罗非鱼性别调控基因的mRNA差异显示
摘要
1．运用银染mRNA差异显示方法分析罗非鱼性别差异基因
建立非同位素银染mRNA差异显示方法，分离性别相关基因。本文从罗非鱼的性腺提取的总RNA为模板，用5’dT_11N锚定引物和两个随机引物以及一个长引物组合进行DDRT-PCR扩增。经10％聚丙烯酰胺凝胶电泳分离后，用银染方法显示差异DNA条带，在直观下从凝胶中回收差异条带并进行再扩增，琼脂糖电泳检测，得到四个差异片段，回收产物克隆入PUCm-T载体，送生物公司测序，将序列与Internet网上多种公共生物信息数据库进行比较，分析未知基因的结构与功能。在本实验中共得到四个差异基因，其中两个为奥利並雄性特有，两个为尼罗雄性特有，经分析这四个基因在以前的研究中均未见有报道，所以其应为新基因。
2．罗非鱼中SRY基因同源序列的比较分析
SRY是哺乳动物性别决定因子。本文中根据人的SRY基因来设计一对引物，它特异扩增正常男性SRY基因含保守区在内约600bpDNA片段，用引物对奥利亚、尼罗罗非鱼的基因组进行扩增，在雌雄间均出现了相同的三条带，回收600bp大小的片段，克隆入PUCm-T载体，送生物公司测序。序列结果经ClustalW软件进行同源性分析，显示其在雌雄个体间和两个物种间无差异。
1.Analyzing Differential Sex Gene of Tilapia Using a Silver Staining Based
DDRT-PCR Protocol.
To develop mRNA differential display polymerase chain reaction (DD-PCR) method with silver staining , isolate and clone sexal differential genes in Tilapias. Total RNA were extracted from the gonads of two types of Tilapias.The cDNA copies of differential expressed mRNA species were amplified by DD-PCR using an anchored primer 5' dTuN, combined with two of arbitrary' primers and one long primer, respectively. The DNA bands on gel were displayed by silver stain method. After running on 10% polyacrylamide gels, the differential displayed DNA bands were visually recovered from the gel and reamplified by PCR. Test by agrose gel ,we get four differential bands. The reamplified products were cloned into pUCm-T vector, and send to biotech compare for sequence analyzing. Speculate the structure and fuction of the differential gene by comparing its sequence with multiple public bioinformation data. Four gene were found in this research , two of them were come from the O.reochromis male, two of them were come from the O.niloticus male, these genes were considerated as new genes after many types of analyses
2.compare of SRY sequence between different type Tilapias
The Sex-determining region Y (SRY) is a strong candidate for the mammalian testis determining factor. In this article using the primers which specially amplify the 600bp fragments of human SRY gene ,we studied O.reochromis aureus and O.niloticus . The results showed that both males and females all have three band in these tilapias . the amplified product which are about 600bp were cloned into pUCm-T vector and were sepuenceed . these sequence of Tilapias were analyzed by clustalW method . the result showed there was no difference between male and female, no differences were between these two species. .
建立非同位素银染mRNA差异显示方法，分离性别相关基因。本文从罗非鱼的性腺提取的总RNA为模板，用5’dT_11N锚定引物和两个随机引物以及一个长引物组合进行DDRT-PCR扩增。经10％聚丙烯酰胺凝胶电泳分离后，用银染方法显示差异DNA条带，在直观下从凝胶中回收差异条带并进行再扩增，琼脂糖电泳检测，得到四个差异片段，回收产物克隆入PUCm-T载体，送生物公司测序，将序列与Internet网上多种公共生物信息数据库进行比较，分析未知基因的结构与功能。在本实验中共得到四个差异基因，其中两个为奥利並雄性特有，两个为尼罗雄性特有，经分析这四个基因在以前的研究中均未见有报道，所以其应为新基因。
2．罗非鱼中SRY基因同源序列的比较分析
SRY是哺乳动物性别决定因子。本文中根据人的SRY基因来设计一对引物，它特异扩增正常男性SRY基因含保守区在内约600bpDNA片段，用引物对奥利亚、尼罗罗非鱼的基因组进行扩增，在雌雄间均出现了相同的三条带，回收600bp大小的片段，克隆入PUCm-T载体，送生物公司测序。序列结果经ClustalW软件进行同源性分析，显示其在雌雄个体间和两个物种间无差异。
1.Analyzing Differential Sex Gene of Tilapia Using a Silver Staining Based
DDRT-PCR Protocol.
To develop mRNA differential display polymerase chain reaction (DD-PCR) method with silver staining , isolate and clone sexal differential genes in Tilapias. Total RNA were extracted from the gonads of two types of Tilapias.The cDNA copies of differential expressed mRNA species were amplified by DD-PCR using an anchored primer 5' dTuN, combined with two of arbitrary' primers and one long primer, respectively. The DNA bands on gel were displayed by silver stain method. After running on 10% polyacrylamide gels, the differential displayed DNA bands were visually recovered from the gel and reamplified by PCR. Test by agrose gel ,we get four differential bands. The reamplified products were cloned into pUCm-T vector, and send to biotech compare for sequence analyzing. Speculate the structure and fuction of the differential gene by comparing its sequence with multiple public bioinformation data. Four gene were found in this research , two of them were come from the O.reochromis male, two of them were come from the O.niloticus male, these genes were considerated as new genes after many types of analyses
2.compare of SRY sequence between different type Tilapias
The Sex-determining region Y (SRY) is a strong candidate for the mammalian testis determining factor. In this article using the primers which specially amplify the 600bp fragments of human SRY gene ,we studied O.reochromis aureus and O.niloticus . The results showed that both males and females all have three band in these tilapias . the amplified product which are about 600bp were cloned into pUCm-T vector and were sepuenceed . these sequence of Tilapias were analyzed by clustalW method . the result showed there was no difference between male and female, no differences were between these two species. .
引文
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[2] 李美玉，范庆红．哺乳动物性别决定基因的研究进展．莱阳农学院学报，2003，20(2)：135-137．
[3] 王扬才，陆开宏．罗非鱼的性别决定与控制．水产科技[J]，2003(3)：17-20．
[4] 杨超，聂刘旺．两栖爬行动物性别决定的研究进展．安徽师范大学学报：自然科学版[J]，2003，26(2)：169-172．
[5] 童金苟，朱嘉濠，等．鱼类性别决定的遗传基础研究概况．水产学报[J]，2003，27(2)：169-176．
[6] 王慧超，朱勇，等．黑腹果蝇的性别控制．遗传[J]，2003，25(1)：97-101．
[7] 刘良国，赵俊，等．鱼类的性别决定与人工控制．水产科学[J]，2003，22(2)：42-45．
[8] 赵光强．植物的性、性染色体及性别决定．生物学通报[J]，2002，37(12)：19-21．
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