CN100554421C - 斑节对虾细胞周期蛋白b的基因序列 - Google Patents
斑节对虾细胞周期蛋白b的基因序列 Download PDFInfo
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Abstract
本发明从近源物种细胞周期蛋白B的基因的CDS序列的保守区设计的兼并引物,从对虾的性腺中提取总RNA,并用PCR法扩增,克隆得到对虾细胞周期蛋白B的基因的表达序列。通过对细胞周期蛋白B基因的克隆,了解基因的组成情况及鱼类生长发育周期过程,并通过对该基因纯化蛋白的获得,可以促进鱼体细胞发育,缩短鱼类发育周期,对提高经济效益有一定的促进作用。
Description
技术领域
本发明涉及一种斑节对虾细胞周期蛋白B基因序列。
背景技术
细胞周期是指连续分裂细胞从一次有丝分裂结束到下一次有丝分裂结束所经历的整个过程。在这个过程中,细胞遗传物质复制并加倍,且在分裂结束时平均分配到两个子细胞中去。细胞周期又可以分为间期和有丝分裂期。从一次有丝分裂结束到下一次有丝分裂开始的时期就是间期。这一期以DNA合成为标志,经过分裂期,加倍的染色体和其他细胞组分被平均分配到两个完全一样的子细胞中。通过分裂,形成了一个新细胞。细胞周期能够严格按照间期到有丝分裂期的顺序运转是与相关调控基因的有序表达分不开的。这一基因可以诱导未成熟细胞进入有丝分裂期,并发现在有丝分裂期唯一需要合成的基因蛋白就是这个周期蛋白,这是一个起关键作用的细胞分裂的调节蛋白。
细胞周期蛋白B为有丝分裂期周期蛋白,在间期到有丝分裂期交界处发挥功能,诱导细胞分裂。细胞周期蛋白与细胞衰老关系密切,细胞周期蛋白B降解受阻,细胞不能退出有丝分裂期,影响细胞衰老。
发明内容
本发明的目的是提供一种斑节对虾细胞周期蛋白B的基因序列,其可促进鱼虾体细胞发育,缩短鱼虾类发育周期。
本发明是通过以近源物种细胞周期蛋白B的基因的CDS序列的保守区设计的兼并引物,并用PCR法扩增,结合RACE技术克隆到斑节对虾细胞周期蛋白B的基因的全表达序列。
本发明从GenBank中下载近源物种(如人、牛、斑马鱼、日本对虾等)细胞周期蛋白B同源基因CDS序列,利用Clustal W软件进行多序列比对,确定保守区,根据保守区序列设计兼并引物,扩增片段大小约为225bp。引物设计后采用β-乙睛亚磷酰胺化学法进行DNA合成得到以下引物序列:
F:5’ATWGCWAGYAAATAYGAAGARATGTA 3’
R:5’TCCATIARRTAYTTKGCWARIGTATG 3’。
提取斑节对虾总RNA,使之与反转录引物(oligo-dT接头引物)混合,进行反转录,合成第一链cDNA作为模板。用上述兼并引物进行PCR扩增,所扩增的PCR产物用1.2%的琼脂糖凝胶电泳进行检测,从凝胶中纯化回收目的产物。然后将纯化的PCR产物克隆到pMD-18T载体中,转化大肠杆菌JM-109感受态细胞,挑取阳性克隆,提取质粒DNA。经简并引物PCR检测后,将具有插入片段的质粒DNA用M13通用引物进行双向测序。
根据已得到的第一链cDNA片段序列设计特异性引物,利用cDNA末端快速扩增技术(Rapid Amplification of cDNA ends,RACE)对目的基因的3′和5′末端进行PCR扩增。在3′端的快速扩增中,由基因引物和接头引物进行PCR反应,扩增片段大小约为1000bp。所述的基因引物和接头引物序列为:
基因引物:5’TCGGGGACTTCGCATACATC 3’
接头引物:5’GGCCACGCGACTAGTAC 3’。
经3′端的快速扩增后的序列进行5′端的快速扩增,利用末端转移酶和dCTP在cDNA末端加上poly(C)尾后,以加尾后的cDNA作为模板,利用外侧特异性引物和oligodG进行第一次PCR扩增,所得PCR产物再利用内侧引物和oligodG进行第二次PCR扩增。所述引物序列为:
oligo-dG:5’GGGGGGGGGGGGGGGH 3’
基因外侧引物:5’GCAAAGGTATGTTGAGAAGC 3’
基因内侧引物:5’GTGTAAGGGAAGGGGATAGG 3’。
经末端快速扩增技术进行PCR所得到的PCR产物在1.2%的琼脂糖凝胶电泳上进行分离检测后,将PCR产物纯化后,克隆到pMD-18T载体中,转化大肠杆菌JM-109感受态细胞,挑取阳性克隆,用M13引物对质粒DNA进行测序,测序所得序列再与兼并引物扩增所得的序列利用Clustal W软件进行拼接即可得到目的基因。
通过对细胞周期蛋白B基因的克隆,了解基因的组成情况及鱼类生长发育周期过程,并通过对该基因纯化蛋白的获得,可以促进鱼体细胞发育,缩短鱼类发育周期,对提高经济效益有一定的促进作用。
