卵黄萤荧光素再生酶基因的克隆与序列分析

Cloning and sequence analysis of Luciola ovalis luciferase Regenerate Enzyme

目的克隆和分析荧光素再生酶基因(LRE)。方法通过GeneBank中已知的荧光素再生酶基因保守区段设计引物,利用5’RACE(rapid-amplification of cDNA ends)和3’RACE技术克隆了来自云南省西双版纳州的卵黄萤(Luciola ovalis)荧光素再生酶基因cDNA和全基因序列。通过GeneBank、National Center for Biotechnology Information和ProDom at the ExPASy Server软件和数据库进行序列分析。结果卵黄萤荧光素再生酶的cDNA序列和基因序列存在2个不同碱基位点,但是它们编码的荧光素再生酶是相同的。卵黄萤荧光素再生酶基因全长(从起始密码子到终止密码子)为1131bp,包含5个外显子4个内含子,其cDNA序列为1008bp,包含924bp的荧光素酶基因开放阅读框和84bp的3’UTR序列。卵黄萤荧光素酶基因的开放阅读框编码1个307个氨基酸的蛋白质。它与北美萤火虫(Photinus pyralis)荧光素再生酶在碱基序列和氨基酸序列上分别有61.8%和53.3%的相似性。结论成功地克隆了荧光素再生酶的cDNA和基因序列,为其在基因工程中的应用奠定了基础。

Objective To clone luciferase regenerate enzyme （LRE） gene and analyze its sequence. Methods Primers designed according to the conservative fragments of LRE gene obtained from the GeneBank, were used to clone the eDNA and full-length DNA sequences of the LRE gene of Luciola lateralis from Xishuangbanna, Yunnan Province by 5＇ RACE and 3＇ RACE techniques. The sequences were then analyzed by using the GeneBank, National Center for Biotechnology Information, and ProDom at the ExPASy Server. Results Two different nucleotides, which encoding a same LRE were detected between the DNA and cDNA sequences of Luciola lateralis LRE. Luciola ovalis LRE DNA was 1131-bp long, containing 5 exons and 4 introns, and its eDNA was 1008-bp long, containing a 924-bp Open Reading Frame （ORF） and a 84-bp 3＇ UTR. The ORF of the luciferase encoded a 307-residue protein. The eDNA and protein sequences of Luciola ovalis LRE showed 61.8% and 53.3% similarity respectively with North American firefly Photinus pyralis. Conclusion We have successfully cloned the LRE gene of firefly Luciola ovalis, which can be further used in gene engineering.