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ATP

Adenosine triphosphate

C. elegans

Caenorhabditis elegans

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Complementary DNA

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Cyan fluorescent protein

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Cyclic guanosine monophosphate

DiI

1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate

DNA

Deoxyribonucleic aciddNTPs

Deoxynucleotide triphosphates

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Forkhead box-containing protein, O sub-family

FRET

Fluorescence Resonance Energy Transfer

GFP

Green fluorescent protein

KQT

K+ channel related to QT interval

LB

Luria-Bertani

NGM

Nematode growth medium

PCR

Polymerase chain reaction

rGC

Receptor guanylyl cyclase

RNA

Ribonucleic acid

TRP

Transient receptor potential

YC

Yellow cameleon

YFP

Yellow fluorescent protein-3-

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C. elegans +®˟Ɍ 1 mm *˹ŉȘƁ*ğįɴǷƁ*ɚʂ%9®
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Kuhara et al. 2008; Glauser et al. 2011; Schild and Glauser 2013; Ohta et al. 2014)ȀũʬƁ&+̆*Ŀġ:ǉ¥%̃ɩ; C. elegans +̆*ȊȀũè˒%ˆö*̃ɩȀũ)ȹç:ʅç%:*ȀũʬƁ)ˤ=:ȵɕęʰ;$9Ȁũþňd}&$ AFD Ƈʒd}

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et al. 2018)ASJ Ƈʒd} Ȁũ>þň:&G [jMʪ& cGMP T
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:(Sonoda et al. 2016)
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$11(ȘȕȧŴá>ǎ

$:KCNQ ˌ©Ľn@x*ıȣ+ŽɳȤ6$?

?ŽƮɭ6˅ʅƁ˴ɦ>ŭʭõĚ&$Ȯ8;$: (Wang et al. 1996; Neyroud et al.
-10-1997; Biervert et al. 1998; Charlier et al. 1998; Jentsch 2000; Singh et al. 2003)C.
elegans )+KCNQ ĥIDy\|f*t{N

ĿġKQT (K+ channel

related to QT interval) ĥ\|f&ć¡8;$:C. elegans Pgy)+
KQT ĥ\|f>Qb: 3 #*ˌ©Ľ kqt-1kqt-2kqt-3

Ŀġ$

9

!KQT-1 & KQT-3 +êŀþňd}ǞǓÜȇþň

*ˌ©ĽșȎ*d}ɱ%ȦȔKQT-2 +ɱ%ȦȔ:&ĩċ;$: (Wei etal. 2005)ɱ%+ KQT ĥIDy\|f+ƛµRCM>Ûź: Ca2+RCM*·˅)˖ʎ(Ŵá>ǎ$:KQT-2 & KQT-3 +Čǅȧ( Ca2+/I
{U}³Ŀȧ(ɱ.* Ca2+ǸÉ*Ûź>ˁ$ƛµRCM>Ûź$: (Nehrke et al. 2008)]a%+KCNQ \|f+´ņ­ȀƇþƁøȵɕɇd}̍TG d}̎%ȦȔ­ȀƇþƁ*Ûź)˖ʎ(Ŵá>ǎ$: (Abd-Elsayed et al. 2015) (ĥIDy\|f

'*7

8çȎ)

)$Ȁũ̇ê>Ûź$:

$ KCNQ+;1%)

ĩċ;$(1.6 C. elegans

C. elegans *˗ȘǑ+*șĢ)7"$ñȑʅç>:ɋɗ&˰ěʅ
ç>:ɋɗ)Õ

;:*ʅçǚŬ)+Ȗĭ*˔ɎȈũ)ˤ=:ȵɕęʰ)7"$Ûź;$:&
h

£*Ȯʏ

ˤ˔Ɏþň8Ȳ;$:Cș* CB4856 Ǒ+̆*ɛ)˰1:˰ěʅç>Ȳ*)ō$oVa-11-ș* N2 Ǒ+ñȑʅç>Ȳ˰ěʅç>Ȳ(hCș CB4856 Ǒ&o

Vaș N2 Ǒ*;8*ʅçǚŬ*ˈ>ǬŃ:ˌ©Ľ>ˌ©ŀȧʗǌ)
79w]mN&<õĚˌ©Ľĳĥ+d}sp\b Y þň®>Qb: npr-1 ˌ©Ľ)Ŀġ (de Bono and Bargmann 1998)oVaș N2 Ǒ+˰ěʅç>Ȳ(*)ō$N2 )$ npr-1 ˌ©Ľ>g]MADaıȣ®+˰ěʅç>Ȳ* npr-1 ıȣ®*˰ěʅç>Ȳȣš+˔Ɏþň®%: GCY-35 :+ GCY-36 *Ǟɫǟƥĥıȣ)7"$ƎĠ
;: (Cheung et al. 2004)GCY-35 & GCY-36 + AQRPQRURX Ƈʒd}
%ȦȔ (Cheung et al. 2004; Gray et al. 2004)*)

: GCY-35 & GCY-36 *Ǟɫ! URX ˔Ɏþňd}˰ěʅç)ˤ$: (Cheung etal. 2004; Zimmer et al. 2009)URX ˔Ɏþňd})$˔Ɏ+˔Ɏþň® GCY-35/36 )7"$þň; (Cheung et al. 2004; Gray et al. 2004)êŀTc
pV&L|]pɖą>$ RMG ġd})ȩƞȵɕƄĩ:URX ˔Ɏþňd}
ADL >ĉ3˾ˑ*©ˇ;

8*˔ɎƄĩ+ RMG ġd}>$*Ƈʒd}*ȵɕǷç>ıê: (Fenk and deBono 2017)npr-1 ıȣ®>̉˔ɎȈũ%̃ɩ:& ADL êŀþňd}
*AVITbnE{)ō:ȵɕƀɃ̃ɩ:&*ƀɃƸ:ADL *ȵɕƀɃȈũ*­ƲĈ)ɚʂ¹®+ȹç:&ɤ+ɚʂɴʱ

­:­˔ɎȈũ%Ƹ:&AVITb

8;:AVITbnE{8Õǯ;:ſˍƁ*nE{%:4ADL *ȵɕƀɃ*Ƹ)79AVITbȈũ*̉˰ěʅçȐƈ

8AVITbȈũ*­ñȑʅç)Tna:&ɤ)URX & RMG >

8;$:*7-12-

$˔ɎƄĩ

ADL *ȵɕǷç>ˣƞȧ)Ûź:&)7"$˰ěʅçÛź;$: (Fenk and de Bono 2017)

1.7 C. elegans * TRP \|f*Ŵá

TRP \|f+ƞʘÜȇɦʒčʒʑʒĖʒȀũ('11(ƇʒÜȇ*þň6*Ƅĩ©ˇ)ˤ="$: (Montell 2005)TRP \|f
;ɴ®

ĲȠ*Üȇ>þň:þň®&$Ǟɫ:Īą5ĳ

TDUDiF*ʑɒɪ)"$þň;ÈƄĩ±,:ÈþňƄĩ©ˇɕʰ%+bpT)7

ɒɪÍ*XIbz]XU|>$ TRP \|f>ˢÿʑɒɪ>ǷƁê:*7)TRP \|fƇʒƄĩ*þň®&$%+(ƇʒƄĩ©ˇ*ʎ\|f&$Ǟɫ:±5ĳ
TRP \|f+ C. elegans )5¸Ŀ;$Ŀġ$

9TRPV \|f>Qb: osm-9 ˌ©Ľ+Ƈʒd}%ȦȔǺʾĠ)ō:ſˍʅç6éȎʪ)ō:êŀʬƁ>Ûź$:&Ȯ8;$: (Colbert et al.

1997)OSM-9 +˹ȀũƇþƁ* TRP \|f&$ĩċ;$©ˇ*ʎ\|f&$Ǟɫ:&ſˍʅç)5ˤ="$

9ƇʒƄĩ

Ȯ8;$:OSM-9 +Ȍ)ō:9Ƈʒd}%þň;´ņȧȌ*Ƅĩ©ˇ)ˤ=:˖ʎ(\|f&$À (Wittenburg and Baumeister 1999; Glauser et al.
2011; Schild and Glauser 2013)C. elegans * TRPV Ro~d]a%: ocr-2 ˌ©Ľ& osm-9 ˌ©Ľ+ſˍÜȇ.*ûƀ)žʎ(\|f%;8*ˌ©Ľ-13-ASH & ADL Ƈʒd}%Ǟɫ:&%¹®*˰ěʅç>·˅:(de Bono et al. 2002)
OCR-2 *bxcafJ_Boıȣ (OCR-2dn) +OCR-2dn t{\|f>ŲƉĪą6˗Șĥ* OCR-1 (OCR-1wt) &*q_\|f OCR2dn/OCR-1wt 6˗Șĥ* OCR-4 (OCR-4wt) &*q_\|f OCR-2dn/OCR4wt >ŲƉĪą)\|f*Ǟɫ>ǷƁê:&)79Ʒǅ*șó
ȣš>ŭʭ&ɤ

8;$: (Jose et al. 2007)TRP \|f+ɚʂ*­ȀɥƁ)5ˤ$:­ȀɥƁ>Ûź$:Ȁũþňd} ADL )$3 Ȼ́* TRP \|fRo~d]a ADL *ȀũƄĩ©ˇ):

ʎ\|f&$Ǟɫ$: (Ujisawa et al. 2018)TRP \|f>Qb
: 3 #*ˌ©Ľ%: ocr-2, osm-9 & ocr-1 ˌ©Ľ>ĆƼ)g]MADa
ocr-2 osm-9; ocr-1 ˖ıȣ®+­ȀɥƁ)ȣš>Ȳ (Ujisawa et al. 2018)
*ȣš+˖ıȣ®)

: ADL Ȁũþňd}*ȀũÜȇ)ō:ȵ

ɕƀɃ*­)7: (Ujisawa et al. 2018)3 Ȼ́* TRP \|fRo~d]a
ADL *ȀũƀɃ)ˤ:&+ʡ/8;$:*Ʋ%;;* TRP \|fRo~d]a* ADL Ȁũþňd})
;$(

-14-

:Ŵá+ĆŃ

2. ɖǎ

2.1 kqt-2 ıȣ®):­Ȁ̇êȣš

ɚʂ C. elegans +­Ȁ)ō$ɥƁ>5#&ĩċ;$:­ȀɥƁ&+˗ȘǑ%: N2 Ǒ> 25 %̃ɩŷ) 2 )ȹ&ǣȄ:*)ō15 %̃ɩŷ) 2 )ȹ&ȘĿ:Ȕʦ%:̍ĝ 1̎ (Ohtaet al. 2014)8)ɷčǼ&)ɚʂ+­Ȁ̇êȔʦ>5#&Õ"$̍ĝ 1̎ (Ohta et al. 2014; Okahata et al. 2016)­Ȁ̇êȔʦ&+±,

25 %̃ɩɚʂ>ƭƼˣ 15 )ȹ&)7"$ 2 %ȘĿ%Ćǚ) 15 %̃ɩɚʂ>ƭƼˣ 25 )ȹ&)7"$ 2 %ǣȄ:Ȕ
ʦ%: (Ohta et al. 2014; Okahata et al. 2016)­ȀɥƁȔʦ+˾ˑ* ASJ Ȁũþňd}

Ȁũ>þňCV>Õǯɱ6ȵɕþň:&)7"$­ȀɥƁ

CV>

Ûź;: (Ohta et al. 2014)8)ɉĽ

˾ˑ* ASJ Ȁũþňd}*ȵɕǷç)ų˺>
8ȵɕ.V_Cbt{>$ų˺>

ɱ

$:&

8ɉĽɉĽ
Õ"$(Sonoda et al. 2016)Ʋ%­Ȁ̇ê)ˤ=:zIdWy)+ǆȮ(ȉĳǤ;$:­Ȁ̇ê)ˤ=:Ʊʐˌ©Ľ>ĆŃ:4)ˆö)(=;

