反义探针检测得出,gsdf mRNA在***每个精小囊外侧的体细胞中观察到强烈且特异性的信号,高表达在Steroid细胞区域,而在生殖细胞内没有发现强信号。
因此,本发明用荧光原位杂交的方法发现鞍带石斑鱼gsdf基因的表达定位在***的sertoli细胞,可以作为sertoli细胞的标记基因。
实施例3鞍带石斑鱼gsdf基因重组质粒在体注射斜带石斑鱼
(1)构建过表达质粒
用上游引物序列如SEQ ID NO:5(具体为ttaaacttaagcttggtaccgccaccatgtcctttacgttcatc)和下游引物序列如SEQ ID NO:6(具体为gccctctagactcgagttactctgtgccgggctgct)扩增出gsdf序列。将pcDNA4.0载体用KpnI和XhoI双酶切后,用in-Fusion的方法将载体和扩增出的gsdf序列连接,得到pcDNA4.0-gsdf重组质粒(重组表达载体)。
用脂质体包封pcDNA4.0-gsdf重组质粒,脂质体通过使用DOTAP(Sigma,用于形成水合模覆盖在脂质体表面,便于体内细胞吸收,使重组质粒进入细胞,实现过表达)和胆固醇(Sigma)的薄膜水合形成。脂质体的制备从我们之前的研究,进行了修改。简言之,将DOTAP和胆固醇加热至70℃,然后在65℃下进行2小时水浴并在25℃下进行10分钟超声波处理。将混合物用100nm膜过滤器,然后将pcDNA4.0空载质粒和pcDNA4.0-gsdf质粒在25℃下通过30分钟超声波封装。构建体的浓度为20μg/μL。
(2)在体注射
将鞍带石斑鱼分为两组。实验组(n=50):gsdf过表达质粒(pcDNA4.0-gsdf);对照组(n=50):空质粒(空pcDNA4.0)。两组均按(1mL质粒/kg体重)的比例,每周注射,收集性腺组织和血清样品。
性腺组织:麻醉处死,剪开腹部,取出性腺。一部分放于波恩氏液中,固定过夜,更换为70%的酒精;一部分放于冻存管中,立即放于液氮。
血清样品:尾静脉抽血,放于1.5mL离心管中,平放静置2-3h后,5000g,4℃,离心10min,取上清,用干冰转移至-80℃冰箱,备用。
(3)组织学分析
①石蜡包埋:将波恩氏液固定后保存于70%酒精的样品经脱水、透明和浸蜡处理,将处理完成的组织样品转移至装有已融化石蜡的小纸盒内,室温静置,凝固后即可使用。
②切片:包埋好的组织石蜡块在切片机上以5μm的厚度切片,用30%的酒精展片后,在42℃水浴中完全展片,42℃烘烤过夜。
③伊红染色:将染好的玻片,置于水平台面,用中性树胶封片,风干后观察。
结果:
组织学切片观察,在pcDNA4.0-gsdf过表达质粒短期注射45天时的性腺变化。实验组每七天腹腔注射一次gsdf过表达质粒,而对照组则注射pcDNA4.0空载质粒,45天时分别随机捞取7条鱼,性腺取样并进行组织学切片观察。由图3发现,45天时,对照组为普通的卵巢结构,而实验组有大量的性原细胞产生,说明了GSDF有促进鞍带石斑鱼转雄的作用。
因此,可以将鞍带石斑鱼gsdf基因及其重组表达载体在制备具有鞍带石斑鱼性转变效果的诱导剂方面应用。
本发明不局限于上述特定的实施方案范围内,上述实施方案仅仅是为了能够对本发明的使用过程进行详细地说明,而且有相等功能的生产方法和技术细节也属于本发明内容的一部分。事实上,本领域技术人员根据前文的描述,就能够根据各自需要找到不同的调整方案,这些调整都应在本文所附的权利要求书的范围内。
序列表
<110> 南方海洋科学与工程广东省实验室(湛江)
广东海洋大学
<120> 一种鞍带石斑鱼gsdf基因及其应用
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 633
<212> DNA
<213> 鞍带石斑鱼(Epinephelus lanceolatus)
<400> 1
atgtccttta cgttcatcgt catgatgatg cttctgggct cttcagtggt gattgcattt 60
gtcttgcagc catccaggga agaacctgca gcctctgcta actctccagt ttaccatcac 120
aggtgtgagt cattgcagtc catgaggaag ggtctcctca gggctctcaa cttgcaggct 180
gagccacagc tgcctgttgg tgggctggac ggtgtcagag agcagtggag gaagaccttc 240
aggaccctca ctcacagagc taaggacact gcagttccag cagtgtctgt gtcacctgat 300
gctgggaatg gaacgagcct ggagtgctgc tccatggctt ctgagatctt tatgaaagat 360
ctgggatggg acaactgggt gatccatcct gccagcctta ccatcgttca ctgtgcaatc 420
tgtaaccctg atctgaacac tgtgcaatgt ccatcatccc atgccaacgt ccaagaagct 480
gactcacagg tgtcctgttg tcagcccacc tcccaggaaa tggtgcctgt tctctacgtg 540
gatgaattca gcaccctggt catttcctcc gtgcagctga cccgcagctg tggctgtggg 600
cctggcaccc tccagcagcc cggcacagag taa 633
<210> 2
<211> 210
<212> PRT
