植物科学最前線 7:124 (2016)᳜≀◊✲࡟࠾ࡅࡿ㸰ගᏊບ㉳㢧ᚤ㙾ࡢά⏝
ᰩཎ኱㍜ྡྂᒇ኱Ꮫ኱Ꮫ㝔⌮Ꮫ◊✲⛉JST࣭ERATO ᮾᒣࣛ࢖ࣈ࣍ࣟࢽࢡࢫࣉࣟࢪ࢙ࢡࢺ
ࠛ464-8602 ឡ▱┴ྡྂᒇᕷ༓✀༊୙⪁⏫
Daisuke Kurihara

Utility of two-photon excitation microscopy for plant researcher
Key words: auto-fluorescence, clearing, fluorescent protein, two-photon
Graduate School of Science, Nagoya University
Higashiyama Live-Holonics Project, ERATO, JST
Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan㸯㸬ࡣࡌࡵ࡟ ⏕యࡢ࡞ࡾࡓࡕࡸ⏕࿨⌧㇟ࢆ᫂ࡽ࠿࡟ࡍࡿୖ࡛㸪┤᥋⣽⬊ࡸศᏊࢆほᐹ㸪ゎᯒ࡛ࡁࡿ㢧ᚤ㙾ᢏ⾡ࡣ௒᪥Ḟ࠿ࡏ࡞࠸ᢏ⾡࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽ㸪⏕యࡢ῝㒊࡛㉳ࡇࡗ࡚࠸ࡿ⏕࿨⌧㇟ࢆゎᯒࡍࡿࡓࡵ࡟ࡣ㸪㢧ᚤ㙾ࡢᢏ⾡ⓗไ㝈࠿ࡽࡇࢀࡲ࡛ᶵᲔⓗ࡟ษ∦ࢆస〇ࡍࡿ࡞࡝㸪ࢧࣥࣉࣝ࡟യࢆࡘࡅ࡚ほᐹࡍࡿᚲせࡀ࠶ࡗࡓࠋࡋ࠿ࡋ㸪❧యⓗ࡞⏕యࡢᵓ㐀ࢆᢕᥱࡍࡿࡓࡵ࡟ࡣ㸪୕ḟඖ᝟ሗࡀᚲせ࡟࡞ࡿࡀ㸪ᶵᲔⓗ࡟ษ᩿ࡋࡓษ∦࠿ࡽ୕ḟඖࡢ❧యᵓ㐀ࢆ෌ᵓ⠏ࡍࡿࡢࡣ㠀ᖖ࡟ᅔ㞴࡛࠶ࡿࠋࡲࡓ㸪ࢲ࢖ࢼ࣑ࢵࢡ࡞⏕࿨⌧㇟ࢆ⏕ࡁࡓࡲࡲゎᯒࡍࡿࡓࡵ࡟ࡶ㸪࡞ࡿ࡭ࡃയࢆࡘࡅࡎ㸪ࡑࡢࡲࡲ┤᥋ほᐹ࡛ࡁࡿࡇ࡜ࡢ㔜せᛶࡣゝ࠺ࡲ࡛ࡶ࡞࠸ࠋࡑࡢࡼ࠺࡞୰㸪⏕యࡢ῝㒊ࢆ┤᥋ほᐹࡍࡿࠕ῝㒊࢖࣓࣮ࢪࣥࢢࠖࢆ㐩ᡂࡍࡿࡓࡵ࡟㸪㏆ᖺ㸪2 ගᏊບ㉳㢧ᚤ㙾࡜࿧ࡤࢀࡿ㢧ᚤ㙾ᢏ⾡ࡀά⏝ࡉࢀ࡚࠸ࡿࠋᮏ✏࡛ࡣ㸪2 ගᏊບ㉳㢧ᚤ㙾ࡢ≉ᚩࢆ⤂௓ࡋ㸪᳜≀◊✲࡟ᐇ㝿࡟⏝࠸ࡿ㝿ࡢὀពⅬࢆ㏙࡭ࡓ࠸ࠋ㸰㸬2 ගᏊບ㉳㢧ᚤ㙾ࢆ⏝࠸ࡓ᳜≀⤌⧊ほᐹ㸰࣮㸯㸬῝㒊࢖࣓࣮ࢪࣥࢢࡀྍ⬟࡛࠶ࡿ 2 ගᏊບ㉳㢧ᚤ㙾ࡢࡼࡃ▱ࡽࢀ࡚࠸ࡿ≉ᚩࡣ㸪⏕యࡢ῝㒊࢖࣓࣮ࢪࣥࢢࡀྍ⬟࡞ࡇ࡜࡛࠶ࢁ࠺ࠋඹ↔Ⅼ㢧ᚤ㙾࡛⏝࠸ࡽࢀࡿග※ࡀ㸪⣸እࡸྍどග࣮ࣞࢨ࣮࡛࠶ࡿࡇ࡜࡟ẚ࡭࡚㸪2 ගᏊບ㉳㢧ᚤ㙾࡛⏝࠸ࡽࢀࡿග※ࡣ㸪ಸ㏆࠸Ἴ㛗ࡢ㏆㉥እගࣃࣝࢫ࣮ࣞࢨ࣮࡛࠶ࡿࠋ㛗Ἴ㛗ࡢග※ࢆ⏝࠸ࡿࡓࡵ㸪⏕యෆ࡛ࡢᩓ஘ࡢᙳ㡪ࢆཷࡅ࡟ࡃࡃ㸪ග※ࡢ῝㒊฿㐩ᛶࡀ㧗࠸ࡇ࡜ࡼࡾ㸪῝㒊࢖࣓࣮ࢪࣥࢢ
ࡀྍ⬟࡜࡞ࡿࠋື≀◊✲ศ㔝࡟࠾࠸࡚ࡣ㸪1,064 nm ࡢ࣮ࣞࢨ࣮ࢆග※࡜ࡋࡓ 2 ගᏊບ㉳㢧ᚤ㙾࡟ࡼࡾ㸪⏕ࡁࡓ࣐࢘ࢫࡢ⬻ࢆ⾲㠃࠿ࡽ 1,600 µm㸪ࡍ࡞ࢃࡕ 1.6 mm ࡶࡢ῝㒊ࡲ࡛ほᐹࡍࡿࡇ࡜࡟ᡂຌࡋ࡚࠸ࡿ㸦Kawakami et al. 2015㸧ࠋ ࡑࢀ࡛ࡣ㸪᳜≀⤌⧊࡟࠾࠸࡚ࡣ࡝࠺࡛࠶ࢁ࠺࠿ࠋࣔࢹ᳜ࣝ≀࡛࠶ࡿࢩࣟ࢖ࢾࢼࢬࢼ࡟࠾࠸࡚㸪᭱ࡶほᐹࡀ⾜࠸ࡸࡍ࠸⤌⧊࡜ࡋ࡚ࡣ㏱᫂࡞⤌⧊࡛࠶ࡿ᰿ࡀᣲࡆࡽࢀࡿ࡛࠶ࢁ࠺ࠋᅗ 1 ᕥࡣ⣽⬊᰾
ࢆ H2B-mClover ࡛ᶆ㆑ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼࡢ᰿ࢆඹ↔Ⅼ㢧ᚤ㙾࡛ほᐹࡋࡓീ࡛࠶ࡿࠋ㏱᫂࡞⤌⧊