DNA wCMACʗǌ*ɖǎ>ÚȚ˗ȘǑ N2 Ǒ> 23 %̃ɩŷ) 17 ) 4 ƼˣȀũTnaˮ)ȦȔıç:ˌ©Ľ&$79 ¹*ˌ©Ľñ˳;$: (Sugi et al. 2011)ȦȔıç-15-

ʏ8;ˌ©Ľ*!ıȣ®

(=; (Ohta et al. 2014)*

Ŀġ:ɋɗ*­ȀɥƁ_Va!ǈȰȼ%+­ȀɥƁ)ȣš

ʏ8;( " 14 ɋɗ)ˤ$­Ȁ̇ê_

("̍ĝ 2A, B, Cĝ 3A, B, C̎25 %̃ɩŷ) 15 ) 0Va>

8 8 Ƽˣɡ2 *­ȀÜȇ>
̃ɩŷ 2 *­ȀÜȇ>

ȘĿȓ>ȁŃɖǎ˗ȘǑ+ 25 :& 5 %*ȘĿȓ>Ȳ15 ) 3 Ƽˣɡ&

30 %8 Ƽˣɡ& 44 %*ȘĿȓ>Ȳ̍ĝ 2A, B, C̎;)ō$\g*`kbPcY̍dhs-4(tm2418)̎*ıȣ®+ 25 ̃ɩŷ 2 *­Ȁ
Üȇ>:&˗ȘǑ N2 )ǧ/̉ȘĿȓ>Ȳȣš

ʏ8;̍ĝ 2A̎

25 %̃ɩŷ) 15 ) 3 Ƽˣɡ$ 2 *­ȀÜȇ>
M\̍clec-67(ok2770)̎*ıȣ®):&C-type$­ȘĿȓ>Ȳȣš

ʏ8;

I  D y \ | f ̍ kqt-2(ok732) ̎ \ g  * ` k b Pc  Y ̍ dhs4(tm2418)̎G [jMʪ α Ro~d]a̍gpa-7(pk610)̎*ıȣ®)ȘĿȓ>Ȳȣš

$̉

ʏ8;̍ĝ 2Bĝ 4B̎8)25 ̃ɩŷ 15 )

8 Ƽˣɡ$5 C-type M\̍clec-67(ok2770)̎*ıȣ®+ 2 *­ȀÜȇ>:&ǣȄ:ȣš>Ȳ ̍ĝ 2C̎Cation transporting ATPase̍catp-3(ok1612)̎TV_Cp_AYˤ˄[jM̍cpr-1(ok1344)̎\g*`kbPcY̍dhs-4(tm2418)̎G [jMʪ α Ro~d]a̍gpa7(pk610)̎˙ŗɖąƁ[jMʪ̍mtl-1(tm1770)̎ATP ɖą[jM̍pmp1(ok773)̎IDy\|f̍kqt-2(ok732)̎Iu˔FV_ãǩÕʗ˓Ɏ
̍T28C12.4(tm1013)̎+ 25 ̃ɩŷ 15 ) 8 Ƽˣɡ$ 2 *­ȀÜȇ>
:&˗ȘǑ)ǧ/$̉ȘĿȓ>Ȳȣš>Ȳ̍ĝ 2Cĝ 4C̎1
15 %̃ɩŷ25 ) 08 Ƽˣɡ 2 *­ȀÜȇ>
-16-ɖǎ˗ȘǑ+15 ̃ɩŷ 2 *­ȀÜȇ>

:& 92 %*ȘĿȓ>Ȳ25 ) 3 Ƽˣɡ

& 26 %8 Ƽˣɡ& 4 %*ȘĿȓ>Ȳ̍ĝ 3A, B, C̎;)ō$
DNA ɖąʲÏĚĽ̍dmd-7(ok2276)̎*ıȣ®)$ȘĿȓ*­ʏ8;

̍ĝ 3A̎15 %̃ɩŷ) 25 ) 3 Ƽˣɡ&G [jMʪ α Ro~d]a̍gpa-7(pk610)̎*ıȣ®)$̉ȘĿȓ>Ȳȣš

ʏ8;̍ĝ

3B̎
KQT ĥIDy\|f%: kqt-2 ıȣ®+ 25 ̃ɩŷ 15 ) 3
Ƽˣ:+ 8 Ƽˣɡ2 *­ȀÜȇ>

:&ȘĿȓƸ:ȣš>Ȳ̍ĝ 4B, C̎Ʋ%15 :+ 25 ̃ɩŷȀũTna) 2 ­ȀÜȇ>

$5 kqt-2 ıȣ®+ȣš>Ȳ("̍ĝ 4Aĝ 5A̎1

15 ̃ɩŷ 25 ) 3 Ƽˣ:+ 8 ƼˣȀũTna2 *­ȀÜȇ>$5kqt-2 ıȣ®+­Ȁ̇êȣš>Ȳ("̍ĝ 5B, C̎;1%)­ȀɥƁ)ˤ=:ˌ©Ľ&$cGMP ³ĿƁ\|f>ȵɕƄĩ©ˇɕʰ*ˌ©Ľĳƭñ˳;$:cGMP ³ĿƁ\|f+ƇʒƄĩ©ˇ)$Ƈʒd}*ɲ˵¬>ɮÕǘ:ʎ(I\H\|f%9*ˢÿ)7"$d}>ǷƁê:Ʋ%­ȀɥƁ)ˤ$Ƈʒd}*ǷƁ>ƎÛ:\|f[jMʪ*ʗǌ+˅?%(%
ǈȰȼ%+ɸȧ)ȵɕǷç>ƎÛ:&
%: KQT-2 *ʗǌ>(Ȯ8;$:IDy\|f&)

kqt-2(ok732) *­Ȁ̇êȣš

kqt-2 ˌ©Ľ*ıȣ)7:5*%:>ʡ/:4)XIbA%:kqt-2(tm642) *­Ȁ̇ê_Va>("ok732 A& tm642 A+ KQT-2 \|f*vA>Qb:Ĵ
-17-ˑÕ*FKZ
:&ǟƥ$:ıȣ%:4 (Wei et al. 2005)cıȣ%

ȲĔ;$:;8*ıȣ®*­Ȁ̇ê_Va>

25 %̃ɩŷ 15 ) 3 Ƽˣɡ2 *­ȀÜȇ>
kqt-2(ok732) &Ćǚ)­Ȁ̇ê

("ɖǎ:&kqt-2(tm642)+Ƹ:ȣš>Ȳ̍ĝ 6̎8)kqt-

2(ok732) *­Ȁ̇êȣš+˗Șĥ* kqt-2 ˌ©Ľ> kqt-2(ok732) )őÉ:&)7"$ȣš*ęŻ)Ȁũʏ8;̍ĝ 7̎£*ɖǎ

8kqt-2 + 25 ıê:ˮ*­Ȁ̇ê)ž˼*ˌ©Ľ%:&

8 15 

ȲĔ;

2.2 IDy\|f KQT-2 +Ƈʒd}%ȦȔ:ˆö*Ȱȼ8IDy\|f KQT-2 +ɱ%*2ȦȔ:&ĩċ;$ (Wei et al. 2005)%kqt-2 ıȣ®*­Ȁ̇êƸ*ȣšɱ*Ǟɫȣš)7"$ŭʭ;:&ɤkqt-2(ok732) )$ɱȏȣȧp

{[%: ges-1p )79ɱȏȣȧ) kqt-2 cDNA >ȦȔ­Ȁ̇ê>ʡ/

(8kqt-2(ok732) *­Ȁ̇êȣš+ɱȏȣȧ) kqt-2 cDNA>ȦȔ$5ęŻ("̍ĝ 8̎;1%*ǈȰȼŅ*ʗǌ)79C.

elegans *­ȀɥƁ+Ƈʒd}&ɱ%Ûź;$:&ĩċ;$

: (Ohta et al. 2014)*4Ƈʒd}ȏȣȧp{[%: osm6p >Ț$ kqt-2(ok732) %ȏȣȧ) kqt-2 cDNA >ȦȔ­Ȁ̇ê_Va>
("*ɖǎkqt-2(ok732) *­Ȁ̇êƸ*ȣš+Ƈʒd}ȏȣȧ) kqt-2 cDNA >ȦȔ:&)7"$ęŻ̍ĝ 8̎-18-Ǡ)kqt-2

ȦȔ:Ƈʒd}>ĆŃ:4)kqt-2 ˌ©ĽȜǊ*ǸɌ 9 kb *p{[˽Ħ*Ǹ)PgyȜǊ* kqt-2 ˌ©Ľ* 1
8 12 ȢFKZ1%ȢFKZĉ1;˒×& GFP >#(pVxb> Aguan D. Wei òİ79Õ$Ǒ*ȦȔɒɪʗǌ>KQT-2::GFPőÉ;˗Ș

("&<GFP ʃÈ+ɚʂ*˾ˑƇʒd}

%ǖÔ;̍ĝ 9A, B̎ˆö*ʢƯ)7:&kqt-2 +ȵɕ%+ȦȔ(&;$(Wei et al. 2005)Ƈʒd}* kqt-2 ˌ©Ľ*ȦȔr:

+ȦȔ˿ũ

ɱ795ŮāɫƁ>ɤ

1 kb

8 9 kb * kqt-2 ˌ©Ľ*p

{[)7:Ƈʒd}*ȦȔ>ʡ/1 kb
ĽȜǊ*p{[*Ǹ) kqt-2 *̏ȢFKZ

8 9 kb * kqt-2 ˌ©

8 12 ȢȨ*FKZ

GFP >ʄąpVxb>˗ȘǑ)őÉƇʒd}*ȦȔr>Ń˘ê&<kqt-2::gfp +ǂ5ȯp{[̍1 kb̎%ȦȔr"̍ĝ 10A, B̎*&8kqt-2 ˌ©Ľ*ǸɌ 9 kb p{[˽Ħ*)+ kqt-2 ˌ©Ľ*ȵɕɒɪ*ȦȔ>ƎÛ:˒×

Ŀġ:&

;8)kqt-2::gfp +HV*Ŕˑ*Çȿ1%ǝȐȽʭˑ*Ƈʒd}%5ȦȔ

ªů$:Ŕ

8KQT-2ȦȔ:˾ˑ*d}˾ˑÇȿ1%ª-$:Ƈʒd}%:&;̍ĝ 9A, B, D̎KQT-2
4)kqt-2 ıȣ®)ȲĔǞɫ$:˾ˑƇʒd}>ƟŃ:

:ȏŃ*Ƈʒd}*Ǟɫ>ʅçr%ʗǌ(: AWA Ƈʒd}&ˤ:ĖʒƀɃ_Va>

("̍ĝ 11A, B, C̎Ì®ȧ)+ǧʵȧňƻ)ʅçʗǌ>
AWC Ƈʒd}

ȲĔʏ8;̍ĝ 9C̎KQT-2::GFP >ȚȦȔɒɪʗǌ
+ƇʒǇȿ

Ű

-19-

AWA +UAX\>þň:Ėʒd}&$AWC +rWA`kb>þň:Ėʒd}&$Ȯ8;$: (Bargmann et al. 1993; Colbert
and Bargmann 1995)AWA Ėʒd})$éȎʪ+̐ęɲʨˁĥ*þň®[jM)7"$þň; (Sengupta et al. 1996)G [jM>ǷƁê˫CHʾˆ\|f%: TRP \|f̍OSM-9̎>ǷƁê:(Colbert et al. 1997)osm-9 ıȣ®+ AWA *éƄĩ©ˇ)ȣš>Ȳ&8

fJ_BoQa&$°Ț̍ĝ 11A̎˗ȘǑ+UAX\)ō$êŀʬƁʅç>Ȳ*)ōosm-9 ıȣ®+ʬƁʅç

ʏ8;(

"

kqt-2 ıȣ®+˗ȘǑ&Ćǚ*ʇȔĥ>Ȳ̍ĝ 11A̎Ćǚ)kqt-2 ıȣ®
)