附图说明
图1为本发明在卵巢不同发育时期的表达;
First:卵巢发育第一阶段(卵巢还未发育);Second:卵巢发育第二阶段(卵巢开始启动发育);Fourth:卵巢发育第四阶段(卵巢发育完成,准备终止);cyclin B:细胞周期蛋白B。
具体实施方式
下面通过以下具体实施方式对本发明作进一步阐述,但是本发明的内容完全不局限于此。
1.总RNA的提取
取鲜活健康斑节对虾(体重约300g)在实验室内暂养2d后(水温约24℃,气泵充气),解剖虾体取出性腺约100mg,放入1mL Trizol(Gibco,Japan)中进行匀浆,按照试剂盒使用说明提取总RNA。
2.cDNA第一链的合成
取斑节对虾总RNA 5μg与反转录引物(oligo-dT接头引物)1μL(10pmol/L)混合,70℃加热5min后,立即放置冰上,然后加入5×buffer,2.5mmol/L dNTP混合液,RibonucleascInhibitor,M-MLV反转录酶,反应体系为25μL。反应过程为42℃60min,70℃15min,最后放入-80℃保存备用。
3.兼并引物设计依据,引物合成的方法
从GenBank中下载近源物种(如人、牛、斑马鱼、日本对虾等)细胞周期蛋白B同源基因CDS序列,利用Clustal W软件进行多序列比对,确定保守区,根据保守区序列设计兼并引物,扩增片段大小约为225bp。引物设计后采用β-乙睛亚磷酰胺化学法进行DNA合成,得到以下引物序列:
F:5’ATWGCWAGYAAATAYGAAGARATGTA 3’
R:5’TCCATIARRTAYTTKGCWARIGTATG 3’。
4.细胞周期蛋白B基因cDNA全序列的克隆
4.1第一链cDNA用兼并引物进行PCR扩增
以近源物种细胞周期蛋白B基因CDS序列的保守区设计的兼并引物,扩增片段大小约为225bp。
以上述合成的第一链cDNA作为模板,用兼并引物进行PCR扩增,反应体系为:10x PCR反应缓冲液5μL,25mmol/L MgCl2 3μL,2.5mmol/L dNTP 2μL,10nmol/L引物dTF和dTR各2μL,Taq酶1.25U,用PCR水将反应体系补充至50μL。反应条件为:1个循环,94℃变性5min;35个循环:94℃变性45s,56℃退火45s,72℃延伸45s;1个循环,72℃延伸10min;4℃保温。所扩增的PCR产物用1.2%的琼脂糖凝胶电泳进行检测,从凝胶中纯化回收目的产物。然后将纯化的PCR产物克隆到pMD-18T载体中,转化大肠杆菌JM-109感受态细胞,挑取阳性克隆,提取质粒DNA。经简并引物PCR检测后,将具有插入片段的质粒DNA用M13通用引物进行双向测序。
4.2第一链cDNA的快速扩增技术进行PCR扩增
根据已得到的cDNA片段序列设计特异性引物。利用cDNA末端快速扩增技术(RapidAmplification ofcDNAends,RACE)对目的基因的3′和5′末端进行PCR扩增。
在3′RACE中,由基因引物和接头引物进行PCR扩增。
引物序列为:
基因引物:5’TCGGGGACTTCGCATACATC 3’
接头引物:5’GGCCACGCGACTAGTAC 3’。
反应体系同兼并引物扩增体系。
反应条件:1个循环,94℃变性5min;35个循环:94℃变性45s,58℃退火30s,72℃延伸1min;1个循环,72℃延伸10min;4℃保温。
在5′RACE中,利用末端转移酶和dCTP在cDNA末端加上poly(C)尾后,以加尾后的cDNA作为模板,利用外侧特异性引物和OligodG进行第一次PCR扩增,所得PCR产物取1μL作为模板,再利用内侧引物和OligodG进行第二次PCR扩增。
引物序列为:
oligo-dG:5’GGGGGGGGGGGGGGGH 3’
基因外侧引物:5’GCAAAGGTATGTTGAGAAGC 3’
基因内侧引物:5’GTGTAAGGGAAGGGGATAGG 3’。
反应体系同兼并引物扩增体系。
反应条件:
第一次PCR反应:1个循环,94℃变性5min;35个循环:94℃变性45s,58℃退火30s,72℃延伸1min;1个循环,72℃延伸10min;4℃保温。
第二次PCR反应:1个循环,94℃变性5min;35个循环:94℃变性45s,59℃退火30s,72℃延伸1min;1个循环,72℃延伸10min;4℃保温。
所得到的PCR产物在1.2%的琼脂糖凝胶电泳上进行分离检测后,将PCR产物纯化后,克隆到pMD-18T载体中,转化大肠杆菌JM-109感受态细胞,挑取阳性克隆,用M13引物对质粒DNA进行测序,测序所得序列再与兼并引物扩增所得的序列利用Clustal W软件进行拼接即可得到目的基因。