BSJ-Review 7:124 (2016)

植物科学最前線 7:125 (2016)࡛ほᐹࡀ⾜࠸ࡸࡍ࠸⤌⧊࡛࠶ࡾ࡞ࡀࡽ㸪⥔⟶᮰ࡢ⣽⬊᰾ࡲ࡛ࡣ࠿ࢁ࠺ࡌ࡚ほᐹ࡛ࡁ࡚࠸ࡿࡀ㸪ᑐ≀ࣞࣥࢬ࠿ࡽ㐲࠸ഃࡢ᰾ࡣ඲ࡃほᐹ࡛ࡁ࡚࠸࡞࠸ࠋほᐹࡋ࡚࠸ࡿࢩࣟ࢖ࢾࢼࢬࢼࡢ᰿ࡢ┤ᚄࡣ 100µm ࡛࠶ࡿࡓࡵ㸪ඹ↔Ⅼ㢧ᚤ㙾࡛ほᐹ࡛ࡁ࡚࠸ࡿ
ࡢࡣ᰿⾲㠃࠿ࡽ 50 µm ࡯࡝࡛࠶ࡿࠋࡑࡇ࡛ྠࡌ᰿
ࢆ 2 ගᏊບ㉳㢧ᚤ㙾࡛ほᐹࡍࡿ࡜㸪ᑐ≀ࣞࣥࢬ࠿ࡽ㐲࠸ഃࡢ⣽⬊᰾ࡲ࡛඲࡚ほᐹ࡛ࡁ࡚࠸ࡿࡇ࡜ࡀ
ศ࠿ࡿ㸦ᅗ 1 ྑ㸧ࠋ࣮࢜࢟ࢩ࣐࣮࣮࡛ࣥ࢝࠶ࡿ
DR5rev::3xVenus-N7 ࢆྠ᫬࡟ほᐹࡍࡿ࡜㸪
2 ගᏊບ㉳㢧ᚤ㙾࡛ほᐹࡋࡓ᪉ࡀ㸪࡝ࡢ㡿ᇦࡲ࡛ DR5 ࡀⓎ⌧ࡋ࡚࠸ࡿ࠿ࡣࡗࡁࡾ࡜㆑ูࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿ㸦ᅗ 1㸧ࠋ㸰࣮㸰㸬㛗Ἴ㛗ග※ࢆ⏝࠸ࡿࡇ࡜࡛⮬ᐙ⺯ගࡀ㍍ῶࡍࡿ ᳜≀⤌⧊࡟࠾࠸࡚㸪2 ගᏊບ㉳㢧ᚤ㙾ࢆ⏝࠸ࡿ
฼Ⅼ࡜ࡋ࡚ࡣ㸪῝㒊࢖࣓࣮ࢪࣥࢢࡀ⾜࠼ࡿࡔࡅ࡛ࡣ࡞ࡃ㸪᳜≀⣽⬊≉᭷ࡢ⮬ᐙ⺯ගࢆ㍍ῶ࡛ࡁࡿⅬࡀᣲࡆࡽࢀࡿࠋ᳜≀⣽⬊ࡢ⮬ᐙ⺯ග࡜ࡋ࡚ࡣ㸪ගྜᡂ࡟㛵ࢃࡿⴥ⥳యࡢࢡࣟࣟࣇ࢕ࣝࡀࡲࡎᣲࡆࡽࢀࡿࡀ㸪ࡑࢀ௨እ࡟ࡶ⣽⬊ቨ࡟Ꮡᅾࡍࡿࣜࢢࢽࣥࡶ㢧ᚤ㙾ほᐹࡢጉࡆ࡟࡞ࡿ⮬ᐙ⺯ගࡢせᅉ࡜࡞ࡿࠋࢡࣟࣟࣇ࢕ࣝࡣᙉ࠸㉥Ⰽࡢ⮬ᐙ⺯ගࢆⓎࡍࡿࡓࡵ㸪㉥Ⰽ⺯ගࢱࣥࣃࢡ㉁ࡢほᐹࡢ㝿࡟ጉࡆ࡜࡞ࡿࠋࡲ
ࡓⴥ⥳యࢆᣢࡓ࡞࠸᰿ࡸⰼ⢊࡞࡝ࢆほᐹࡍࡿሙྜ࡛ࡶ㸪⣽⬊ቨ࡟ࣜࢢࢽࣥࡀᏑᅾࡋ㯤⥳Ⰽࡢ⮬ᐙ⺯ගࢆⓎࡍࡿࡓࡵ㸪GFP ࡞࡝ࡢ⥳Ⰽ⺯ගࢱࣥࣃࢡ㉁ࢆ⏝࠸࡚Ⓨ⌧㔞ࡢప࠸┠ⓗࢱࣥࣃࢡ㉁ࢆほᐹࡍࡿ㝿࡟㸪⮬ᐙ⺯ග࡜ࡢ㆑ูࡀᅔ㞴࡜࡞ࡗ࡚ࡋࡲ࠺
㸦Mizuta et al. 2015㸧ࠋࡲࡓ㸪⥳Ⰽ࠶ࡿ࠸ࡣ㉥Ⰽ⺯ᅗ ඹ↔Ⅼ㢧ᚤ㙾ീ࡜  ගᏊບ㉳㢧ᚤ㙾ീࡢẚ㍑
⣽⬊᰾ࢆ RPS5Apro::H2B-tdTomato㸪࣮࢜࢟ࢩࣥᛂ⟅ࢆ DR5rev::3xVenus-N7 ࡛ᶆ㆑ࡋ࡚࠸ࡿࠋୖẁࡣ⦪᩿㠃㸪⥺࡛♧ࡋ࡚࠸ࡿ㒊ศࡢᶓ᩿㠃ࢆୗẁ࡟♧ࡍࠋࢫࢣ࣮ࣝࣂ࣮ࡣ 30 µmࠋ
Kurihara et al. (2015)ࡼࡾᨵኚࡋ࡚㌿㍕ࠋ

