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樊连春, 谢京, 汪洋, 桂建芳. 银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A1的cDNA克隆[J]. 水生生物学报, 2000, 24(6): 573-581.
引用本文: 樊连春, 谢京, 汪洋, 桂建芳. 银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A1的cDNA克隆[J]. 水生生物学报, 2000, 24(6): 573-581.
FAN Lian-chun, XIE Jing, WANG Yang, GUI Jian-fang. CONSTRUCTION OF OOCYTE cDNA LIBRARIES OF GYNOGENETIC SILVER CRUCIAN CARP AND GONOCHORISTIC COLOR CRUCIAN CARP AND CLONING OF THEIR CYCLIN A1 cDNAs[J]. ACTA HYDROBIOLOGICA SINICA, 2000, 24(6): 573-581.
Citation: FAN Lian-chun, XIE Jing, WANG Yang, GUI Jian-fang. CONSTRUCTION OF OOCYTE cDNA LIBRARIES OF GYNOGENETIC SILVER CRUCIAN CARP AND GONOCHORISTIC COLOR CRUCIAN CARP AND CLONING OF THEIR CYCLIN A1 cDNAs[J]. ACTA HYDROBIOLOGICA SINICA, 2000, 24(6): 573-581.

银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A1的cDNA克隆

CONSTRUCTION OF OOCYTE cDNA LIBRARIES OF GYNOGENETIC SILVER CRUCIAN CARP AND GONOCHORISTIC COLOR CRUCIAN CARP AND CLONING OF THEIR CYCLIN A1 cDNAs

  • 摘要: 分别取行天然雌核发育繁殖的银鲫和两性生殖的彩鲫的卵母细胞为材料,提取总RNA,分离mRNA,进而反转录合成cDNA并定向插入λgtll Sfi-Not克隆载体,经体外包装构建了银鲫与彩鲫卵母细胞的表达型cDNA文库。测试结果表明库容量分别达到3.1×106(银鲫)和1.6×106(彩鲫)。进一步人工合成Cyclin A1保守引物,采用PCR扩增文库的方法,克隆了银鲫(1616bp)与彩鲫(1626bp)的Cyclin A1全长cDNA。序列分析结果表明:两种鱼编码区长度均为1173bp,起始于一个包含在脊椎动物起始密码子ANNATG基元内ATG的单一开放读码框,编码391个氨基酸;5'-端非编码区长度也同为70bp,3-'端非编码区长度略有不同,银鲫为373bp,而彩鲫则为383bp;二者3'-端均带有AATAAA的Poly(A)加尾信号以及24bp(银鲫)和27bp(彩鲫)的Poly(A)尾巴。比较银鲫、彩鲫和金鱼与人、爪蟾Cyclin A1氨基酸序列同源性的结果表明,Cyclin A1在人、爪蟾与鱼类之间具有较高同源性;而在银鲫、彩鲫和金鱼之间,Cyclin A1仅在周期蛋白框外存在5个氨基酸的差异,且这些差异均是由个别碱基的变异造成的。

     

    Abstract: Total RNAs were isolated from maturated oocytes of gynogenetic silver crucian carp (Carassius auratus gibelio) and gonochoristic color crucian carp (Carassius auratus color variety). After mRNA purification, double strand cDNAs were synthesized by using MMLV reverse transcriptase, inserted into λgtll Sfi-Not cloning vector, and packaged with packaging extracts. Therefore, the expressive cDNA libraries were constructed from both fish oocytes. The titration of libraries were 3.1×106 for silver crucian carp and 1.6×106 for color crucian carp respectively. A fast technique system for cloning full-length cDNA was also established through PCR-based method. Using the data from Gene Bank, we designed and synthesized conservative primers of cyclin A1 in carp, and cloned the full length cyclin A1 cDNAs of silver crucian carp and color crucian carp from their oocyte cDNA libraries. The length of cyclin A1 cDNAs is 1680 bp for silver crucian carp and 1627 bp for color crucian carp, and both have a same 70 bp of 5’ untranslated regions followed by a single open reading frame of 1173 bp. Both of the open reading frames are initiated from the consensus sequence ANNATG flanking translational start sites in vertebrates, and can be translated into proteins that consist of 391 amino acids. The differences mainly exist in 3’ untranslated regions, in which there are 373 bp for silver crucian carp, and there are 383 bp for color crucian carp. Pairwise comparison of the deduced amino acid sequences of the gynogenetic and gonochoristic crucian carp cyclin A1 with homologues from human, Xenopus and other fish revealed a high evolutionary conservation. Only 5 amino acid differences exist among three cyclin A1 of silver crucian carp, color crucian carp and goldfish, and they should be resulted from the variation of single nucleotide.

     

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