$AWC Ėʒd}*rWA`kb)ō:êŀʬƁ_Va>
("̍ĝ 11B̎fJ_BoQa&$AWC Ėʒd})$rWA`kb*éƄĩ©ˇ)ˤ=: cGMP ³ĿƁ\|f tax-4

ˌ©Ľ*ıȣ®>°Ț˗ȘǑ+rWA`kb)ō:êŀʬƁʅçʏ8;*)ōtax-4 ıȣ®+ʬƁʅç ʏ8;(ȣš>Ȳ;)ō$kqt-2 ıȣ®+˗ȘǑ&Ćǚ)rWA`kb)ō$Ǣš(êŀʬƁʅç>Ȳ̍ĝ 11B̎;8*ɖǎ

8KQT-2 *Ǟɫɒɪ+ AWA d}& AWC d}%+(āɫƁɤ8;%AWA & AWC£Ĳ*˾ˑ*ȏŃ* 6 ō*Ƈʒd} (ADL, ASH, ASI, ASJ, ASK, AWB) >ʫɹʃÈ%Ǐɹ: DiI Ǐɹǰ>Ț$ KQT-2 *ȦȔɒɪ>ʡ/kqt-2::gfp>őÉɚʂ> DiI Ǐɹ˾ˑ*Ƈʒd}>ʕŌ&<DiI )7"$Ǐɹ;: ADL & ASK Ƈʒd})ʏ8;̍ĝ 9A, B̎kqt-2 ˌ©Ľ$KQT-2::GFP *ȦȔ˾ˑ*Ą 1 ō*Ƈʒd}%: ADL-20-

& ASK %ȦȔ:&ɖǎ+kqt-2 ıȣ®*­Ȁ̇êȣšƇʒd}%ȏȣȧ) kqt-2 cDNA >ȦȔ:&)79ęŻ:&8) kqt-2 ıȣ®* AWA & AWC Ėʒd}*ĖʒƀɃǢš%:&ɖǎ&ɶ

$:̍ĝ 8 ĝ 11A, B̎KQT-2 *Ƈʒd}*ɒɪÍŕġ>ʡ/:
4kqt-2 ȜǊ*p{[*Ǹ) kqt-2 cDNA & GFP >ƙÉpVxb>¯ʌŕġʗǌ>
Ê®%ȦȔ
*ʃÈ

ʏ8;

("*ɖǎKQT-2::GFP +Ƈʒd}ȏ)Ƈʒd}*ƇʒǇȿ&ɒɪ®%Ű GFP

ʏ8;̍ĝ 9D̎#19KQT-2 +Ƈʒd}*ƇʒǇȿ&ɒɪ®))ŕġ$:&ɤ

8;:1GFPȦȔ$:d}ƭĮã$*ȕȜ&$+*ŕġʗǌ)Ț DNA QVaM
a+kqt-2 ˌ©Ľ*ˢĻQbǸ 9 kb p{[*ȩ) kqt-2 cDNA::gfp>ʄą5*%:4Pgy DNA )ˁšĿġ: kqt-2 ˌ©Ľ*C
a˒×>ĉ?%(*4Pgy DNA * kqt-2 ˌ©Ľ*Ca
*˒×)+#ĉ1;$:&ɤ*d}%*ȦȔ>ƎÛ:4* DNA ˒×8;:̍ĝ 9D̎

2.3 kqt-2 ıȣ®*­Ȁ̇êȣš+ ADL Ƈʒd}ȏȣȧ) kqt-2 ˌ©Ľ>ȦȔ:&%ęŻ:

kqt-2 ıȣ®*­Ȁ̇ê Ƹ:ȣš+Ƈʒd}ȏȣȧ) kqt2 cDNA >ȦȔ:&)7"$ęŻ̍ĝ 8̎%KQT-2
-21-

­Ȁ̇ê

>Ûź:Ƈʒd}>ĆŃ:4)ɒɪȏȣȧVK}ń̈>
("ADL Ƈʒd}ȏȣȧp{[%: sre-1p & ASK Ƈʒd}ȏȣȧp{[%: sra-7p >Ț$;;*d}
ȏȣȧ) kqt-2 cDNA > kqt-2 ıȣ®)ȦȔ­Ȁ̇ê_Va>̍ĝ 12̎
*ɖǎkqt-2 ıȣ®*­Ȁ̇ê

("Ƹ:ȣš+ ADL Ƈʒd}

ȏȣȧ) kqt-2 cDNA >ȦȔ:&)7"$ęŻASK Ƈʒd}
%+ęŻ("̍ĝ 12̎ADL Ƈʒd}+ 1-octanol 6AVIT

bnE{('ſˍƁ*êŀȎʪ>þň:Ƈʒd}&$Ȯ8;$:41-octanol )ō: kqt-2 ıȣ®*ſˍƁ*ʅç_Va>

("̍ĝ 13A, B̎1-octanol +˾ˑ* ADLASHAWB Ƈʒd}%þň;
Ȯ8;$: (Troemel et al. 1995; Troemel et al. 1997; Hart et al. 1999)

:&

fJ_BoQa&$ADL & ASH ): 1-octanol *þňƄĩ©

ˇ)ˤ=: TRP \|f OSM-9 &ADL & AWB ): 1-octanol *þňƄĩ©ˇ)ˤ=: cGMP ³ĿƁ\|f TAX-4 *ıȣ®>Ț$ 1-octanol )ō:ſˍʅç_Va>

("̍ĝ 13A, B̎*ɖǎ˗ȘǑ+ 1-octanol )ō

$ſˍʅç>Ȳ*)ōfJ_BoQa%: osm-9 ıȣ®
& tax-4 ıȣ®%+ſˍʅç*ȣš ʏ8;̍ĝ 13A̎kqt-2 ıȣ®%+˗ȘǑ)ǧ/1-octanol )ō:ſˍʅç*­ʏ8;̍ĝ 13A̎;8*ɒɪȏȣȧVK}ń̈&ſˍʅç_Va*ɖǎ)

8KQT-2 + ADL Ƈʒd}$­Ȁ̇ê&ſˍʅç*Ûź)ˤ=:&ȲĔ;

;1%)25 %̃ɩ˗ȘǑ)ȀũÜȇ>
d}Í*ɒɪÍITDyȈũ

:&ADL ƇʒȀũıê)ƀ$ıê:&

-22-

ĩċ

;$: (Ujisawa et al. 2018)*&8 ADL +Ȁũþňd}%

8;$:* ADL Ȁũþňd}*ȀũƀɃƁ

:&ɤ

̃ɩȀũ)

>ʡ/:4)ITDyC`BO[%: YC3.60 > ADL³Ŀ:Ȁũþňd}ȏȣȧ)ȦȔITDyCzUNʗǌ>"25 ̃ɩŷ*˗ȘǑ%+17 Ƈʒd}Í)(8 23 )Ȁũıêˮ) ADL

:IzH*ʃÈʷũǧɌ 910 ̌ıê̍ĝ

14A, B̎;)ō15 ̃ɩ*˗ȘǑ%+ 25 ̃ɩ*˗ȘǑ795 ADLȀũþňd}*Ȁũıê)ō:ITDyȈũıê
*ʃÈʷũǧ*ıêʏ8;̍ĝ 14A, B̎£*ɖǎ$ ADL Ȁũþňd}*ȀũƀɃƁıê:&­Ɍ 45 ̌

8̃ɩȀũ)7"ȲĔ;

25 ̃ɩŷ* kqt-2 ıȣ®* ADL Ȁũþňd}*ȀũƀɃƁ>ITDyCzUNʗǌ%ʡ/&<17 

8 23 *Ȁũıê)ō

$kqt-2 ıȣ®* ADL %+˗ȘǑ)ǧ/ȀũƀɃƁ*ȵɕǷç*­ʏ8;̍ĝ 15A, B̎kqt-2 ıȣ®* ADL Ȁũþňd}*ȀũƀɃƁ*­ȣš+ADL Ƈʒd}ȏȣȧ) kqt-2 ˌ©Ľ>ȦȔ:&7"$ęŻ̍ĝ 15A, B̎( 815 %̃ɩ kqt-2 ıȣ®%+ADL*ȀũƀɃƁ)ȣš+ʏ8;("̍ĝ 16A, B̎£* kqt-2 ıȣ®*I

TDyCzUN*ɖǎ+kqt-2 ıȣ®

25 ̃ɩŷ)­Ȁ̇êȣš>Ȳ15 ̃ɩŷ* kqt-2 ıȣ®+Ǣš(­Ȁ̇ê>Ȳ&&ɶ$:



£*ɖǎ

8IDy\|f KQT-2 + 25 ̃ɩ¹®)

$ ADL Ȁũþňd}*ȀũƇȮƄĩ©ˇ&­Ȁ̇ê>Ûź$:&

-23-

ȲĔ;

2.4 KQT ĥIDy\|f KQT-3 + KQT-2 &ûō*Ŵá>ǎē́* KQT ĥIDy\|f%: KCNQ \|f+ǷƁê:&)7"$ȵɕǷç>ƎÛ:&Ȯ8;$:

ıȣ®%+Ȁũıê)ō: ADL *ȵɕǷç*­%KQT-2'*7ƅ)û$ kqt-2

ʏ8;̍ĝ 15A, B̎)$ ADL *ȵɕǷç>Ûź:>ʡ/:4)

ADL Ƈʒd}% kqt-2 cDNA >ˆàȦȔ˗ȘǑ*Ȁũ³Ŀȧ(ITDyȈũıê>ȁŃkqt-2 ˌ©Ľ> ADL %ˆàȦȔ˗ȘǑ)$ITDyCzUNʗǌ>

("&<ADL *ȵɕǷçˆǷƁê:ʇȔĥ

ʏ8;̍ĝ 17A, B̎#19IDy\|f KQT-2 +˗ȘǑ* ADL )$ȵɕǷç>ǷƁê:À>5#&ē́* KCNQ2 & KCNQ3 +q_\|f>ŲƉ:&(Wang et al. 1998)*4KQT-2 51ʍą®>ŲƉKQT-2

ȲĔ;

ĩċ;$:* KQT ĥIDy\|f&

fJ_BoL}[&$ÀāɫƁɤ8;C. elegans + kqt-1kqt-2 & kqt-3 * 3 Ȼ́* KQT ĥIDy\|f>Qb$:%kqt-1 ıȣ®& kqt-3 ıȣ®*­Ȁ̇ê_Va>("kqt-1 ıȣ®+­Ȁ̇ê)ȣš+ʏ8;(Ȁ̇êƸ:ȣš

kqt-3 ıȣ®%+­ʏ8;̍ĝ 18A-Fĝ 19̎kqt-3 + ADL >ĉ3ʍƭ*Ƈʒd}%ȦȔ$:&%KQT-3"ĩċ;$: (Wei et al. 2005)

Ȁũıê)ō: ADL Ƈʒd}*ȵɕǷç)ˤ="$:>ʡ/:4)kqt-3 ıȣ®*ITDyCzUNʗǌ>

("

kqt-3 ıȣ®* ADL Ƈʒd}*Ȁũıê)ō:ITDyȈũıê>-24-ȁŃ&<˗ȘǑ&ǧ/ɒɪÍITDyȈũ*Ƹ

ʏ8;̍ĝ 20A,

B̎
kqt-2 ıȣ& kqt-3 ıȣ*ˌ©ŀȧÄ¬ˤ¶>ʡ/:4)kqt-2; kqt-3
˖ıȣ®>¯ʌADL *ȀũƀɃƁ*ITDyCzUNʗǌ&­Ȁ̇ê_Va>

("kqt-2; kqt-3 ˖ıȣ®* ADL Ȁũþňd}*ITDyCzUNʗǌ*ɖǎkqt-2 ıȣ®*Ȁũıê)ō: ADL *ȵɕǷç*­+ kqt-3 ıȣ)7"$ƎĠ;kqt-3 ıȣ®&Ćǚ*ʇȔĥ>Ȳ̍ĝ 20A, B̎8)kqt-2; kqt-3 ˖ıȣ®*­Ȁ̇ê_Va*ɖǎ
kqt-2 ıȣ®*­Ȁ̇êȣš+ kqt-3 ıȣ)7"$ƎĠ;̍ĝ 19̎;8*ˌ©ŀȧʗǌ