ගࢱࣥࣃࢡ㉁ࢆほᐹࡍࡿ௨እࡢሙྜ࡟ࡶ㸪ࢡࣟࣟࣇ࢕ࣝࡸࣜࢢࢽࣥ࡞࡝ࡣ⺯ගࢱࣥࣃࢡ㉁ࡢບ㉳ගࢆ྾཰ࡋ࡚ࡋࡲ࠸㸪༑ศ࡟┠ⓗࡢ⺯ගࢱࣥࣃࢡ㉁ࢆບ㉳࡛ࡁ࡞࠸ࡓࡵ㸪ບ㉳ගࡢࣃ࣮࣡ࢆᙉࡃࡍࡿᚲせࡀ࠶ࡾ㸪⣽⬊࡟ࢲ࣓࣮ࢪࢆ୚࠼ࡿ࡞࡝ࡢᜍࢀࡶ࠶ࡿࠋࡇࡢࡼ࠺࡟㸪⮬ᐙ⺯ගࢆᢚ࠼࡚㢧ᚤ㙾ほᐹࢆ⾜࠺ࡇ࡜ࡣ㠀ᖖ࡟㔜せ࡞ࡇ࡜࡛࠶ࡿࠋ
 2-1 ࡛㏙࡭ࡓࡼ࠺࡟㸪ඹ↔Ⅼ㢧ᚤ㙾࡟ẚ࡭࡚ 2 ගᏊບ㉳㢧ᚤ㙾࡟ࡼࡾほᐹࡍࡿ㝿ࡣ 700 nm ~1,100 nm ࠶ࡿ࠸ࡣࡼࡾ㛗Ἴ㛗ࡢ࣮ࣞࢨ࣮ࡀ⏝࠸ࡽࢀ࡚࠸ࡿࠋ㛗Ἴ㛗㡿ᇦࡣࢡࣟࣟࣇ࢕ࣝࡸࣜࢢࢽ
ࣥࡢ྾཰ࢫ࣌ࢡࢺࣝ࠿ࡽࡣࡎࢀࡓ࡜ࡇࢁ࡟࡞ࡿࡓࡵ㸪㛗Ἴ㛗ࡢ࣮ࣞࢨ࣮ࢆ↷ᑕࡋࡓሙྜ࡛ࡶ㸪᳜

BSJ-Review 7:125 (2016)

植物科学最前線 7:126 (2016)≀ࡢ⮬ᐙ⺯ගࡣ๻ⓗ࡟㍍ῶࡉࢀࡿࠋ2 ගᏊບ㉳㢧ᚤ㙾ࡢሙྜ࡛ࡶ㸪800 nm ࡜࠸ࡗࡓẚ㍑ⓗ▷࠸Ἴ㛗ࡢ࣮ࣞࢨ࣮ࢆ↷ᑕࡋࡓሙྜ㸪ࢡࣟࣟࣇ࢕ࣝ㸪⣽⬊ቨࡢ⮬ᐙ⺯ග୧᪉࡜ࡶ᳨ฟࡉࢀࡿࡀ㸪1,000 nm࡜࠸ࡗࡓࡼࡾ㛗Ἴ㛗ࡢ࣮ࣞࢨ࣮ࢆ↷ᑕࡋࡓሙྜ㸪⮬ᐙ⺯ගࡣ᳨ฟࡉࢀ࡟ࡃࡃ࡞ࡿ㸦Mizuta et al.
2015㸧ࠋࡇࡢࡓࡵ㸪ࡼࡾᙅ࠸࣮ࣞࢨ࣮࡛ࡶ⺯ගࢱࣥࣃࢡ㉁ࡀ᳨ฟ࡛ࡁࡿࡓࡵ㸪⣽⬊࡬ࡢࢲ࣓࣮ࢪࡶ㍍ῶ࡛ࡁࡿࠋࡲࡓ㸪ࡇࢀࡲ࡛ࢡࣟࣟࣇ࢕ࣝࡢ⮬ᐙ⺯ග࡜㔜࡞ࡿࡓࡵ㑊ࡅࡽࢀ࡚ࡁࡓ㉥Ⰽ⺯ගࢱࣥ
ࣃࢡ㉁ࡶ฼⏝࡛ࡁࡿࡓࡵ㸪⺯ගࢱࣥࣃࢡ㉁ࡢ㑅ᢥ⫥ࡢᖜࡶᣑࡀࡿࠋ

