JP6758624B2 - トマト耐暑性変異体及びその作出方法 - Google Patents
トマト耐暑性変異体及びその作出方法 Download PDFInfo
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Description
[1] 野生型と比較して高温条件下における種子を有する果実の形成能を向上させる遺伝的変異を、トマト植物に導入することを含む、耐暑性トマト植物の作出方法。
この遺伝的変異は、受託番号FERM BP-22278で表されるJHT06系統に由来するものであることが好ましい。
この遺伝的変異は、高温条件下における、花粉生存率、並びに種子を有する果実の形成比率及び収量を向上させるものであることが好ましい。
この遺伝的変異は、野生型と比較して葉の葉緑素含量の増加をさらにもたらすものであってもよい。
この遺伝的変異は、例えば、後述の表3に記載の少なくとも1つの遺伝子における非同義変異又は遺伝子欠損である。その非同義変異は、前記遺伝子に対応した表3に記載のアミノ酸変異を引き起こすヌクレオチド変異であってもよいし、前記遺伝子に対応した表3に記載のSNP変異であってもよい。
本方法では、前記変異を導入したトマト植物を35℃〜40℃の温度に曝露することを含むことも好ましい。
この耐暑性トマト植物の例は、受託番号FERM BP-22278で表されるJHT06系統又は前記変異を保持するその派生株である。
本発明は、トマトの夏期高温条件における着果効率の増加をもたらす遺伝子変異を利用した、夏期の着果不良が改善された耐暑性トマト植物の作出方法に関する。
(1)栽培法
本実施例及び後述の実施例においてトマトの栽培は温室で以下のとおり行った。連結ポット(信和株式会社)に培養土を入れ、種子を培養土上に静置した後、上部から培養土を薄くかけて播種した。土の乾燥を防ぐために灌水を毎日行い、週に一度、灌水と同時に施肥した。バイブレーターによって花を振動させることで、受粉の補助を行った。
筑波大学(日本)においてトマト品種マイクロトム(Solanum lycopersicum, cv. Micro-Tom)のエチルメタンスルホン酸(EMS)処理によって作製した変異誘発集団を、夏期の高温なガラス温室内で栽培し、耐暑性候補系統の選抜を行った。着果の優れる91系統を選抜し、採種を行った。さらに翌年、選抜された91系統を12粒ずつ播種し、夏期の高温な温室内で同様に栽培し、耐暑性について再評価を行った。その結果、高温下での着果性に優れる16系統を選抜した。いずれの選抜試験においても、開花時の最高気温はトマトの着果に大きな影響を与えるとされる35℃を超えていたことから、十分な高温ストレスが付加されたと判断した。
(i)高温条件下での着果率、果実収量及び一果実当たりの重量の調査
上記(2)に記載したようにして高温条件下で栽培中の各トマト系統について、開花数、総着果数、着果率、果実収量、及び一果実当たりの重量を調べた。さらに、種子の有無により果実を分類し、それぞれの分類群における着果率、果実収量、及び一果実当たりの重量を算出した。
着果率(%)=着果数/開花数×100
図4に示すとおり、JHT06系統は野生型に比べ、栽培開始後から平成25年10月3日までに開花した花の数が少なかった。しかしJHT06系統の着果率及び総着果数は、野生型に比べて高い値を示した(図5A及びB)。この結果から、JHT06系統は、開花する花は少ないが、開花した花は高い確率で果実を形成できることが示された。
(1)高温条件下での花粉稔性試験
高温条件下でJHT06系統が種子を有する果実を形成する能力が高いことの原因として、高温条件下でも高い稔性を維持する花粉を形成している可能性が考えられた。そこで、高温条件下で生育させたJHT06系統と野生型系統について、細胞呼吸や代謝活性を測定するために用いられる2,3,5-トリフェニルテトラゾリウムクロライド(TTC)により花粉を染色し、花粉の生存率を算出した。なおTTC染色はミトコンドリアの還元反応に基づく細胞生存活性の指標であり、生存細胞(ここでは生存花粉)はTTCにより赤く染色されるが、非生存細胞は染色されない。
花粉生存率(%)=染色花粉数/総花粉数×100
そこで次に、温度条件を35℃に制御できる閉鎖系植物栽培室を利用して、JHT06系統及び野生型マイクロトムの花粉稔性に対する温度条件の影響を温度制御条件下で調査した。