8kqt-3 +ˌ©ŀȧ) kqt-2 *Ǹ%­Ȁ̇ê& ADL *ȵɕǷç>Ûź:&ȲĔ;*ɖǎ+ KQT-2}[&$À&
ADL Ƈʒd})KQT-3 *fJ_BoL¤ʟ&ɶ$:£*ɖǎ

8KQT-2 +$KQT \|f*fJ_BoL}[&$À&)7"$ADL *ȀũƇȮ>Ûź:āɫƁ

ɤ

8;

2.5 TRP \|f OCR-1 + ADL Ƈʒd}*ȀũƀɃƁ*ʧ*ÛźĚĽ&$ÀIDy\|f KQT-2 + ADL Ȁũþňd}%­Ȁ̇ê>Û
źADL Ƈʒd}*ȀũƀɃƁ>Ǣ)Ûź:&)KQT-2 &ƴȮ* ADL Ȁũþňd})-25-

ȲĔ;Ǡ:ȀũƄĩ)ˤ=:ÕĽ&*ˌ©ŀȧ(ˤ¶>ʡ/ˆö*ʗǌ

8ADL %ȦȔ: TRP \|f%: OSM-9OCR-1 & OCR-2 +­ȀɥƁ)ˤOSM-9OCR-1 & OCR-2>$ ADL Í*ȀũƄĩ©ˇ(=;$:&ĩċ;$:(Ujisawa et al. 2018)1osm-9 ıȣ®& ocr-2 osm-9; ocr-1 ˖ıȣ®+­Ȁocr-1 ıȣ®& ocr-2 ıȣ®*Ąñȑ*ıȣ®%+­ȀɥɥƁ)ȣš>Ȳ" (Ujisawa et al. 2018)%ocr-1 ıȣ®& ocr-2 ıƁ*ȣš+ʏ8;(ȣ®*Ąñȑ*ıȣ®)ˤ$­Ȁ̇ê_Va& ADL Ȁũþňd}*ȀũƀɃƁ*ITDyCzUNʗǌ>

("*ɖǎocr-1 ıȣ®& ocr-2 ıȣ®+­Ȁ̇ê)ȣš>Ȳ(" ̍ĝ 21A, B̎kqt-2 ıȣ®

*­Ȁ̇êȣš+ ocr-1 ıȣ)7"$ƎĠ;̍ĝ 22̎*&
̇ê)

8­Ȁ

$ocr-1 ıȣ+ kqt-2 ıȣ)ō$ˌ©ŀȧ)Ä¬%:&ȲĔ;#19­Ȁ̇ê)ˤ$OCR-1 & KQT-2 +ˌ©ŀȧ)Ćɕʰ
%¯Ț$:&ɤ

8;:̍ĝ 22̎

TRP \|f+ǷƁê:&ɮÕǘ>ŭʭ˹ˋƑƁ˫CH
\|f&$Ȯ8;$:4TRP \|f*Ǟɫǟĸĥ*ıȣ®%+ȵɕǷç­:&ɤ

8;:ocr-1 ıȣ®*ITDyCzUN*ɖǎƅ)û$Ȁũıê)ō$ ADL Ƈʒd}*ɒɪÍITDyȈũ˗ȘǑ)ǧ/$Ƹ:ȣšʏ8;̍ĝ 23A, B̎8)osm-9; ocr-1 ˖ıȣ®& ocr-2; ocr-1 ˖ıȣ®*ITDyCzUN>
ocr-1 ıȣ®)$ ADL *ȀũƀɃƁ

("ɖǎƸ:ȣš+osm-9 ıȣ1+

ocr-2 ıȣ)7"$ƎĠ;̍ĝ 24A, Bĝ 25A, B̎£*ɖǎ

8osm-9

ıȣ& ocr-2 ıȣ+ocr-1 ıȣ)ō$ˌ©ŀȧ)Ä¬%:&

ȲĔ;

-26-

#19OCR-1 + OCR-2 & OSM-9 *ƎÛĚĽ&$ǞɫOCR-1OCR2 & OSM-9 +ˌ©ŀȧ)Ćɕʰ%¯Ț$:&ɤ8;:Ǡ)kqt-2; ocr-1 ˖ıȣ®*ITDyCzUNʗǌ>("*ɖǎkqt-2 ıȣ®*ȀũÜȇ)ō: ADL *ȵɕǷç*­+ ocr1 ıȣ)7"$ˑÕȧ)ƎĠ;ocr-1 ıȣ®& kqt-2 ıȣ®* ADL *ȀũƀɃƁ*ˣ*ʇȔĥ>Ȳ̍ĝ 26A, B̎£*ĳ˖ıȣ®)ȀũƀɃƁ*ITDyCzUNʗǌ

: ADL *8ADL Ȁũþňd}Í)

$TRP \|f%: OCR-1 +IDy\|f KQT-2 &+Ùɕʰ%
ADL Ƈʒd}*Ȁũıê)ō:ȵɕǷç>Ûź$:&ɤ

2.6 ˔ɎƄĩ+ ADL >:­Ȁ̇ê)ų˺>

;1%*ʗǌ

8;:

:8 kqt-2 ıȣ®+­Ȁ̇ê)ȣš>5#&;kqt-2 ıȣ®*­Ȁ̇ê*ʇȔĥʗǌ>˅4:(ȲĔ%ƅ)û$

kqt-2 ıȣ®*­Ȁ̇ê+̃ɩŋĵħĢ̍NGM pa̎*Ĵ)7"$ȣ(:ʇȔĥʏ8;ó

8Ɖʂ1%ȩŵ 3.5 cm *ŋĵħĢ̍ȩŵ 3.5 cm p

a̎%̃ɩŷ) 2 *­ȀÜȇ>̀ɾ(­Ȁ̇ê*ŝ+ʏ8;($5˗ȘǑ& kqt-2 ıȣ®*ˣ%"&<

˗ȘǑ& kqt-2 ıȣ®>ȩŵ

6 cm *ŋĵħĢ̍ȩŵ 6 cm pa̎%̃ɩŷ) 2 *­ȀÜȇ>:&kqt-2 ıȣ®%+˗ȘǑ)ǧ/$̀ɾ)­Ȁ̇ê̍ĝ 27̎#19̃ɩȀũ)ã

Ƹ:ȣš

ʏ8;$̃ɩħĢ*Ĵ)ʭĚ:

*Ȗĭ

-27-

ĚĽ

kqt-2 ıȣ®*­Ȁ̇êȣš)ų˺>

$:&ȲĔ;

1%* kqt-2 ıȣ®*­Ȁ̇ê_Va&ITDyCzUNʗǌȀũþňd}­Ȁ̇ê>Ûź:&8ADL

ȲĔ;̍ĝ 12ĝ 15A, B̎

ADL +ſˍƁ*êŀȎʪ>þň:Ėʒd}&$5Ȯ8;$9ſˍȎʪ%:AVITbnE{)ō: ADL Ƈʒd}*ȵɕƀɃƁ+˔ɎȈũ*ų˺>þ:&ĩċ;$: (Fenk and de Bono 2017)

Ì®ȧ)+˔Ɏþňd}%: URX Ƈʒd}+ RMG ġd}
>$ˣƞȧ) ADL *ȵɕǷç>ıê:&%ʏ#ȵɕęʰr"$: (Fenk and de Bono 2017);8*Ƅĩ>5&)ȩŵ 3.5 cm

pa&ȩŵ 6 cm pa*˔ɎȈũ*ˈȔĥ)ų˺>$:āɫƁ>ɤ

kqt-2 ıȣ®*­Ȁ̇ê*ʇ

ˆö*Ȱȼ8ŋĵpa*˔

ɎȈũ+ɚʂ*̆%:Ĵɱɽ)7"$ų˺;:&ĩċ;$:(Gray et al. 2004)%­Ȁ̇ê_Va%°Ț$:ȩŵ 3.5 cm pa
&ȩŵ 6 cm pa%˔ɎȈũȣ(:>ʡ/Ì®ȧ)+Èŀȧ)˔

ɎȈũ>ȁŃ%:ʊɡ>Ț$̆%:Ĵɱɽ

ī8;ŋĵ*˔ɎȈũ>ȁŃ̍ĝ 28̎­Ȁ̇ê_Va*ƨ¯paQ&Ćǚ)Ĵɱɽīş;̃ɩŋĵħĢ%ʔ)ó>ș1ƭƼˣŷ)ʔ>ý9˩̃ɩp
a>jnBy%Ŋˡ;8*pa> 24 Ƽˣ

8 64 Ƽˣ˸ɡ̃ɩpa*ŋĵħĢ)īş;Ĵɱɽ*˔ɎȈũ>ȁŃ*ɖǎșóŷ 52 Ƽˣ

8 64 Ƽˣŷ)

$ȩŵ 6 cm pa*Ĵɱɽ*˔ɎȈũ+ŁŃ$Ɍ 15 %*˔ɎȈũ>Ȳ*)ōȩŵ 3.5 cm p
a%+ŋĵħĢ)īş;Ĵɱɽ*˔ɎȈũ+Ɍ 10 %%"̍ʇ 1̎
-28-

˔ɎƄĩ

­Ȁ̇ê)ų˺>

ň)ȣš>5#ıȣ®)

:āɫƁ

ɤ

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kqt-2 ˖ıȣ®)ǧ/$­Ȁ̇ê_Va) $­ȘĿȓ>Ȳ̍ĝ 34̎
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Ûź:­Ȁ̇êǈȰȼ*Șȕŀȧʗǌ&ˌ©ŀȧʗǌ)79KQT-2 *ȦȔɔɟ*!ADL Ƈʒd}ʏ8;Ȁũıê)ō:ȵɕǷç>ıê:&)7"$­Ȁ̇ê>Ûź:&Ȳ;KQT ĥIDy\|f%: KQT-2 & KQT-3 *ıȣ®*­Ȁ̇ê_Va*ɖǎ
3 + ADL Ƈʒd}%­Ȁ̇ê>Ûź:&

8KQT-2 & KQT-ȲĔ;8)ADL

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8

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*ȵɕǷç>Ûź:&8kqt-

2 ˌ©Ľ+˗ȘǑ N2 > 23 %̃ɩŷ) 17 ) 4 ƼˣȀũTna:
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ĮãKQT-3 &ʍą®>ŲƉ$KQT-2/KQT-3 q_\|f>ŲƉ

\|f*ǷƁ>ƎÛ:āɫƁ
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8;:kqt-2 ıȣ®%+KQT-

ŲƉ;ǷƁê KQT-3 t{\|f

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8;:8)kqt-2 ˌ©Ľ> ADL %ˆàȦȔ:&

KQT-2/KQT-3 q_\|f
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)KQT-2 +Ȗĭ*ıê)ƀ$ȦȔ˘>ıê:&)79KQT-3 *ǷƁ*ʡɇ>:[jMʪ&$À&ȲĔ;KQT-2

ʡɇÀ&)79ADL Ȁũþňd}*ȵɕǷç;ƱȀũȖĭ.*̇êzIdWy

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ocr-1 ˖ıȣ®*­Ȁ̇ê&ITDyCzUN*ʗǌ

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ADL %ȦȔ: TRPV \|f%: OSM-9OCR-2 & OCR-1

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:&) TRP \|f& KQT ĥIDy

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8;:

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Ujisawa et al. 2018)8)endu-2 ıȣ®+Ȁũıê)ō: ADL Ȁũþňd
}*ȵɕǷç­:ȣš

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Ŀȓ:&˗ȘǑ)ǧ/$ȘƸ:ȣš>ȲADL *Ȁũıê)ō:ȵɕǷç­:ȣš