㸰࣮㸱㸬1 Ἴ㛗ྠ᫬ບ㉳ከⰍ࢖࣓࣮ࢪࣥࢢࡀྍ⬟࡛࠶ࡿ ࡶ࠺୍ࡘࡢ 2 ගᏊບ㉳㢧ᚤ㙾ࡢ≉ᚩ࡜ࡋ࡚㸪୍ࡘࡢບ㉳Ἴ㛗࡛ከⰍࡢ⺯ගࢱࣥࣃࢡ㉁ࢆບ㉳ࡍࡿࡇ࡜ࡀྍ⬟࡜࡞ࡿࡇ࡜࡛࠶ࡿࠋ⺯ගࢱࣥࣃࢡ㉁ࡢᛶ㉁࡜ࡋ࡚㸪2 ගᏊບ㉳࡛ࡁࡿἼ㛗ࡣ 1 ගᏊບ㉳Ἴ㛗ࡢ༢⣧࡟ಸ࡟࡞ࡿࢃࡅ࡛ࡣ࡞ࡃ㸪ບ㉳ࢫ࣌ࢡࢺࣝࡣᣑࡀࡿഴྥ࡟࠶ࡿࠋࡑࡢࡓࡵ㸪1 ගᏊບ㉳ࡍ࡞ࢃࡕඹ↔Ⅼ㢧ᚤ㙾࡛ほᐹࡍࡿሙྜ㸪⥳Ⰽ⺯ගࢱࣥࣃࢡ㉁ࡣ 488 nm㸪㉥Ⰽ⺯ගࢱࣥࣃࢡ
㉁ࡣ 561 nm ࡜࠸࠺ࡼ࠺࡟㸪␗࡞ࡿἼ㛗ࡢ࣮ࣞࢨ࣮ࡀᚲせ࡛࠶ࡾ㸪
2 Ⰽࢆྠ᫬࡟ほᐹࡍࡿࡓࡵ࡟ࡣ㸪࣮ࣞࢨ࣮ࢆ 2 ࡘ↷ᑕࡋ࡞ࡃ࡚ࡣ࡞ࡽ࡞࠸ࠋࡋ࠿ࡋ㸪2 ගᏊບ㉳ࡢሙྜ㸪900 ~ 1,000 nm ࡢἼ㛗࡛⥳Ⰽ㸪㉥Ⰽ⺯ගࢱࣥࣃࢡ㉁࡜ࡶ࡟ບ㉳࡛ࡁࡿࡓࡵ㸪୍ᗘࡢ࣮ࣞࢨ࣮↷ᑕ࡛ከⰍ࢖࣓࣮ࢪࣥࢢࡀྍ⬟࡜࡞ࡿࡓࡵ㸪⣽⬊࡬ࡢࢲ࣓࣮ࢪࡀ㍍ῶ࡛ࡁࡿࠋᅗ 2 ࡣ mTFP1, sGFP, Venus, mApple ࡜࠸ࡗࡓỈⰍ࠿ࡽᶳⰍࡲ࡛ࡢ 4 ✀㢮ࡢ⺯ගࢱࣥࣃࢡ㉁ࢆࡑࢀࡒࢀⓎ⌧ࡍࡿⰼ⢊ࢆ㞟ࡵ࡚㸪ࡵࡋ࡭࡟ཷ⢊ࡋࡓᵝᏊ࡛࠶ࡿࠋບ㉳Ἴ㛗 990 nm ࡢ 2 ගᏊບ㉳㢧ᚤ㙾ほᐹ࡟ࡼࡾ㸪 4 Ⰽ඲࡚ࡢ⺯ගࢱࣥࣃࢡ㉁ࡀ᳨ฟ
࡛ࡁ࡚࠸ࡿࡇ࡜ࡀศ࠿ࡿࠋ ከⰍ࢖࣓࣮ࢪࣥࢢࢆࡍࡿ㝿ࡢὀពⅬ࡜ࡋ࡚ࡣ㸪࡝ࡢ⺯ගࢱࣥࣃࢡ㉁ࢆ㑅ࡪ࠿࡜
࠸࠺Ⅼࡀᣲࡆࡽࢀࡿࠋఱ✀㢮ྠ᫬࡟ほᐹࡋࡓ࠸࠿࡟ࡼࡿࡀ㸪2 ✀㢮ࡢሙྜࡣ㸪⥳Ⰽ࡜ᶳⰍ࠶ࡿ࠸ࡣ㉥Ⰽ⺯ගࢱࣥࣃࢡ㉁ࡀ
Ⰻ࠸ࠋࡇࢀࡽࡢ⤌ࡳྜࢃࡏ࡛ࡣ㸪950 ~1,000 nm ࡜࠸ࡗࡓ㛗Ἴ㛗ࡢບ㉳ගࢆ⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿࡓࡵ㸪⮬ᐙ⺯ගࢆᢚ࠼࡞ࡀࡽほᐹࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋࡋ࠿ࡋ㸪ᶳⰍ㸪㉥Ⰽ⺯ගࢱࣥࣃࢡ㉁ࡣ< 1,000 nm ࡢບ㉳ග࡛ࡣ྾཰ຠ⋡ࡀప࠸ࡓࡵ㸪2 ✀㢮ࡢ┠ⓗࢱࣥࣃࢡ㉁ࡢ࠺ࡕ㸪Ⓨ⌧㔞ࡢ㧗࠸ࢱࣥࣃࢡ㉁ࢆᶳⰍ㸪㉥Ⰽ⺯ගࢱࣥࣃࢡ㉁࡜⼥ྜࡋ㸪Ⓨ⌧㔞ࡢప࠸┠ⓗࢱࣥࣃࢡ㉁ࢆ⥳Ⰽ⺯ගࢱࣥࣃࢡ㉁࡟⼥ྜࡍࡿ࡜࠸࠺ᅗ ከⰍࡢⰼ⢊ࢆཷ⢊ࡋࡓࡵࡋ࡭