JHT06系統において葉の緑色が濃いという特徴が認められたことから、葉緑素含量の指標であるSPAD値を測定し、野生型とJHT06系統を評価した。
JHT06系統について全ゲノム配列を決定し、変異遺伝子の解析を行った。JHT06系統のゲノムDNAの抽出には、DNA精製キットMaxwell(R) 16 DNA Purification Kits(Promega)を用いた。JHT06系統の若い葉を6枚程度切除し、1.5mLチューブに入れ液体窒素によって凍結した。凍結した葉をマイクロペッスルを用いて十分にすり潰した後、上記キット中の溶解バッファーを含むカートリッジに粉砕した葉を入れ、カートリッジを核酸・タンパク質自動精製装置Maxwell(R) 16にセットして、ゲノムDNAの抽出を行った。抽出したゲノムDNAは滅菌水400μLに溶解し、ゲノムDNAが全量1μgになるようにバルクで調整し、次世代シークエンサー HiSeqTMシーケンスシステム(Illumina)を用いて、JHT06系統の全ゲノム配列を決定した。
実施例4でSNPsの存在が確認された表3中の4つの遺伝子(JHT06系統の変異型遺伝子)について、実施例4と同様の方法により、再度、配列決定及び変異解析を実施した。併せて対応する野生型遺伝子(野生型マイクロトム由来)の塩基配列も決定した。Solyc06g005540.1.1の変異型遺伝子について決定されたCDS配列及びコードされるアミノ酸配列をそれぞれ配列番号71及び72に、対応する野生型遺伝子のCDS配列及びコードされるアミノ酸配列をそれぞれ配列番号73及び74に示す。Solyc06g005930.1.1の変異型遺伝子について決定されたCDS配列及びコードされるアミノ酸配列をそれぞれ配列番号75及び76に、対応する野生型遺伝子のCDS配列及びコードされるアミノ酸配列をそれぞれ配列番号77及び78に示す。Solyc06g071730.2.1の変異型遺伝子について決定されたCDS配列及びコードされるアミノ酸配列をそれぞれ配列番号79及び80に、対応する野生型遺伝子のCDS配列及びコードされるアミノ酸配列をそれぞれ配列番号81及び82に示す。Solyc04g076040.2.1の変異型遺伝子について決定されたCDS配列及びコードされるアミノ酸配列をそれぞれ配列番号83及び84に、対応する野生型遺伝子のCDS配列及びコードされるアミノ酸配列をそれぞれ配列番号85及び86に示す。
Claims (7)
- 野生型と比較して高温条件下における種子を有する果実の形成能を向上させる遺伝的変異を、トマト植物に導入することを含む、耐暑性トマト植物の作出方法であって、
前記変異が、受託番号FERM BP-22278で表されるJHT06系統に由来し、かつ、表3に記載の4つの遺伝子における、非同義変異又は遺伝子欠損であって以下のa)〜d)である変異:
a) 配列番号64で表されるアミノ酸配列を基準として定められるアミノ酸変異D61Yを引き起こす塩基変異、
b) 配列番号66で表されるアミノ酸配列をコードする遺伝子又はそのアミノ酸配列に対して90%以上の配列同一性を示すアミノ酸配列からなる機能性タンパク質をコードする遺伝子の、欠損、
c) 配列番号68で表されるアミノ酸配列をコードする遺伝子又はそのアミノ酸配列に対して90%以上の配列同一性を示すアミノ酸配列からなる機能性タンパク質をコードする遺伝子の、欠損、及び
d) 配列番号70で表されるアミノ酸配列を基準として定められるアミノ酸変異*102Cを引き起こす塩基変異であって、但し*は終止コドンの生成を示す、塩基変異
を含む、方法。 - 前記変異が、高温条件下における、花粉生存率、並びに種子を有する果実の形成比率及び収量を向上させる、請求項1に記載の方法。
- 前記変異が、野生型と比較して葉の葉緑素含量の増加をさらにもたらす、請求項1又は2に記載の方法。
- 前記変異を導入したトマト植物を35℃〜40℃の温度に曝露することを含む、請求項1〜3のいずれか1項に記載の方法。
- 請求項1〜4に記載の方法により作出される、前記変異が導入された耐暑性トマト植物。
- 受託番号FERM BP-22278で表されるJHT06系統又は前記変異を保持するその派生株である、請求項5に記載の耐暑性トマト植物。
- 請求項5又は6に記載の耐暑性トマト植物を育種親として、他のトマト植物と交配し、子孫植物として前記変異を有するトマト植物を取得することを含む、耐暑性トマト植物の育種方法。
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