ʏ8;*­Ȁ̇êȣš& ADL *ȀũıêƼ*ȵɕǷç*ȣš+ ADLd}%ȏȣȧ) kqt-2 ˌ©Ľ>ȦȔ:&)7"$ęŻ8)
ADL %ȦȔ: TRPV \|f*˖ıȣ®̍ocr-2 osm-9; ocr-1̎%+­ȀɥƁ*Ƹȣš

ʏ8;ADL *ȵɕǷç­:ȣš5Ȳ&ĩċ;$: (Ujisawa et al. 2018);8* endu-2 ıȣ®& kqt-2 ıȣ®$ TRPV\|fıȣ®*­ȀɥƁ­Ȁ̇ê*ʇȔĥ& ADL *ȀũƀɃƁ*ITD
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8ADL Ȁũþňd}*Ȁũıê)ō:­ȀɥƁ­Ȁ̇ê*Ƹ)#(
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8*˔ɎƄ

ĩ+ RMG ġd}>$ ADL êŀþňd}*AVITbn
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ADL Ƈʒd}%ɗą;:&)7"$­Ȁ̇êþň;ȀũƄĩ

Ûź;$:āɫƁɤ

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%+(AVITbnE{6 1-octanol ('*êŀȎʪ>þň:&ĩċ;$: (Troemel et al. 1995; Troemel et al. 1997; Jang et al. 2012)

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:&9 (Fenk and de Bono 2017)ǈȰȼ*ʗǌ

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24 ¹%:ɚʂ C. elegans +˧8;ƭ*Ƈʒd}%ʍƭ*ȖĭƄĩ>þň;8*Ƅĩ>ìÙ6ɗą:ȵɕęʰŲƉ;$:&ɤ8;:ka>ĉ3̉ɁçȎ%+8)ɼĴ(Ƅĩ˘>ˉÖ)Óȕ:4*ʍ˲(ȵɕęʰ>ǙƉ$:ɯȵɕɋ>ǙƉ:¹*ȵɕɒɪ)Éâ;:ʍƭ*Ƅĩ

ǽɚ:&('*7

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8;:4

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15 %̃ɩ˗ȘǑ& 25 %̃ɩ kqt-2 ıȣ®+ ADL *ȵɕǷç*­ʏ8;2 *­ȀÜȇ>

ADL *ȵɕǷç:&̉ȘĿȓ>Ȳ#19

­:&)7"$­Ȁ̇ê-35-Ƹ:āɫƁɤ

8;: (ɕǷç8kqt-3 ıȣ®& ocr-1 ıȣ®+Ȁũıê)ō: ADL *ȵƸ:)5ˤ=8kqt-3 ıȣ®+­Ȁ̇êƸocr-1 ıȣ®

+Ǣš(­Ȁ̇ê*ʇȔĥ>Ȳ8)gcy-35 ıȣ®%+ ADL *Ȁũıê)ō:ȵɕǷç+Ǣš­Ȁ̇ê)ˤ$­ȘĿȓ>Ȳ*4

ADL Ƈʒd}+­Ȁ̇ê>Ûź:&ɤ8;:­Ȁ̇ê*zIdWy)+ ADL )ã

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Ujisawa et al. 2016)ɉĽ*ıȣ®gsp-4 ıȣ®*­Ȁ̇êȣš+ kqt-2 ıȣ)
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®ɰ*˔Ɏþňd} URX )35 *ıȣ®%+­Ȁ̇ê*­$˔Ɏþň®&$À GCY-

ʏ8;gcy-35 ıȣ+ kqt-2 ıȣ®*­Ȁ̇ê*Ƹ& ADL *ȵɕǷç*­>ƎĠ120 %& 10 %˔ɎȈũ%̃
ɩ gcy-35 ıȣ®*­Ȁ̇ê+ı=8( "Ȉũ%̃ɩƲkqt-2 ıȣ®+ 10 %˔Ɏ

20 %˔ɎȈũ̃ɩŷ* kqt-2 ıȣ®795­Ȁ̇ê:ʇȔĥ>Ȳ̍ĝ 32̎*&
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:&

ȲĔ;*Tp(ȵɕęʰ>ĆŃ;1%­ȀɥƁ)

ˤ=:ˌ©Ľ6­ȀɥƁ>Ûź:ɔɟ+ʍƭĆŃ;$:Ȁũ̇êzȊ"ę*ʗǌ)79Ȁũ̇ê)+ KQT ĥ

IDy\|fˤ="$

9KQT ĥIDy\|f+ ADL %­Ȁ̇

ê>Ûź:&=IdWy)ˤ$+ĩċ

OCR-1"1ADL %+ TRP \|f OSM-9OCR-2

Ȁũıê)ō:ȵɕǷç>Ûź$>ʧ)Ûź:&9OCR-1ȲĔ;8)KQT ĥIDy\|f:Ȁũ̇ê+̃ɩ˔ɎȈũ)7"$ų˺>þ:&Dy\|f

=Ûź"KQT ĥI­Ȁ̇ê>Ûź: ADL Ȁũþňd}*Ǹ)+ URX

˔Ɏþňd}

Ŀġ$

9ȀũƄĩ& URX

8*˔ɎƄĩ

Ȁũþňd}%ɗą;:&)7"$­Ȁ̇ê:&ɤ

OSM-9OCR-2

ADL˔ɎȈũ)³Ŀ

8;:

8;:çȎ+š)ʍƭ*ȖĭƄĩ>þ&9ƄĩÓȕ$:&ɤ̉ɁçȎ%+ʍ˲%:4ʍƭ*ȖĭƄĩ>©ˇ:ȵɕęʰ*zIdWy>ʗƺ:)+Ƽˣ>ʎ:&ɤ

8;:ǈȰȼ%+Tp({`çȎ%:ɚʂ C. elegans >Ț$ȀũƄĩ&˔ɎƄĩ&ȣ(: 2 #*ƇʒƄĩ*ɗą)ˤ=:ȵɕęʰ{`>ơĕŷ*ń̈
ɋ>Ț$ʗǌ>˅4:&%̉ɁçȎ1%¸Ŀ;ȵɕęʰ)ĩ*ìÙ6ɗą*Ĩǈõȕ*ȕʗ)ɞ

:&ɤ

-38-

8;:

:Ƅ

5.

ɚʂ C. elegans *̃ɩǰħĢ6 Buffer *ɔƉɁ+ The Nematode Caenorhabditis
Elegans )Ÿ" (Wood and Researchers 1988)

°Țɚʂɋɗ

N2 Bristol England, KHR181 kqt-2(ok732), kqt-2(tm642), CX4544 ocr-2(ak47), CX4652
ocr-2(ak47) osm-9(ky10), ZK64 kqt-3(aw1), KHR182 kqt-3(aw1); kqt-2(ok732),
CX4533 ocr-1(ok132), KHR183 ocr-1(ok132); kqt-2(ok732), KHR184 ocr-2(ak47); kqt2(ok732), CX6448 gcy-35(ok769), KHR185 gcy-35(ok769); kqt-2(ok732), RB1415 catp3(ok1612), VC693 cgt-1(ok1045), RB2095 clec-67(ok2770), RB1262 cpr-1(ok1344),
FX2418 dhs-4(tm2418), RB1772 dmd-7(ok2776), FX2468 F58E6.7(tm2468), NL795
gpa-7(pk610), RB1373 gpdh-1(ok1558), RB883 kqt-2 (ok732), CZ1758 max-1(ju142),
FX1770 mtl-1(tm1770), FX830 pgp-9(tm830), RB908 pmp-1(ok773), T28C12.4(tm1013),
CX0010 osm-9(ky10), FK127 tax-4(p678), VC1149 C25B8.4&kqt-1(ok413), KHR186
tax-4(p678); kqt-2(ok732), FX05415 gsp-4(tm5415), KHR187 gsp-4(tm5415); kqt2(ok732), kqt-2(ok732); Ex[pKDK66, pAK62], kqt-2(ok732); Ex[kqt-2 genomic gene,
pKDK66, pAK62], kqt-2(ok732); Ex[ges1-p::kqt-2, pKDK66, pAK62], kqt-2(ok732);
Ex[osm-6p::kqt-2, pKDK66, pAK62], N2; Ex[pMSK029 kqt-2p(1 kb)::kqt-2 genomicgene(1st to 12th exon)::gfp, pRF4], N2; Ex[pMSK030 kqt-2p(2.3 kb)::kqt-2 genomicgene(1st to 12th exon)::gfp, pRF4], N2; Ex[pMSK031 kqt-2p(4.6 kb)::kqt-2 genomicgene(1st to 12th exon)::gfp, pRF4], N2; Ex[kqt-2 cDNA::gfp], kqt-2(ok732); Ex[sra-39-

7p::kqt-2 cDNA pAK62, pKDK66], kqt-2(ok732); Ex[sre-1p::kqt-2 cDNA, pAK62,pKDK66], N2; Ex[sre-1p::yc3.60, pRF6], kqt-2(ok732); Ex[sre-1p::yc3.60, pRF6], kqt2(ok732); Ex[sre-1p::kqt-2 cDNA, sre-1p::yc3.60], N2; Ex[sre-1p::kqt-2 cDNA, sre1p::yc3.60],

kqt-3(aw1);

kqt-2(ok732)

Ex[sre-1p::yc3.60],

kqt-3(aw1); Ex[sre-1p::yc3.60], ocr-1(ok132); Ex[sre-1p::yc3.60], ocr-2(ak47); Ex[sre-1p::yc3.60], osm9(ky10); ocr-1(ok132); Ex[sre-1p::yc3.60], ocr-2(ak47); ocr-1(ok132); Ex[sre1p::yc3.60], ocr-1(ok132); kqt-2(ok732); Ex[sre-1p::yc3.60], osm-9(ky10)ocr-2(ak47);
Ex[sre-1p::yc3.60].

ɚʂ*̆&$°ȚĴɱɽ

OP50 Ǒ: ɚʂ*̆&$ NGM pa)īş̀ż˞ʾˆŬń®̀ż˞ (OLYMPUS SZX12) (Olympus Corporation, Japan)ʾˆŬń®ʃÈ̀ż˞ (NIKON SMZ18) (Nikon Corporation, Japan)
Olympus IX81 ̀ż˞ (Olympus Corporation, Japan)Ë ȋ ȉ ̀ ż ˞ ̉ Ƈ ũ ` B _ M [  ¡  (FV1000-IX81 with GaAsP PMT)
(Olympus Corporation, Japan)

ɚʂ>̃ɩ:*)°ȚħĢħĢ*¯ʌ)+ÊɴçħĢ¯ʌʊɡ MEDIA CLAVE10 (INTEGRA Biosciences,
Switzerland) >ȚNematode Growth Medium (NGM) Plate (1,000 ml): 3 g *-40-

NaCl20 g *¦ˏŋĵ̍¦ˏ̂đśǗǑŬ¨ȴƵǈ̎2.5 g * Bacto Peptone(Becton Dickinson, USA)975 ml * H2O > MEDIA CLAVE10 *Í˚)É;20
Õ 121 %ãȌȄɽ*ŷ 60 )¸Ȁ$

8 1 ml * MgSO4 ȃǻ (1M)1 ml * CaCl2 ȃǻ (1 M)1 ml * Cholesterol ȃǻ (5 mg/ml in EtOH)25 ml* Potassium Phosphate ȃǻ (1 M, pH 6.0) >ã

;>ȩŵ 6 cm *T|) 14 ml:+ 3.5 cm *T|) 6 ml #˵çÕǳǞ̍FH-10SS, ǑŬ¨ȴkIƵǈ̎%Õǳ$Ğ4AmTÉ9 LB (Luria-Bertani) pa

H2O 500 mlLB Broth Lennox (Becton Dickinson, USA) 10 g¦ˏŋĵ̍¦ˏ̂đśǗǑŬ¨ȴƵǈ̎10 g >ʖnVQ('*ɥȌňĘ)É;$ǽąH
aMo%ãȌȄɽ*ŷƧƔ(T (100 mg/ml) 1,000 µl >ã