ᕤኵࡀᚲせ࡛࠶ࡿࠋࡲࡓ㸪3 ✀㢮ྠ᫬࡟

LAT52pro::mTFP1,ほᐹࡋࡓ࠸ሙྜࡣ㸪ඹ↔Ⅼࢩ࢔ࣥⰍ⺯ග

LAT52pro::mApple ࡛ࡑࢀࡒࢀᶆ㆑ࡋࡓⰼ⢊ࢆࡵࡋ࡭࡟ཷ

ࢱࣥࣃࢡ㉁ࡀࡼࡃ⏝࠸ࡽࢀ࡚࠸ࡿࡀ㸪950⢊ࡋ㸪2 ගᏊບ㉳㢧ᚤ㙾࡟ࡼࡾほᐹࡋࡓࠋࢫࢣ࣮ࣝࣂ࣮

nm ௜㏆࡛ࡣ࡯࡜ࢇ࡝ບ㉳ࡉࢀ࡞࠸ࡓࡵ㸪ࡣ 100 µmࠋKurihara et al. (2015)ࡼࡾᨵኚࡋ࡚㌿㍕ࠋ

LAT52pro::sGFP,

LAT52pro::Venus,

BSJ-Review 7:126 (2016)

植物科学最前線 7:127 (2016)1 Ἴ㛗ྠ᫬ບ㉳ࡣ㞴ࡋ࠸ࠋⴭ⪅ࡢሙྜ㸪ࢩ࢔ࣥⰍ⺯ගࢱࣥࣃࢡ㉁࡜⥳Ⰽ⺯ගࢱࣥࣃࢡ㉁ࡢ㛫ࡢⰍ࡛࠶ࡿ TFP ࢆࡼࡃ⏝࠸࡚࠸ࡿ㸦ᅗ 2㸧ࠋ ࡲࡓ㸪ከⰍࡢ⺯ගࢱࣥࣃࢡ㉁ࢆྠ᫬࡟ほᐹࡍࡿሙྜ㸪᳨ฟഃ࡟ࡶẼࢆ㓄ࡿᚲせࡀ࠶ࡿࠋTFP㸪
GFP㸪YFP ࡜࠸ࡗࡓỈⰍ㸪⥳Ⰽ㸪㯤Ⰽ⺯ගࢱࣥࣃࢡ㉁ࡣ⺯ගࢫ࣌ࢡࢺࣝࡀ㏆᥋ࡋ࡚࠸ࡿࡓࡵ㸪⺯ගࣇ࢕ࣝࢱ࣮࡛༊ูࢆࡘࡅࡿࡇ࡜ࡀᅔ㞴࡛࠶ࡿࠋࡑࡢሙྜ㸪ࣉࣜࢬ࣒࡛ศගᚋ㸪ከᩘࡢ᳨ฟჾ࡛
ࡑࢀࡒࢀࡢἼ㛗ࡢࢩࢢࢼࣝࢆ᳨ฟࡋ࢖࣓࣮ࢪࣥࢢࡍࡿࢫ࣌ࢡࢺࣝ࢖࣓࣮ࢪࣥࢢࡀ᭷⏝࡛࠶ࡿࠋඛ࡟♧ࡋࡓᅗ 2 ࡣࢫ࣌ࢡࢺࣝ࢖࣓࣮ࢪࣥࢢ࡛᧜ീࡋࡓࡶࡢ࡛࠶ࡿࡀ㸪mTFP1㸪sGFP㸪Venus ࡀࡑࢀࡒࢀ␗࡞ࡿⰍ࡜ࡋ࡚㆑ู࡛ࡁࡿࠋ

㸱㸬㏱᫂໬࡟ࡼࡾࡉࡽ࡞ࡿ῝㒊࢖࣓࣮ࢪࣥࢢ࡬
 2-1 ❶࡟࠾࠸࡚ 2 ගᏊບ㉳㢧ᚤ㙾ࡣ῝㒊࢖࣓࣮ࢪࣥࢢࡀྍ⬟࡛࠶ࡿࡇ࡜ࢆ㏙࡭࡚ࡁࡓࡀ㸪2 ගᏊບ㉳㢧ᚤ㙾ࢆᣢࡗ࡚ࡋ࡚ࡶ㸪᳜≀⤌⧊ࡣ῝㒊࢖࣓࣮ࢪࣥࢢࡀ㞴ࡋ࠸⤌⧊࡛࠶ࡿࠋᅗ 3 ࡣࢩࣟ࢖ࢾࢼࢬࢼࡢⴥࡢ⣽⬊᰾ࢆ㸪ඹ↔Ⅼ㢧ᚤ㙾࡜ 2 ගᏊບ㉳㢧ᚤ㙾ࢆ⏝࠸࡚ほᐹࡋࡓࡶࡢ࡛࠶ࡿࠋඹ↔Ⅼ㢧ᚤ㙾࡛ࡣ㸪ⴥࡢẼᏍࡸ⾲⓶⣽⬊࡜࠸ࡗࡓ⾲㠃࠿ࡽ 1~2 ᒙ㸪῝ࡉ࡟ࡋ࡚ 30 ~ 40 µm ࡋ࠿ࢩࢢࢼࣝࡣ᳨ฟ࡛ࡁ࡚࠸࡞࠸㸦ᅗ 3, ඹ↔Ⅼ㸧ࠋ୍᪉㸪2 ගᏊບ㉳㢧ᚤ㙾࡟࠾࠸࡚ࡶ㸪ඹ↔Ⅼ㢧ᚤ㙾࡜ྠ⛬ᗘࡢ῝ࡉࡲ࡛ࡋ࠿㸪⣽⬊᰾ࡢࢩࢢࢼࣝࡣ᳨ฟ࡛ࡁ࡞࠸㸦ᅗ 3, 2 ගᏊ㸧ࠋࡑࡢࡓࡵ㸪2 ගᏊບ㉳㢧ᚤ㙾ࢆᣢࡗ࡚ࡋ࡚ࡶ㸪ⴥࡢෆ㒊࡟࠶ࡿ⥔⟶᮰ࡣほᐹ࡛ࡁ࡞࠸ࠋᅗ 1 ࡛♧ࡋࡓࡼ࠺࡟㸪2 ගᏊບ㉳㢧ᚤ㙾ࢆ⏝࠸ࡿ࡜㸪┤ᚄ 100 µm ࡢ᰿࡛ࡣ඲⣽⬊᰾ࡀ㸪ࡲࡓࡵࡋ࡭ࡢ୰࡟࠶ࡿ⬇⌔ࡢほᐹ࡛ࡣ㸪175µm ࡢ῝㒊࡟࠶ࡿ⬇ங᰾ࢆࡣࡗࡁࡾほᐹࡍࡿࡇ࡜ࡀ࡛ࡁࡿ㸦Ỉከ࡜ᰩཎ 2015㸧ࠋᅗ ࢩࣟ࢖ࢾࢼࢬࢼࡢⴥ࡟࠾ࡅࡿඹ↔Ⅼ㢧ᚤ㙾ീ࡜  ගᏊບ㉳㢧ᚤ㙾ീ⣽⬊᰾ࢆ UBQ10pro::H2B-mClover ࡛ᶆ㆑ࡋ࡚࠸ࡿࠋᕥᅗࡣ xz ᖹ㠃࡬ࡢᢞᙳᅗ࡛࠶ࡿࠋୖഃࡀᑐ≀ࣞࣥࢬࡀᏑᅾࡍࡿഃ࡛࠶ࡿࠋྑᅗࡣ xy ᖹ㠃࡬ࡢᢞᙳᅗ࡛࠶ࡿࠋ࣮࢝ࣛࢫࢣ࣮ࣝࡣᑐ≀ࣞࣥࢬ࠿ࡽࡢ㊥㞳ࢆ♧ࡍࠋࢫࢣ࣮ࣝࣂ࣮ࡣ 100 µmࠋKurihara et al. (2015)ࡼࡾᨵኚࡋ࡚㌿㍕ࠋ ࡇࢀࡽࡢ㐪࠸ࡣఱ࡟㉳ᅉࡍࡿࡢ࡛࠶ࢁ࠺࠿ࠋࡑࡢ㐪࠸ࡣ⤌⧊ࡢᵓ㐀࡟࠶ࡿࠋࡑࡶࡑࡶ㸪⏕య⤌⧊ࡢ῝㒊ࡀほᐹࡋ࡟ࡃ࠸ࡢࡣ㸪⏕య⤌⧊ࡀᆒ୍࡛ࡣ࡞ࡃ㸪ᒅᢡ⋡ࡢ␗࡞ࡿࡉࡲࡊࡲ࡞≀㉁ࡀධࡾ