5.对斑节对虾细胞周期蛋白B基因的测定
所得到的PCR产物在1.2%的琼脂糖凝胶电泳上进行分离检测后,将PCR产物纯化后,克隆到pMD-18T载体中,转化大肠杆菌JM-109感受态细胞,挑取阳性克隆,用3730测序仪对质粒DNA进行测序。测序结果用BLAST软件(http://www.ncbi.nim.nih.gov/)进行同源性测定,来确定为斑节对虾细胞周期蛋白B基因同源序列。比对结果为:
Score E
Sequences producing significant alignments: (Bits) Value
gi|54660743|gb|AAV37462.1|cyclin B[Marsupenaeus japonicus] 528 1e-148
gi|10955|emb|CAA41255.1|cyclin B[Patella vulgata]>gi|11617... 3199 e-86
gi|52851368|dbj|BAD52077.1|cyclin B2[Anguilla japonica]>gi... 305 2e-81
gi|10337|emb|CAA33513.1|unnamed protein product[Spisula sol... 303 9e-81
gi|42542462|gb|AAH66507.1|Cyclin B2[Danio rerio]>gi|282778... 302 1e-80
gi|68363504|ref|XP_709645.1|PREDICTED:similar to Cyclin B2 iso 301 3e-80
根据Iroe1995年的报道,只要两个序列间用BLAST软件比对后E-Value值小于0.005,则两个序列之间具有绝对的同源性,即证明序列为同一种基因序列。从对比结果看斑节对虾目的基因序列BLAST比对后,与其他物种的细胞周期蛋白B的E值绝对小于0.005,所以证明所克隆的目的基因是细胞周期蛋白B的同源序列。
6.斑节对虾细胞周期蛋白B基因在不同卵巢发育时期的表达
按照上述RNA提取,cDNA合成的方法,取不同发育阶段的卵巢来检测细胞周期蛋白B基因对卵巢发育的影响。以β-肌动蛋白(β-actin)作为内参照物来调节各个阶段模板即cDNA的量,使其所含的模板数一致,从而进行PCR反应,将所得到的PCR产物在1.2%的琼脂糖凝胶电泳上进行分离检测基因的表达量的高低。所用的引物序列为:
β-actin:
F:5’TTGCTACATCGCCCTTGACT 3’
R:5’TGTGGACGGTTTCCTGAATA 3’
基因特异性引物:
F:5’TCGCATACATCACAGACAAA 3’
R:5’TGAGAAGCATCAACAGAGCC 3’
图1所示的结果中斑节对虾细胞周期蛋白B在发育阶段表达量最高,卵巢发育第四阶段即卵巢发育完成准备终止时的表达量也较高,而在卵巢发育的第一阶段即未开始发育时的表达量非常低,说明斑节对虾细胞周期蛋白B基因直接影响卵巢的发育。
序列表
<110>中国水产科学研究院南海水产研究所
<120>斑节对虾细胞周期蛋白B的基因序列
<150>CN200610037559.7
<151>2006-9-7
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tga 1262
*
ggtggtcgcc atttaaaaag taaatattgt tcatgttgaa agaatggggt tgtttttgta 1322
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accaccagtt tttttttttt tttttaacaa gagtagtttt ggggggcagc atgtaccccc 1442
cagagtgggt ggtttcggca tccctccatt catcgtggtc ttcattgtat taaaataatt 1502
ttaggcagga caaaaatggt tatttgaaga aaggaaccag caaagaagta tatgcaatgt 1562
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taataaacca gttttatcca aaaaaaaaaa aaaaaa 1658
<210>2
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193 199 205