8Ɍ 50 )Ð1$

8Am;>ȩŵ 9 cm *T|) 12 ml #Õǳ$Ğ4

LB ǻ®ħĢ̍200 ml̎
H2O 200 mlLB Broth Lennox (Becton Dickinson, USA) 4 g >zUDyȗ)É;$ǽąHaMo%ãȌȄɽC. elegans *Òɖ¸Ŀ)Ț Freezing Solution (2,000 ml)
NaCl 10.85 gKH2PO4 9.8 gNa2HPO4 6 gGlycerol 300 g1 M NaOH ȃǻ 5.6 ml>zUDyȗ)ã

ŷ) H2O % 2,000 ml 1%zVA]p;>Ha-41-

Mo%ãȌȄɽŷ) 1 M MgSO4 ȃǻ 1.3 ml >ã



M9 buffer (1000 ml)
KH2PO4 3 gNa2HPO4 6 gNaCl 5 gH2O 1000 ml >ǽąHaMo%ãȌȄɽŷ) 1 M MgSO4 ȃǻ 1 ml >ã

­ȀɥƁ_Vaȩŵ 3.5 cm :+ȩŵ 6 cm * NGM pa%̆%: E. coli OP50 >îÕ
 C. elegans >ʗǌ)Ț1 1+ 2 ¹®# OP50)ī8;Ʊ

 2 % (w/v) NGM pa)ɡ (P0)Ą̃ɩȀũ% 1224 Ƽˣ̃ɩɌ
100 ¹*ó>ș1P0 >˩öó (F1)Ɖʂ)(:1%15 *Īą+132150 Ƽˣ25 *Īą+ 5267 Ƽˣ̃ɩpa* F1 ¹®

Ɖ

ʂ1%Ɖ˟ŷNGM pa*ŋĵħĢ½>Ǫ*) 20 Õ˸ɡ20 Õ
ŷ 2 *ÐʁŪ (CRB-41A, Hitachi, Japan) )ȹ$ 48 Ƽˣ˸ɡ2 % 48
Ƽˣ˸ɡŷ15 ) 1 Ƶ˸ɡȘ$:ɚʂ&ǣ?%:ɚʂ>ƭ1 ę*A]XC)+ 3 pa£ȚƆȣ(:Ƶ) 3 ę£ʙ 9 pa£>Ȃ¾ (Ujisawa et al. 2014)

­Ȁ̇ê_Va­Ȁ̇ê_Va+ˆö*ʢƯ>÷ɤ)$(" (Ujisawa et al. 2014)ȩŵ 3.5 cm :+ȩŵ 6 cm * NGM pa%̆%: E. coli OP50 >îÕ)

 C. elegans >ʗǌ)Ț12 ë*Ɖʂ (P0) >ǂØ*Ȁũ% 1520-42-Ƽˣ̃ɩɌ 100 ¹*ó>ș1P0 > 3 ë£ɡĪą+Ɍ 100 ¹*ó
>ș31%ƭƼˣ̃ɩ­Ȁ̇ê*ʗǌ)îÕ(˘*ó>ș1ŷ¢:4) P0 >ý9˩ó>Ơ

8Ɖʂ)(:1%ǂØ*Ȁũ%̃ɩ

15 *Īą+Ɖʂ)(:1% 132150 Ƽˣ̃ɩ25 *ĪąǂØ*Ȁũ+ 5267 Ƽˣ̃ɩǂØ*Ȁũ%Ɖʂ)(:1%̃ɩŷ2 ȢȨ*Ȁ
ũ*CK}r[)ȹ0358 Ƽˣ˸ɡ2 ȢȨ*Ȁũ%̃ɩ
ŷ0358 Ƽˣɕ"8NGM pa*ŋĵħĢ½>Ǫ*) 20 Õ˸ɡ20 Õŷ 2 *ÐʁŪ (CRB-41A, Hitachi, Japan) )ȹ$ 48 Ƽˣ˸ɡ2 % 48 Ƽˣ˸ɡŷ15 ) 1 Ƶ˸ɡȘ$:ɚʂ&ǣ?%1 ę*A]XC)+ 3 pa£ȚƆȣ(:Ƶ) 3 ę

:ɚʂ>ƭ

£ʙ 9 pa£>Ȃ¾ (Ujisawa et al. 2014)

kaT]M)7:HV*ýŹƜą=ɚʂɋɗ* L4 (P0) > 1 #*pa) 3050 ë*$ 30 *ȀũT]M> 8 Ƽˣpa)ɡF1ɣƵƉʂ)("¹®> 23 ë#ƱƉʂ)(:1%̃ɩF1

Ɖʂ)("8p

8HV (F1) >ʏ#Ô23 ë#zC_BNpa̍ħĢ*ŽaˑÕ)Ĵɱɽ>*5*̎)ȹ)Ɯą=ɚʂɋɗ*˱˯Ć®>Ɍ 4 ë*˒*pa> 35 ǍȚƆɣƵƜ
ą=˱˯Ć®> 1 ë#Ʊpa)ȹǠ¢
̃ɩ˯

Ô$:

ȱʛ

-43-

Ɖʂ)(:1%ǟĸıȣ>ƗǛ&Ɗ˒ (±: kqt-2(ok732))˱ ˯ Ć ® * kqt-2(ok732) (P0) & N2; ExUDA17[pAK62 AIYp::gfp, pKDK66 ges1p::gfp] (P0) *HV&˒ʃÈ>Ȧ: F1 ¹®#19ıȣ̍deletion̎
q_&(:˱˯Ć® (F1) >ñ˳ɴŇþɉ:&ıȣĥ*t{˗Șĥ*t{5+q_>Ɩ"ɚʂȦȘ: (F2)F2 ¹®> 1 ë#T|)ȹƽó>ș1ŷɣƵ F2
Ɲý*Pgy>Ț$ PCR >8ɚʂ 1 ¹®ȜǊ*Pgy>

(ıȣĥ*t{q_˗Șĥ*t{>ʏÕˌ©ŀȧ)+1/4 *ȱȓ%ıȣĥ*t{>Ɩ"ɋɗ
Ȕ;:*ɋɗ>Ț$5 ũƊ˒>

(";> 5 ęɠ9ʼ&Å"ıȣ̍deletion̎£Ĳ*ıȣ+˗Șĥ)ɡƢ=:ǂŷ)­Ȁ̇ê_Va>(ȣš

Ǥ"$:>ȱʛ

kqt-2 ıȣ®*­Ȁ̇êȣš>ˌ©ĽőÉ)79ęŻ:ń̈
kqt-2 ˌ©ĽÊ˟>˗ȘǑ*Pgy>˜ĥ&$ PCR ǰ>Ț$p{[˽Ħ>ĉ3 DNA ưȍ>ĮŢ˵ǨǴç>

("Ȩȧ*ib>Ö9ÔIy>Ț$PƓÔɍȎ>ý9˩ɔɟȏȣȧ( kqt-2 ıȣ®*ęŻń̈)+ Aguan D. Wei òİ79Õ$ kqt-2 cDNA *Ǹ)ȏȣȧp{[>ƙÉpVxb>ȚőÉ:ˌ©Ľ&C
UEMTwIges-1p::nls::gfp ȃǻ& AIYp::gfp ȃǻ>ǽąCU
EMTȃǻ>JV˛>Ț$ kqt-2 ıȣ®*ȘǥŚ)ǳÉCUE
MT¹®
Ȕĥ

8Ș1;ɚʂ*!wI>Ɩ#¹®>ɋɗêʇ˗ȘǑ&ĆȺũ1%ęŻ$:-44-­Ȁ̇ê_Va>("_Va*ˮ)+­ȀÜȇ*ÞƵ) GFP *ʃÈ>Ǆ$:¹®*2>Ɍ 30 ë/pa)(:7)˰4;> 3 pa£Ȃ¾ʗǌ>

("ÕĽȘȎŀȧʗǌ(1) PCR ȃǻ&RwRCM*ȀũpNyEx Taq (Takara Bio, Japan) >Ț PCR1 µl * 10 × Ex Taq Buffer0.8 µl * 2.5 mM dNTP Mixture ȃǻȨȧ*ˌ©Ľ˽Ħ>ĮŢ:4*nG

bpCw&iVpCw>Ą 10 µM% 0.5 µl #0.05 µl * Ex Taq_pa&(: DNA > 100 ng £)(:7)ã

Ê˘ 10 µl )(:7

) dH2O >É;$7ǽąRwR

C M   LifeECO (Hangzhou Bioer Technology, China)  :  + LifeTouch(Hangzhou Bioer Technology, China) *ȀũpNy+94 > 2 Õ94 >
15 ȸ58 > 30 ȸ72 ̍30 ȸ/1 kb̎> 1 RCM&42 ęɠ9ʼ
15 %¸ĿKOD -Plus- Neo (ǋǵɏƵǈ) >Ț PCR5 µl * 10 × PCR Buffer for KOD -Plus- Neo5 µl * 2 mM dNTPs ȃǻ3 µl * 25
mM MgSO4 ȃǻȨȧ*ˌ©Ľ˽Ħ>ĮŢ:4*nG

bpCw

&iVpCw>Ą 10 µM % 1 µl #1 µl * KOD -Plus- Neo_pa> Genomic DNA 200 ng/50 µlPlasmid DNA 50 ng/50 µl )(:7ãÊ˘ 50 µl )(:7)

) dH2O > 200 µl \}o)É;$7ǽąR  w  RC M  LifeECO (Hangzhou Bioer Technology, China)  :  +-45-LifeTouch (Hangzhou Bioer Technology, China) *ȀũpNy+2 V_]p:+ 3 V_]p*pNy>Ț2 V_]p*Īą+94 > 2 Õ
98 > 10 ȸ68 ̍30 sec/1 kb̎> 1 RCM&35 ęɠ9ʼ15 %¸Ŀ3 V_]p*Īą+94 > 2 Õ98 > 10 ȸ°Ț:pCw* Tm »)ą=Ȁũ% 30 ȸ68 ̍30 sec/1 kb̎> 1 RCM& 35ęɠ9ʼ15 %¸ĿLA Taq (Takara Bio, Japan) >Ț PCR1 µl * 10 × LA PCR Bufferll (Mg2+ free)0.8 µl * 2.5 mM dNTPs Mixture ȃǻ0.6
0.8 µl * 25 mM MgCl2 ȃǻȨȧ*ˌ©Ľ˽Ħ>ĮŢ:4*nGbpCw&iVpCw>Ą 10 µM % 0.25 µl #0.1 µl * LA Taq_pa&(: DNA > 1 µg £)(:7)ã

Ê˘ 10 µl )(:7

) dH2O > 200 µl \}o)É;$7ǽąRwRCM LifeECO(Hangzhou Bioer Technology, China)  :  + LifeTouch (Hangzhou Bioer
Technology, China) *ȀũpNy+94 > 2 Õ94 > 30 ȸpCw
* Tm »)ą=Ȁũ% 30 ȸ68 ̍1 min/1 kb̎> 1 RCM& 40 ę
ɠ9ʼ15 %¸Ŀ(2) pVxb*¯ʌ)Țƌǰ
In-Fusion ǰ)7:pVxbrM[.* DNA ưȍ*ƙÉÛ˧˓ɎÓȕ5+ PCR )7"$ɚȐ)rM[DNA *Ǉȿ˒×)Ȫ
Ć( 15 ĬĨ>Ȩȧˌ©ĽĮŢȚpCw* 5'Ǉȿ)¡ãȨȧˌ©Ľ>-46-

