Identification And Functional Analysis Of The Arsenic Methyltransferase Gene From Three Species Of Tetrahymena

With the development of the social economy and industry, a large number ofpollutants are released into the environment and cause a real and potential threat tohuman survival and development. With the characteristics such as widespreadpresence, wide application, high hazard, the arsenic has become one ofenvironmental problems which caused a common concern. In nature, the toxicity ofarsenic could be reduced through the pathway of biological metabolism, which issignificant for arsenic bioremediation. At present, he mechanism of arsenicbiotransformation has not been elucidated clearly, and the work of arsenicbioremediation moves slowly. So it is important to discover more arsenic resistantorganisms and research the mechanism of arsenic biotransformation. Someexperiments were done with three Tetrahymena: Tetrahymena shanghaiensis, T.borealis, T.corlissi. In the experiment of As(V) toxic effects on the threetetrahymena, the24h EC50values of As(V) were measured in the early logarithmicphase, and the result rank in this order: T.corlissi (110.09mg/L)> T. shanghaiensis(14.09mg/L)> T. borealis (13.10mg/L). Compared with some other organisms, thethree tetrahymena show a high arsenate resistant ability. Then, I analysised thegenomic data of three species of Tetrahymena, and found three sequences whichwere remarkably similar to other arsenic methyltrasferase genes. and I get thenucleotide sequence and the amino acid sequence of the three new gene by thesoftware AUGUSTUS. Alignment of the amino acid sequences of the three newgene and arsenic methyltrasferase of some other organisms showed a close relationbetween these gene products. The following work found that some genes nearby tothe three arsM genes are highly homologous with T. thermphila. The arsenicbiotransformation related genes are linked gene in some orgnisms such as fungi andbacteria, so it is worth to further study the relationship between the homologousgenes nearby to the three arsM genes and the arsenic metabolism.Subsequently, with the three Tetrahymena, two experiments were ranrespectively to study arsM gene expression and arsenic speciations under thecondition of As(V). The real-time fluorescent quantitative PCR (RT_qPCR)technology was used to study the arsM expression induced by As(V), and the result show that the arsM expression of the three tetrahymena significantly increased atthe present of As(V). At last, the HPLC–ICP–MS was used to analysis the arsenicspeciations in three tetrahymena,and found five forms of arsenic, they are: As(Ⅴ),As(Ⅲ), MMA, DMA and one unknown form in T. shanghaiensis. The analysis oftotal arsenic and four arsenic forms content in the three tetrahymena reflects that thethree Tetrahymena has a high arsenic metabolism and enrichment capability. Theabove results proved the existence of arsenic methylation in the three tetrahymena,combined with prediction results of arsM gene, I consider the arsenic methylation iscatalyzed by the arsenic methyltransferase. Before that, arsenic methylation andarsenic methyltransferase only exsits in Tetrahymena pyriformis in the protozoan.This research not only find three new Tetrahymena with arsenic methylationcapacity, but also elaborate that arsenic methylation is a ubiquitous phenomenon inprotozoa. What’s more, it is conducive to study the mechanism of arsenicbiotransformation and arsenic bioremediation.