Arg Asn Lys Leu Gln Leu Val Gly Val Thr Ala Met Phe Ile Ala Ser
209 215 221
Lys Tyr Glu Glu Met Tyr Cys Pro Glu Ile Gly Asp Phe Ala Tyr Ile
225 231 237
Thr Asp Lys Ala Tyr Ser Lys Ala Glu Ile Arg Lys Met Glu Val Thr
241 247 253
Met Leu Asn Glu Leu Gly Phe Asn Val Ser Tyr Pro Leu Pro Leu His
257 263 269
Phe Leu Arg Arg Asn Ser Lys Ala Gly Ser Val Asp Ala Ser Gln His
273 279 285
Thr Leu Ala Lys Tyr Leu Met Glu Leu Cys Leu Pro Glu Tyr Ser Met
289 295 301
Cys His Tyr Lys Ser Ser Met Ile Ala Ala Ser Ala Leu Cys Leu Ser
305 311 317
Leu Lys Leu Leu Asp Gly Asn Asn Trp Ser Asp Thr Leu Thr Phe Tyr
321 327 333
Ser Arg Tyr Thr Glu Gln Gln Leu Met Pro Val Met Cys Lys Met Ala
337 343 349
Ser Val Val Val Lys Ser Ser Ser Ala Lys Gln Gln Ala Val Arg Gln
353 359 365
Lys Tyr Lys Ala Ser Lys Leu Met Lys Ile Ser Glu Ile Pro Gln Leu
369 375 381
Lys Ser Lys Leu Ile Asn Ser Leu Ala Glu Lys Ser Ala Ser Tyr Ala
385 391 397
Claims (1)
1、一种斑节对虾细胞周期蛋白B基因,其特征在于:它的核苷酸序列及编码相应的氨基酸序列如SEQ ID NO.1所述。
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CNB2007100912290A CN100554421C (zh) | 2006-09-07 | 2007-03-23 | 斑节对虾细胞周期蛋白b的基因序列 |
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CN105219778A (zh) * | 2015-11-10 | 2016-01-06 | 中国水产科学研究院淡水渔业研究中心 | 克氏原螯虾细胞周期相关的cyclinB基因及其编码蛋白的鉴定 |
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Three forms of cyclin B transcripts in the ovary of the kurumaprawn Marsupenaeus japonicus: their molecularcharacterizations and expression profiles during oogenesis. Gao-Feng Qiu, Keisuke Yamano.Comparative BioChemistry and Physiology: B-BioChemistry and Molecular Biology,Vol.141 No.2. 2005 |
Three forms of cyclin B transcripts in the ovary of the kurumaprawn Marsupenaeus japonicus: their molecularcharacterizations and expression profiles during oogenesis. Gao-Feng Qiu,Keisuke Yamano.Comparative BioChemistry and Physiology: B-BioChemistry and Molecular Biology,Vol.141 No.2. 2005 * |
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