Cloning of MT-2 gene and its promoter in Pheretima aspergillum / 中草药

ABSTRACT

Objective: To lay a foundation for attenuating the heavy metal accumulation in Pheretima aspergillum by means of genetic engineering technology in further research, we revealed the transcriptional regulation mechanism of MT-2 gene. Methods: The coding sequence of MT-2 gene was amplified by PCR with specific primers, which were designed according to their known cDNA sequences, and the outcomes were contrastively analyzed after the sequencing process. Prior to the isolation of 5' promoter sequence by genome walking technology, three specific primers were designed based on MT-2 cDNA sequence. Meanwhile, the cis-acting elements of MT-2 gene were analyzed by Promoter Prediction online software. Results: After PCR and sequencing processes, a 2 826 bp coding sequence of MT-2 gene were obtained, four exons and four introns were found to compose the coding area by comparing with the known MT-2 cDNA sequence (accession No.KC787373.1). Besides, after genome walking and Promoter Prediction online analysing, a 1 534 bp promoter region of MT-2 was isolated, which contained not only CAAT box, TAAT box, and other core promoter elements, but also three MRE elements which specifically response to heavy metal involved in regulating the MT-2 expression. Conclusion: The expression of MT-2 gene in P. aspergillum can be induced by heavy metal, and the transcriptional level is achieved by MRE regulatory elements located in MT-2 gene promoter region.