PCR ĮŢĮŢșȎ>˵ǨǴçP

8ɉʌ$5 × In-Fusion HD

Enzyme Premix (Takara Bio, Japan) 2 µl &ɚȐêrM[&CRa>;;100 ng )(:7)ã

Ê˘ 10 µl )(:7 ) dH2O >ǽąɖą

̍In-Fusion ûƀ̑50 15 Õ̎In-Fusion ûƀǻ%ŲʪʲƢ>

(Ȩȧ

M>VMdN

Ligation ǰ)7:pVxbrM[.* DNA ưȍ*ƙÉÚȚ:pVxbrM[&CRa>Ćǚ*Û˧˓Ɏ%Öư˵ǨǴçŷP

8ɉʌ$ɚȐêrM[̑CRa = 1:3 *áą%ǽąrM[&CRa*ǽąȃǻ̑Ligation high Ver. 2 (ǋǵɏƵǈ) = 1:1 *á
ą%ǽąȃǻ> 15 *CK}r[% 30 Õ£˸ɡɖą̍Ligation ûƀ̎Ligation ûƀǻ%ŲʪʲƢ>

(ȨȧM>VMdNFMavT)7:pVxb DNA *Ĵɱɽ.*ŲʪʲƢ-20 )¸Ɇ$:Qm_aX DH10β̍FMavTȚ̎>Ǫ%ȃ

*ˮK}r]a>Ð6$ȃQm_aX

20 µl )pVxb DNA ȃǻ> 12 µl É;ʴǽ$)ǱȾ(7ǳƆ(

8K}r]a*˭ˣ

8É;MicroPulser (Bio-Rad, USA) *i)Ĉ>ȱʛ$ƙÉpulse &ǁ ;u[>ƒ180 µl * LB ǻ®ħĢ%Qm_aX>ęü50 µl100 µl >AmTÉ9 LB pa)137 %ƽħ̅-47-

kaT]Mǰ)7:pVxb DNA *Ĵɱɽ.*ŲʪʲƢ-80 )¸Ŀ$:Qm_aX DH10β >Ǫ%ȃ

ȃ



DH10β &Ȩȧ*pVxbȃǻ>ǽąǽą=ȃǻ> 42 % 45 ȸ
ˣkaT]M>

Ȩȧ*pVxb>Qm_aX)őÉk

aT]Mŷ*ȃǻ>Ǫ),8˸ɡŷMr\%Am
TÉ9 LB pa)ī9Ŧ37 %ƽħ̅ʗǌ)Ț PCR ưȍ&pVxb*¯ʌǰ

kqt-2 ıȣ®*­Ȁ̇êȣš>ęŻ:ń̈)Ț PCR ưȍ+PgyȜǊ
* kqt-2 ˌ©ĽǸ 3.2 kb &Ê$*FKZ>ĉ?

˽Ħ> PCR )79ĮŢ˵ǨǴçŷ)PƓÔ$ɉʌ5*>°Țkqt-2 ıȣ®*ɒɪȏȣȧ(­Ȁ̇êęŻń̈)ȚpVxb+£*Ʋǰ%¯ʌpMSK008 +Kozak ˒×& kqt-2 cDNA*Ǹ) unc-54 ˌ©Ľ* 3’UTR >ĉ?%:
* pMSK008 * kqt-2 cDNA *Ǹ)ɱȏȣȧp{[ges-1p (3.3 kb)Ƈʒd}ȏȣȧp{[osm-6p (2.1 kb)ASK ȏȣȧp{[sra-7p(4.2 kb)ADL ȏȣȧp{[sre-1p (1.2 kb) >;;ƙÉpMSK004
pMSK005pMSK007pMSK024 >¯ʌǈȰȼ%ʕŌ kqt-2::gfp *p
Vxb+ Aguan D. Wei òİ79Õ$*pVxb+ kqt-2

ˌ©Ľ*Ǹ 9 kb p{[˽Ħkqt-2 ˌ©Ľ* 1 ȢȨFKZȨFKZĉ1;PgyȜǊ*ˌ©Ľ˒×*Ǹ) GFP

8 12 Ȣ

őÉ;$

(Wei et al. 2005)*pVxb>Å) Inverse PCR ǰ>Ț$Ȩȧ˒×>ĮŢŷ) self ligation >(p{[˽Ħ>;; 4.5 kb2.3 kb
-48-

1 kb )ɜ4pVxb̍pMSK031pMSK030pMSK029̎>¯ʌ̍ĝ 10A̎
KQT-2 *ŕġʗǌȚ*pVxb%: kqt-2 cDNA::gfp (pMSK028) *¯ʌ)+GFP

ĉ1;$: pPD95.75 >rM[pVxb)Țkqt-2 ˌ©Ľ*Ǹ 9 kb *p{[˽Ħ& kqt-2 cDNA > GFP *Ǹ)ƙÉ*ˮ
kqt-2 cDNA *ɓǡQb>˩$ GFP &*ʄąˌ©Ľ>¯ʌ­˔ɎȈũ*­Ȁ̇ê_Va

̆%: E. coli OP50 >îÕ)

Ɖʂ (P0) >ʍƭë NGM pa)*Ɍ 100 ¹*ó>ș31%ƭƼˣ̃ɩîÕ(ó>ș1ŷ¢>Ơ:4) P0 >ý9˩jnBy% NGM pa>ŊˡJViAƁjD\ʈ*Ĺ. O2 z[BIONIX-3 (nB-3) (SUGIYAMA-GEN, Japan) >É;*ǜ) NGM pa>ɡJVȈũʡɇß>Éÿ¡ʻ)ɡ$
M]p%ŊŏJVȈũʡɇß)79jD\Í* O2[Ċü;O2 z

Ȩȧ* O2 Ȉũ>ȲŷJVȈũʡɇß& NGM pa>M]p%Õ˳O2 Ċü>Va]pjD\ʈ)É;11ó8Ɖʂ)(:1% 25 *CK}r[% 5267 Ƽˣ̃ɩŷ15 *CK}r[)ȹ3 Ƽˣ˸ɡ15 % 3 Ƽˣ̃ɩŷjD\ʈ)É;11NGM
pa*ŋĵħĢ½>)$Ǫ) 20 Õ˸ɡ20 ÕŷjD\ʈ

8

NGM pa>ý9Ô[]j)ȹ$ 2 *ÐʁŪ (CRB-41A, Hitachi,
Japan) % 48 Ƽˣ˸ɡ2 % 48 Ƽˣŷ15 ) 1 Ƶ˸ɡȘ$:
ɚʂ&ǣ?%:ɚʂ>ƭ1 ę*A]XC) 3 paȚƆȣ(:Ƶ) 3 ę£ʙ 9 pa£>Ȃ¾
-49-

˔ɎȈũ*ȁŃ˔ɎȈũ+ OXY-1 SMA (PreSens Precision Sensing GmbH, Germany)>Ț$˔
ɎȈũ>Èŀȧ)ȁŃ˔ɎXR\]p SP-PSt3-NAU-D5-YOP (PreSens
Precision Sensing GmbH, Germany) >ȩŵ 3.5 cm :+ȩŵ 6 cm * NGM pa*̆%:Ĵɱɽ*)ɡ̆>îÕ)

ɚʂ (P0) ɡ$ 60100

ë*ó>ș31% 23 Ƽˣ˸ɡîÕ(ó>ș1ŷ¢>Ơ:4) P0 >ý9˩jnBy% NGM pa>Ŋˡ25 %̃ɩ
8 24 Ƽˣ64 ƼˣŷÈn@Ci

ó>ș?%8åʭÈ>˔ɎXR\]p)$$˔ɎȈũ)ƀʃÈ>ȁŃ:&)7"$Èŀȧ)˔ɎȈ
ũ>ȁŃ˔ɎȈũʙ (PreSens OXY-1 SMA) >Ț$1 ȸ) 1 ę*ˣˬ%˔ɎȈũ>ȁŃó>ș?%8 24 Ƽˣ64 Ƽˣŷ);; 10 ȸˣ*˔ɎȈũ>ţģƭ»>ɅÔ

DiI Ǐɹ
DiI Ǐɹ)79˶Ô$:˾ˑ*d} (ADL, ASH, ASI, ASJ, ASK,AWB) & Ŕ ˑ * d }    (PHA, PHB)Ǐ ɹ  ; :  DiI Ǐ ɹ +WORMATLAS *p aQ (http://www.wormatlas.org/EMmethods/DiIDiO.htm)>÷ɤ)

("DiI *Va]Mȃǻ (5 mg/ml) >É;\}o+AxtC>Ş$ˊÈ-20 %¸Ŀɚʂ> DiI Ǐɹ:ˮ)+ DiI Va]Mȃǻ (5 mg/ml) 0.7 µl > 2 ml * M9 )ȃ $Š˕*! 150 µl >200 µl *\}o)ÕǳǏɹɚʂʍƭë>Ǐɹǻ)É;1 ƼˣȺ
ũŅȀ%ǏɹŷjV^ms]a>°"$Ĵɱɽ
-50-

ī8;ƱNGM pa)ɚʂ>ȹɌ 1 Ƽˣɚʂ>ˀ=:&%ɱ)É9ʺ?
DiI Ǐɹǻ6®)#Ǐɹǻ>ɮɹ100 mM NaN3 ȃǻ>ƭȅɡAJ
Vj]a)ɮɹŷ*ɚʂ>*$IiJV>ʃÈʕŌˌ©ĽőÉˌ©ĽőÉ+;1%)ĩċ;$Ʋǰ>5&)

(" (Mello et al.

1991)ˌ©ĽőÉ*ˮco-injection mix )+ń̈)°Ț: DNA >;; 5
50 ng/µl *Ȉũ%É;CUEMTwI)+ pKDK66 ges1p::nls::gfp > 50 ng/µlpAK62 AIYp::gfp > 30 ng/µl1+ pRF4 rol-6gf > 30
ng/µl *Ȉũ%É;

êŀʬƁȚpa*¯ʌ

20 g *¦ˏŋĵ̍¦ˏ̂đśǗǑŬ¨ȴƵǈ̎1,000 ml * H2O > MEDIA
CLAVE10 (INTEGRA Biosciences, Switzerland) % 20 Õ 121 %ãȌȄɽ*ŷ 60 )¸Ȁ$

8 1 ml * MgSO4 ȃǻ (1 M)1 ml * CaCl2 ȃǻ (1M) 25 ml * Potassium Phosphate ȃǻ (1 M, pH 6.0) >ã

;>ȩŵ 9 cm

*T|) 12 ml #˵çÕǳǞ̍FH-10SS, ǑŬ¨ȴkIƵǈ̎%Õ

ǳ$Ğ4ſˍʅçʗǌſˍʅç*ʗǌ+ˆö*ʢƯ)Ÿ"$(" (Troemel et al. 1997; Kimura et-51-

al. 2010)̆%: E. coli OP50 >îÕ)

Ɖʂ 2, 3 ë>Ɍ 200 ë*

ó>ș1ʗǌ:¹®*Ɖ˟˂ũ>ą=:4)Ɍ 20 Ƽˣŷ)ʔ (P0)>ý9˩ó8Ɖʂ1% 20 %̃ɩſˍʅç*ʗǌȚpa)+ȩŵ 9 cm *êŀʬƁȚpa [2 % agar, 1 mM MgSO4 ȃǻ, 1 mM CaCl2 ȃǻ,
25 mM potassium phosphate ȃǻ (pH 6.0)] >°Țȩŵ 9 cm *êŀʬƁȚpa+ 12 ìȟ (#1-12) )ɁÕ$

20 %̃ɩ$NG i]n@% 1.5 ml \}o)ęüɚʂ

Ũ)ǭ?

Ɖʂ>

*>ȱʛŷ)

Ȇ2>ý9˩8) NG i]n@> 1 ml ãɚʂ>ȱʛŷ)Ȇ2>ý9˩Ćǚ*ƨ¯>5

ũ

Ũ)ǭ?