BSJ-Review 7:127 (2016)

植物科学最前線 7:128 (2016)஺ࡌࡗ࡚సࡽࢀ࡚࠸ࡿ࠿ࡽ࡛࠶ࡿࠋᒅᢡ⋡ࡀ␗࡞ࡿ≀㉁ࡢ⏺㠃࡛ࡣ㸪ගࡀ᭤ࡀࡗ࡚ࡋࡲ࠺ࡓࡵ㸪ᑐ㇟㡿ᇦࡀ῝㒊࡟࡞ࡿ࡯࡝㸪ບ㉳ගࡀ↔Ⅼ࡟ྜࢃ࡞࠿ࡗࡓࡾ㸪⺯ගࢱࣥࣃࢡ㉁࠿ࡽⓎࡏࡽࢀࡿ⺯ගࡀᑐ≀ࣞࣥࢬࡲ࡛ᒆ࠿࡞࠿ࡗࡓࡾࡍࡿࡓࡵ㸪῝㒊ࡢࢩࢢࢼࣝࡣ᳨ฟࡋ࡟ࡃ࠸ࠋ1 ࡘࡢ⣽⬊࡛ࡶ⣽⬊㉁㸦ᒅᢡ⋡ 1.36㸧ࡸ⣽⬊ቨ㸦ᒅᢡ⋡ 1.42㸧࡜ᒅᢡ⋡ࡀ␗࡞ࡿࡓࡵ㸪ඹ↔Ⅼ㢧ᚤ㙾࡛ࡣ㏱᫂࡞᰿ࡢ⤌⧊࡛ࡶ῝㒊࢖࣓࣮ࢪࣥࢢࡀ㞴ࡋ࠸ࡀ㸪≉࡟᳜≀ࡢⴥࡣෆ㒊࡟✵Ẽ㸦ᒅᢡ⋡ 1.00㸧ࢆከࡃྵࢇ࡛࠸ࡿࡓࡵ㸪ࡼࡾ῝㒊࢖࣓࣮ࢪࣥࢢࡀᅔ㞴࡛࠶ࡿࠋඖ᮶㸪ⴥࡣගྜᡂࢆ⾜࠺ჾᐁ࡛࠶ࡿࠋࡑࡢࡓࡵ㸪ⴥࡣෆ㒊࡟⣽⬊࡜ᒅᢡ⋡ࡢ኱ࡁࡃ␗࡞ࡿ✵Ẽࢆྵࡳ㸪ࡲࡓᙧែࡢ␗࡞ࡿ⣽⬊࠿ࡽᵓᡂࡉࢀ࡚࠸ࡿࡓࡵ㸪ྲྀࡾ㎸ࢇࡔኴ㝧ගࢆྛᡤ࡛ᒅᢡࡉࡏⴥࡢෆ㒊࡟㛢ࡌ㎸ࡵࡿࡇ࡜࡟ࡼࡗ࡚㸪ຠ⋡ࡼࡃගྜᡂࢆ⾜ࡗ࡚࠸ࡿ㸦Kumar & Silva 1973㸧ࠋࡑࡢࡼ࠺࡞⤌⧊ᵓ㐀ࡢ≉ᚩ࠿ࡽ㸪ࢩࣟ࢖ࢾࢼࢬࢼ࡛ࡣⴥࡸࡵࡋ࡭ࡀ㠀ᖖ࡟῝㒊࢖࣓࣮ࢪࣥࢢࡢ㞴ࡋ࠸⤌⧊࡛࠶ࡿࠋ ⴭ⪅ࡽࡣ᭱㏆㸪㏱᫂໬ࡢᢏ⾡ࢆ⏝࠸࡚㸪ࡇࡢᅔ㞴࡞᳜≀῝㒊࢖࣓࣮ࢪࣥࢢࢆྍ⬟࡟ࡍࡿᡭἲࢆ㛤Ⓨࡋࡓ㸦Kurihara et al. 2015㸧ࠋ᳜≀ࡢ㏱᫂໬࡛ࡼࡃ⏝࠸ࡽࢀ࡚࠸ࡿࡢࡣ㸪ᢪỈࢡ࣮ࣟࣛࣝࢆྵࢇࡔ㏱᫂໬ᾮ࡛࠶ࢁ࠺ࠋ
ྂࡃ࠿ࡽ⏝࠸ࡽࢀ࡚࠸ࡿ㏱᫂໬ᾮ࡛࠶ࡿࡀ㸪ᢪỈࢡ࣮ࣟࣛࣝࡣ⺯ගࢱࣥࣃࢡ㉁ࢆኻάࡉ
ࡏ࡚ࡋࡲ࠺ࡓࡵ㸪⺯ගほᐹࢆ⾜࠺ࡇ࡜ࡣ࡛ࡁ࡞࠿ࡗࡓࠋࡑࡢࡓࡵ㸪ື≀◊✲ศ㔝࡛㏆ᖺ㛤Ⓨࡉࢀ࡚࠸ࡓ㏱᫂໬ᢏ⾡㸦Hama et al.
2011, Susaki et al. 2014㸧ࢆᛂ⏝ࡋ࡚㸪᳜≀⤌⧊࡟᭱㐺໬ࡋࡓ㏱᫂໬ヨ⸆ ClearSee ࢆ㛤Ⓨᅗ ㏱᫂໬ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼᐇ⏕ᕥࡣ PBS㸪ྑࡣ ClearSee ࡛ฎ⌮ࡋ࡚࠸ࡿࠋ࣐ࢫ┠ࡣ 1mmࠋKurihara et al. (2015)ࡼࡾᨵኚࡋ࡚㌿㍕ࠋ

