Ds轉位子造成水稻EPSPS基因exonization現象之研究

Abstract

轉位子是基因體中可移動的DNA片段，若轉位子插入基因之隱子，可能提供剪接位置而產生選擇性剪接之mRNA，這種因為轉位子插入而產生新生隱子的現象稱為exonization。 本研究目的為模擬轉位子插入基因隱子，觀察是否發生exonization ，因此建構一含有Ac 之5’ 端及3’ 端各約250 bp序列而成為mini Ds，分別以正/反向插入水稻EPSPS（5’-enolpyruvylshikimate-3’-phosphate synthase）基因之7個隱子。全部14個構築中，菸草與阿拉伯芥分別成功轉殖7個與9個構築；在轉基因菸草中，當Ds以正向插入EPEPS基因第6個隱子發現exonization現象，經定序確認是Ds提供一5’ 端donor site而產生選擇性剪接片段，證明轉位子可提供mRNA splicing之donor/acceptor site所需之訊息，因而具有推動演化之潛力。本論文並詳細探討轉位子中界定5’ 端剪接位置之因子。

The maize transposon element Ac/Ds can move to new location within genome. Sometimes it jumps into intron of the inserted gene and offers splicing donor/accepter sites, which can result in alternative splicing. The special alternative splicing event that a new exon is produced by insertion of transposon is termed as “exonization”. The purpose of this study is to assess the frequencies of exonization events when a transposon jumps into the introns of the inserted gene. To this end, a mini Ds, which contains 5’ and 3’ cis-required sequences of Ac transposon, was inserted forward or reversely into each intron of the rice EPSPS gene. For a total number of 14 constructs, 7 were successfully transformed into tobacco and 9 into Arabidopsis. Only one exonization event was observed in the transgenic tobacco containing the construct that the mini-Ds was located forwardly in intron 6 of the rice EPSPS gene. In this case, Ds offers an alternative 5’ donor site for splicing resulted in the exonization. These results indicate that Ds contains the information for defining 5’ donor site and transposon harbors the potential for driving evolution events. Several factors that affect the alternative splicing by transposon in plants were discussed.