<213> 鞍带石斑鱼(Epinephelus lanceolatus)
<400> 2
Met Ser Phe Thr Phe Ile Val Met Met Met Leu Leu Gly Ser Ser Val
1 5 10 15
Val Ile Ala Phe Val Leu Gln Pro Ser Arg Glu Glu Pro Ala Ala Ser
20 25 30
Ala Asn Ser Pro Val Tyr His His Arg Cys Glu Ser Leu Gln Ser Met
35 40 45
Arg Lys Gly Leu Leu Arg Ala Leu Asn Leu Gln Ala Glu Pro Gln Leu
50 55 60
Pro Val Gly Gly Leu Asp Gly Val Arg Glu Gln Trp Arg Lys Thr Phe
65 70 75 80
Arg Thr Leu Thr His Arg Ala Lys Asp Thr Ala Val Pro Ala Val Ser
85 90 95
Val Ser Pro Asp Ala Gly Asn Gly Thr Ser Leu Glu Cys Cys Ser Met
100 105 110
Ala Ser Glu Ile Phe Met Lys Asp Leu Gly Trp Asp Asn Trp Val Ile
115 120 125
His Pro Ala Ser Leu Thr Ile Val His Cys Ala Ile Cys Asn Pro Asp
130 135 140
Leu Asn Thr Val Gln Cys Pro Ser Ser His Ala Asn Val Gln Glu Ala
145 150 155 160
Asp Ser Gln Val Ser Cys Cys Gln Pro Thr Ser Gln Glu Met Val Pro
165 170 175
Val Leu Tyr Val Asp Glu Phe Ser Thr Leu Val Ile Ser Ser Val Gln
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Leu Thr Arg Ser Cys Gly Cys Gly Pro Gly Thr Leu Gln Gln Pro Gly
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Thr Glu
210
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtccttta cgttcatcgt 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttactctgtg ccgggctgct 20
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttaaacttaa gcttggtacc gccaccatgt cctttacgtt catc 44
<210> 6
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
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gccctctaga ctcgagttac tctgtgccgg gctgct 36
<210> 7
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tttttttttt tttttttt 18
Claims (7)
1.一种鞍带石斑鱼gsdf基因,其特征是:其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述鞍带石斑鱼gsdf基因的编码蛋白,其特征是:其氨基酸序列如SEQ IDNO:2所示。
3.权利要求1所述的鞍带石斑鱼gsdf基因的构建方法,其特征是包括以下步骤:
(1)从鞍带石斑鱼中提取总RNA;
(2)以步骤(1)所提取的总RNA为模板,以RNA Oligo dT为引物,其核苷酸序列如SEQ IDNO:7所示,逆转录合成cDNA;
(3)以步骤(2)所合成的cDNA为模板,以SEQ ID NO:3、SEQ ID NO:4所示的序列为上、下游引物,通过PCR扩增,得到鞍带石斑鱼gsdf基因。
4.权利要求1所述鞍带石斑鱼gsdf基因或权利要求2所述的编码蛋白作为雄性sertoli细胞的标记基因方面的应用。
5.一种鞍带石斑鱼gsdf重组表达载体,其特征是:包括权利要求1所述鞍带石斑鱼gsdf基因。
6.根据权利要求5所述的鞍带石斑鱼gsdf重组表达载体,其特征是:所述重组表达载体为pcDNA4.0-gsdf。
7.权利要求6所述鞍带石斑鱼gsdf重组表达载体的制备方法,其特征是包括以下步骤:
(1)从鞍带石斑鱼中提取总RNA;
(2)以步骤(1)所提取的总RNA为模板,以RNA Oligo dT为引物,其核苷酸序列如SEQ IDNO:7所示,逆转录合成cDNA;
(3)以步骤(2)所合成的cDNA为模板,以SEQ ID NO:5、SEQ ID NO:6所示的序列为上、下游引物,通过PCR扩增,获得具有酶切位点的鞍带石斑鱼gsdf基因,与KpnI和XhoI酶切的pcdan4.0载体连接,得到鞍带石斑鱼gsdf重组表达载体pcDNA4.0-gsdf。
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