ࡋࡓ㸦Kurihara et al. 2015㸧
ࠋClearSee ࡛⤌⧊ࡸ⣽⬊ࡢ୰ࢆ⨨᥮ࡋ㸪ࡲࡓ‶ࡓࡍࡇ࡜࡛✵Ẽࢆྲྀࡾ㝖ࡃࡇ࡜ࡀ࡛ࡁࡿࡓࡵ㸪⤌⧊ࡢ୰ࢆᆒ୍໬ࡍ
ࡿࡇ࡜ࡀ࡛ࡁࡿࠋࡲࡓ㸪ClearSee ࡣ᳜≀⣽⬊࠿ࡽࢡࣟࣟࣇ࢕ࣝࢆྲྀࡾ㝖ࡃࡇ࡜࡟ࡼࡾ㸪⮬ᐙ⺯ගᅗ ㏱᫂໬ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼࡢⴥ࡟࠾ࡅࡿඹ↔Ⅼ㢧ᚤ㙾ീ࡜  ගᏊບ㉳㢧ᚤ㙾ീ⣽⬊᰾ࢆ UBQ10pro::H2B-mClover ࡛ᶆ㆑ࡋ࡚࠸ࡿࠋᅗࡢᵓᡂࡣᅗ 3 ࡜ྠࡌ࡛࠶ࡿࠋࢫࢣ࣮ࣝࣂ࣮ࡣ
100 µmࠋKurihara et al. (2015)ࡼࡾᨵኚࡋ࡚㌿㍕ࠋBSJ-Review 7:128 (2016)

植物科学最前線 7:129 (2016)

