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Role of the clock gene period in the circadian rhythm and photoperiodism of the silkmoth Bombyx mori

Ikeda, Kento 京都大学 DOI:10.14989/doctor.k23364

2021.05.24

概要

生物は一日および一年周期の環境変化に適応する能力を持っており、多くの場合、一日のものに対しては概日時計、一年のものに対しては光周性により時間を予測し対応している。概日時計を作り出す遺伝子としていくつかの時計遺伝子が明らかになっており、キイロショウジョウバエにおいて提唱されたモデルでは、これらの時計遺伝子が負のフィードバックループを形成し約24時間の周期を形成する。光周性において日長を計測するのに概日時計が関与していることは広く認められている。

period(per)は代表的な時計遺伝子であり、フィードバックループにおける負の転写因子として知られる。しかし、チョウ目昆虫では、perの存在は確認されているが、発現場所や核内移行、周期性などの特徴がキイロショウジョウバエとは異なる。したがってチョウ目昆虫においてperが他の昆虫と同様に概日時計の形成に関与しているかどうかは分かっていない。また、これまでいくつかの昆虫において、RNA干渉による時計遺伝子の発現抑制により光周性が失われることが報告されている。これらの昆虫の光周性は、幼虫もしくは成虫休眠に関係するもので、いずれも幼若ホルモンにより制御されている。したがって、時計遺伝子の多面発現として幼若ホルモンの系に作用し、休眠に影響した可能性を完全に排除することはできない。

本研究では、カイコガBombyx moriに着目した。カイコガは遺伝子編集技術を用いた解析技術が確立されているため、遺伝子レベルの解析が可能であり、羽化と孵化を概日リズムが支配している。また、幼若ホルモンとは異なる休眠ホルモンによって調節される休眠の誘導に光周性が関わることも報告されている。そこで本研究では、カイコガにおいて遺伝子編集技術によってperのノックアウト系統を作成することにより、これらの問題の解決を試みた。第1章ではperが概日時計の形成に関与しているかどうか、第2章では光周性に関与しているかどうかを検証した。

第1章においては、休眠をもたない系統であるpnd w-1を用いてTALENにより、perノックアウト系統を作成した。元となったpnd w-1において、恒常条件で明瞭な自由継続リズムが羽化と孵化ともに検出された。しかし、perノックアウト系統においてはこの概日リズムが消失していた。また時計遺伝子の時間的な発現の変化を調べると、 ノックアウト系統では、pnd w-1で見られていた時間的な変動が失われていることが分かった。

第2章においては、光周性によって調節された休眠をもつ系統である江浙を用いて TALENにより、perノックアウト系統を作成した。この系統においても行動と遺伝子レベルで概日リズムが消失していることが確かめられた。元となった江浙とperiodノックアウト系統を卵期20℃恒明におき、幼虫期を25℃の長日と短日で飼育したところ、江浙は長日では非休眠卵、短日では休眠卵を産下したが、perノックアウト系統 は、どちらの光周期でも非休眠卵を産下した。さらに、perノックアウト系統を20℃の短日で飼育したところ、半数以上の個体が休眠卵を産下した。すなわち、perノックアウト系統は光周性を失っていたが、休眠卵を産むしくみは保持していた。

これらの結果から、カイコガにおいても per は概日時計形成に重要な分子であると結論した。また、光周性においても per の関わる概日時計が重要な役割を果たすことが明らかになった。

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