ࢆ㍍ῶࡋ㸪㏱᫂ᗘࡶቑࡍ㸦ᅗ 4㸧ࠋࡇࡢࡼ࠺࡟ ClearSee ࡛㏱᫂໬ࡋࡓࢩࣟ࢖ࢾࢼࢬࢼ࡛ࡣ㸪ཌࡉ100 µm ࡢⴥࡢ⾲㠃࠿ࡽ⿬㠃ࡲ࡛඲࡚ࡢ⣽⬊᰾ࢆほᐹࡍࡿࡇ࡜ࡀ࡛ࡁࡿ㸦ᅗ 5, ஧ගᏊ㸧ࠋࡑࡢࡓࡵ㸪㏱᫂໬ฎ⌮ࢆࡋ࡞࠸ⴥ࡛ࡣほᐹ࡛ࡁ࡞࠿ࡗࡓ⥔⟶᮰ࡢ⣽⬊᰾ࡲ࡛ほᐹࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࠋࡲ
ࡓ㸪2 ගᏊບ㉳㢧ᚤ㙾ࡔࡅ࡛ࡣ࡞ࡃ㸪ඹ↔Ⅼ㢧ᚤ㙾࡟࠾࠸࡚ࡶⴥ඲యࡢ⣽⬊᰾ࢆほᐹࡍࡿࡇ࡜ࡀ
࡛ࡁࡿ㸦ᅗ 5, ඹ↔Ⅼ㸧ࠋࡋ࠿ࡋ࡞ࡀࡽ㸪2 ගᏊບ㉳㢧ᚤ㙾ࢆ⏝࠸ࡿ࡜㸪S/N ẚࡢ㧗࠸࢖࣓࣮ࢪࣥࢢࡀྍ⬟࡛࠶ࡿࡋ㸪400 µm ࢆ㉸࠼ࡿࡵࡋ࡭ࡢࡼ࠺࡞ჾᐁ࡛ࡶ୸ࡈ࡜ࡢほᐹࡀྍ⬟࡛࠶ࡿ㸦Kurihara
et al. 2015㸧ࠋ㸲㸬ᒎᮃ ᮏ✏࡛ࡣ㸪᳜≀⤌⧊ࢆ῝㒊࢖࣓࣮ࢪࣥࢢࡍࡿࡓࡵࡢ 2 ගᏊບ㉳㢧ᚤ㙾ࡢ≉ᚩ࠾ࡼࡧὀពⅬ࡟ࡘ࠸࡚⤂௓ࡋ࡚ࡁࡓࡀ㸪2 ගᏊບ㉳㢧ᚤ㙾ࡣほᐹࡍࡿࡔࡅ࡛ࡣ࡞࠸ά⏝ἲࡶ࠶ࡿࠋ2 ගᏊບ㉳㢧ᚤ㙾࡛ࡣ㸪1 ࡘࡢ⺯ගศᏊࡀ 2 ࡘࡢගᏊࢆྠ᫬࡟྾཰ࡋ 2 ගᏊບ㉳≧ែ࡬㑄⛣ࡍࡿࡓࡵ࡟㸪ග※࡜ࡋ࡚ගᏊᐦᗘࢆ㧗ࡵࡿࡇ࡜ࡀ࡛ࡁࡿࣇ࢙࣒ࢺ⛊ࣃࣝࢫ࣮ࣞࢨ࣮ࢆ⏝࠸࡚࠸ࡿࠋࣇ࢙࣒ࢺ⛊ࣃࣝࢫ࣮ࣞࢨ࣮ࡣ▐㛫ⓗ࡞ࣆ࣮ࢡࣃ࣮࣡ࡀ㠀ᖖ࡟㧗࠸ࡓࡵ㸪⣽⬊࡟ᒁᡤⓗࢲ࣓࣮ࢪࢆ୚࠼ࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࠋࡇࡢ≉ᛶࢆ฼⏝ࡋ࡚㸪ࢱࣂࢥᇵ㣴⣽⬊ෆࡢ࢔ࢡࢳࣥ⧄⥔ࡢ୍㒊ࢆᦆയࡉࡏࡓࡾ㸪ཷ⢭༸ࡀ୙➼ศ⿣ࡋ࡚⏕ࡌࡿ 2 ࡘࡢ⣽⬊ࡢ࠺ࡕ 1 ࡘࡢ⣽⬊ࡔࡅ࡟ࢲ࣓࣮ࢪࢆ୚࠼ࡓࡾࡍࡿࡇ࡜ࡶྍ⬟࡜࡞ࡗ࡚࠸ࡿ㸦Hasegawa et al. 2014, Gooh et al. 2015㸧ࠋࡇࡢࡼ࠺࡟㸪⣽⬊ࢆほᐹࡋ࡞ࡀࡽ᫬✵㛫≉␗ⓗ࡟ࣇ࢙࣒ࢺ⛊ࣃࣝࢫ࣮ࣞࢨ࣮࡛ᦆയࢆ୚࠼㸪ࡑࡢᙳ㡪ࢆࣜ࢔ࣝࢱ࢖࣒࡟ほᐹࡍࡿࠕࣛ࢖ࣈࢭࣝゎᯒࠖࡣ㸪᳜≀ࡢⓎ⏕◊✲࡟࠾࠸࡚㠀ᖖ࡟㔜せ࡞ゎᯒἲ࡟࡞ࡗ࡚࠸ࡃ࡛࠶ࢁ࠺㸦Kurihara et al. 2013㸧
ࠋ2 ගᏊບ㉳㢧ᚤ㙾࡟ࡼࡾ῝㒊࢖࣓࣮ࢪࣥࢢࡀྍ⬟࡛࠶ࡿࡓࡵ㸪ࣛ࢖ࣈࢭࣝゎᯒࡀ᳜≀ࡢ῝㒊࡛㉳ࡇࡗ࡚࠸ࡿ⏕࿨⌧㇟࡟ࡶ㐺⏝ྍ⬟࡛࠶ࡿࠋࡲࡔ᳜≀◊✲⪅࡟ࡣ 2 ගᏊບ㉳㢧ᚤ㙾ࡣᬑཬࡋ࡚࠸࡞࠸ࡓࡵ㸪ᮏ✏ࡀ 2 ගᏊບ㉳㢧ᚤ㙾ࢆ౑ࡗ࡚ࡳࡼ࠺࡜࠸࠺ࡁࡗ࠿ࡅ࡟࡞ࢀࡤᖾ࠸࡛࠶ࡿࠋྡྂᒇ኱Ꮫ࡟ࡣ᳜≀⛉Ꮫ᭱ඛ➃◊✲ᣐⅬࢿࢵࢺ࣮࣡ࢡ࡟ࡼࡾᩚഛࡉࢀࡓ஧ගᏊ㢧ᚤ㙾ࡀᑟධࡉࢀ࡚࠾ࡾࠊ᳜≀⛉Ꮫ◊✲⪅࡬ࡢᨭ᥼ࢆ⾜ࡗ࡚࠸ࡿࠋᖖ㥔ࡢࢳ࣮ࣇࢥ࣮ࢹ࢕ࢿ࣮ࢱ࣮ࡀࠊ฼⏝⪅ࡢ◊✲┠ⓗ࡟ྜࢃࡏࡓ㢧ᚤ㙾㑅ᐃ࠾ࡼࡧゎᯒ᪉ἲ࡞࡝ࡢ┦ㄯ࡟ࡢࡾࠊ㢧ᚤ㙾᧯స࡟ࡘ࠸࡚ࡶᢏ⾡ᨭ᥼ࢆ⾜ࡗ࡚࠸ࡿࡢ࡛ࠊࡐࡦྡྂᒇ኱Ꮫࣛ࢖ࣈ࢖࣓࣮ࢪࣥࢢࢭࣥࢱ࣮ࡢ࢙࢘ࣈࢧ࢖ࢺ
㸦http://www.itbm.nagoya-u.ac.jp/liveimagingcenter/index.html㸧ࢆࡈぴ㡬ࡁࠊ฼⏝ࡋ࡚㡬ࡁࡓ࠸ࠋ㸳㸬ㅰ㎡ ᮏ◊✲ࡢ୍㒊ࡣᩥ㒊⛉Ꮫ┬᭱ඛ➃◊✲ᇶ┙஦ᴗ᳜≀⛉Ꮫ᭱ඛ➃◊✲ᣐⅬࢿࢵࢺ࣮࣡ࢡࡢᨭ᥼࡟ࡼࡾྡྂᒇ኱Ꮫࢺࣛࣥࢫࣇ࢛࣮࣐ࢸ࢕ࣈ⏕࿨ศᏊ◊✲ᡤ(WPI-ITbM)࡟࡚ᐇ᪋ࡉࢀࡓࠋ

㸴㸬ᘬ⏝ᩥ⊩
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