*

(ʙ 2 ę

NG i]n@%ɚʂ>Ƕǹȩŵ 9 cm *ŋĵħĢpa*ķɚ)3 ɄƋ)Õ$ɚʂ>ɡNG i]n@>ý9˩ŷ)Ŝ

8 3 ȢȨ*ɚ) 2 ɄƋ)Õ$ 1-octanol >ɡpa>ʋʼɚʂ>ɡ¬ɡ
>wU]M%Ĝ"$ʚ$
ɚʂ

ç

(7

A]XCpa> 12 Õ˸ɡŷſˍʅç)CHCl3 ȃǻ>ƿ˶A]XC+Ù*Ƶ) 3 ę

("12 ìȟ;;*ìȟ)Ŀġ:ɚʂ*ƭ>IDaʚ*ʙɅŬ̍Avoidance index̎)ű$+4$ʙɅ>("̍ĝ 13B̎pa

ķ*wU]M%Ĝ=;ˑÕ̍Ø4)ɚʂ>ɡ¬ɡ̎*¹®ƭ+¯Ǘ*ʿ%¿#āɫƁ

:4IDa);("

Avoidance index = ΣWiNi/N, [Wi: -2.5 (#1), -2.0 (#2), -1.5 (#3), -1.0 (#4), -0.5 (#5), 0.25 (#6), +0.25 (#7), +0.5 (#8), +1.0 (#9), +1.5 (#10), +2.0 (#11), +2.5 (#12) ] (Ni: ;;*ìȟ*ɚʂ*ƭ)(N: Ê$*ìȟ)Ŀġ:ɚʂ*ƭ)-52-éȎʪ)ō:ƀɃ_Va

êŀʬƁ_Va+ Bargmann 8*ʢƯ)Ÿ"$(" (Bargmann et al. 1993)

ȩŵ 9 cm *êŀʬƁȚpa*ƣȦƁʜŭȎʪ>ɡ¬ɡ)wU]M% A
̍A + Attractant̍ʜŭȎʪ̎*Ɔč̎F[g>¬ɡ) C̍C + Control*Ɔč̎ɚʂ>ɡĪƋ>ķɚ%ʚɚʂ> 3 ëɡ20 %ó>ș1
ŷ P0 >ý9˩20 %̆>îÕ)

ǉ¥% F1 >̃ɩ$Ź8;

Ɖʂ¹®̍150300 ¹®/6 cm NGM pa̎>_Va)°Țȩŵ 6 cm
* NGM pa)M9 buffer 1 ml >ã

ŷǾ>˩8) M9 buffer 1 ml >ã

\}o*Ũ)îÕǭ?
ʂŨ)ǭ?

ɚʂ> 1.5 ml \}o)ęüɚ&)Î-Ǿ>˩Ƕǹ̍2 ę̎êŀʬƁȚpa* A ȉ& C ȉ) 1 M NaN3 ȃǻ>;; 1 µl #ȅêŀʬƁȚp
a*ķɚ)ɚʂ> 3 ȉ)Õ$Vv]aVv]aŷ_B]T}Ky

Cp*Çȿ>ɒ4ǩÕ((:1%Ċ"pa* A ȉ)ƣȦƁéȎʪ>ɎƷ 4 µl ȅC ȉ)+Qa&$ȃļ%:F[g>ɎƷ 4 µl ȅpa>ʋʼɚʂ>ɡ¬ɡ>w
U]M%Ĝ"$ʚ$60 Õˣ˸ɡ$ɚʂ>ɴȜ)ʅç_Va*ɓƼˣ)("8pa>ʋʼ11 CHCl3 ȃǻ>ʀ)ȅʀ>ˢ)bnaÍ) 5 Õ£˸ɡ:&)7"$ɚʂ>Ǧ*ŷ4 %ƽCK}raɣƵIDaéȎʪ½&F[g½*¹®ƭ>;;* A (attractant) & C (control) &$ʙȁChemotaxis
Index >Ŭ Chemotaxis Index =̍A ):¹®ƭ  C ):¹®ƭ̎/̍A ):¹®ƭ + C ):¹®ƭ̎%ɅÔǄæƭľ+ 2 ǒ̍paķ*wU-53-]M%Ĝ=;ˑÕ̍Ø4)ɚʂ>ɡ¬ɡ̎*¹®ƭ+¯Ǘ*ʿ%¿#āɫƁ

:4IDa)É;("̎̍ĝ 11C̎

in vivo ITDyCzUNǈȰȼ* ADL * in vivo ITDyCzUN+ˆö*ʢƯ)Ÿ"$(" (Kuhara et al. 2011; Ohnishi et al. 2011)ITDyCzUN&+Èˌ©
ŀ>Ț$ȵɕɒɪÍITDyȈũıê>Ń˘ê:ƍʆ%:ǈȰȼ%+ITDyC`BO[&$ Yellow Cameleon 3.60 (YC3.60) >ȚYC3.60 +N Ǉ) CFPC Ǉ) YFP >5!ķ)ITDyɖąˑ¬>
5#Kz[jMʪ%:ɒɪÍITDyȈũ­Ƽ)YC3.60 ) 440

nm *åʭÈ>$:& 480 nm >mM&:˷ɹ*ʃÈ>Ȧ:&<ɒɪÍITDyȈũ
HƸ:&YC3.60 *ITDyɖą)ITDyC

ɖą:&%Ⱦ®Ǚ˃ıêCFP & YFP ˣ*ʯ˳ƭcgza&ʻ(:*&)440 nm *åʭÈ>$:&CFP *ʃÈʃÈË̊FfLȹç̍FRET̎&Ď,;:FfLȹç)79 535 nm >mM
&: YFP *̋ɹ*ʃÈ>Ȧ:7)(:*̋ɹ&˷ɹ*ʃÈ*ǧ>ý

:&%ɒɪÍITDyȈũ*ıê>Ń˘ê:&%:YC3.60 [jMʪ+ˌ©Ľ)7"$Qb;$:4Ȩȧ&:ȏŃ*ɒɪ% yc3.60
ˌ©Ľ>ȦȔ:pVxb>¯ʌɚʂ*ȘǥŚ)őÉ:&)7"$ʗǌȵɕɒɪ*ITDyȈũıê>ȁŃ:&%:ǈȰȼ%+ ADL d}%ȏȣȧ)ˌ©ĽȦȔ>ʜő:p{[%: sre1 p{[>Ț$ yc3.60 ˌ©Ľ>ȦȔʜő:pVxb [sre-1p::yc3.60-54-

(pTOM63)] >ɚʂ)őÉADL *Ȁũıê)ō:ITDyCzUNʗǌ>

("ʗǌ)+25 :+ 15 %̆îÕ)Ŀġ:ħĢ%̃ɩƉʂ>ȚITDyCzUNʗǌ>

(

űƵ

82Ƶ

Þ1%) 24 × 24 IiJV*)2 % (w/v) AJVj]b>¯ʌAJVj]a*)ITDyCzUNȚ*ɋɗ Ex[sre-1p::yc3.60]>Ɍ 4 ëɡíȥȚƞȭßAAn@ A̍ɀËǑŬ¨ȴƵǈ̎%˾
ˑ&Ŕˑ>ĞŃM9 buffer >Ĥ8 86IiJV>ƜwdK}A%ŏ>$ɿȦ>˦ʗǌ)+ 4 ë*!ƞȭß%1ĞŃ %   ¹ ® > ° Ț    R  p  > Olympus IX81 microscope (Olympus
Corporation, Japan) )ʊ¾;$: ITO JVpa (Tokai Hit, Japan) *)ɡ2 Õ0'ɡDual-View optical system (Molecular Devices, USA) >Ț$ʃÈ>ʕŌʃÈȟÁ+ EM-CCD Iz Evolve 512 (Photometrics,
USA) %1 × 1 ldNEM Gain 10030 ms *˶ÈƼˣ% 1 ȸ&)ƦųJVpa*Ȁũ+IV[yzCbȀũÛźTV_y (Tokai Hit, Japan))7"$ÛźȀũ>ʚ˝;;*ȟÁ*ʃÈŰũ+ MetaMorph
Image Analysis Software (Molecular Devices, USA) )79ʗǌɒɪÍ*ITDyȈũ*Ȫōıê+ YC3.60 * acceptor/donor̍̋ɹʃÈ» / ˷ɹʃÈ»̎ʃÈ*ǧ%ʙɅÊ$*ibjVnB[+ YC3.60 )

$;1%*ĩċ>÷ɤ)(Kuhara et al. 2011);;*ǲ˟)ˉibjVnB
[>ȚIV[yzCbȀũÛźTV_y>Ț$17 →23 →17 *ȀũıêÜȇ>

Ɍ 5 Õˣ ADL Ƈʒd}*ɒɪ®Í*ITDyȈũ*ıê>ȁŃ
-55-Ëȋȉ̀ż˞>ȚʕŌËȋȉ̀ż˞>Ț$ʃÈʕŌ:ÞƵ:+űƵ)Ëȋȉ̀ż˞ƦųȚAJVj]a>¯ʌVCbJV*) 2 % (w/v)*AJVP>¯ʌ100 mM NaN3 ȃǻ> 10 µl ȅ*) 510 ë*Ɖʂ>ɡ
ɚʂ>ĞŃ

8IiJV>*pja>Ëȋȉ̀ż˞ (FV1000-IX81 with GaAsP PMT, Olympus Corporation, Japan) *V_U)
ʕŌ)ˉnB[)ą=$ʃÈȟÁ>ƦųƦų)+ FV10ASW software (Olympus Corporation, Japan) >ȚǈȰȼ*ʕŌ)Ț kqt2::gfp *pVxb+ Aguan D. Wei òİ79Õ$*pV

xb+ kqt-2 ˌ©Ľ*Ǹ 9 kb *p{[˽Ħkqt-2 ˌ©Ľ* 1 ȢȨFKZ8 12 ȢȨFKZĉ1;PgyȜǊ*ˌ©Ľ˒×*Ǹ) GFPőÉ;$ (Wei et al. 2005)*pVxb>Å) Inverse PCR ǰ>Ț$p{[˽Ħ>;; 4.5 kb2.3 kb1 kb )ɜ4pVxb>¯ʌʃÈʕŌ>

("̍ĝ 9A, B, C̎TVFzaʗǌ˗ȘǑ* kqt-2 ˌ©Ľ*p{[˽Ħ 9 kb4.5 kb2.3 kb1 kb * kqt-2::gfp*pVxb>;;˗ȘǑ)őÉʕŌ)+̆îÕ)Ŀġ:ħĢ%̃ɩƉʂ>Ț24 × 24 IiJV*)2 % (w/v) AJVj]b>¯ʌAJVj]a*) kqt-2::gfp >őÉɋɗ>ɡíȥȚƞȭßAAn@ A̍ɀËǑŬ¨ȴƵǈ̎%˾ˑ&Ŕˑ>ĞŃM9 buffer >Ĥ8

86IiJV>ƜwdK}A%ŏ
-56-

>$ɿȦ>˦

Rp> Olympus IX81 microscope (Olympus Corporation,

Japan) *V_U)ɡʃÈ>ʕŌʃÈȟÁ+ EM-CCD Iz
Evolve 512 (Photometrics, USA) %EM Gain 70Exposure time 200 msSj13340 ºōȎW*ǉ¥%ƦųƇʒd}*ɒɪ®% GFPȦ Ȕ  $  : ¹ ® * ţ ģ ʷ ũ » > MetaMorph Image Analysis Software(Molecular Devices, USA) >Ț$ƭ»êɗʙʗǌ­ȀɥƁ_Va+ 8 pa£œ(&5 3 ę*A]XC>

("Ê$*ĝ)ʇʚ;$:ǛȂʞŝ+ standard error of the mean (SEM) >ʇ$:Ê$*`[*ÕƬ+ǢʐÕş:&¤Ńjza]MǖŃ%:Tukey-Kramer ǰ& Dunnett’s ǖŃ1+ÕƬɁ(&¤Ń 2 Ǜǈ)

7: t ǖŃ
̍Welch̎>Ț$ʗǌĳ˖ǧʵ)+ Tukey-Kramer ǰ& Dunnett’s
ǖŃ>Ț$Dunnett’s ǖŃ+ȢŜ*ǔNn&*Np>ǧʵ2ɢǧʵ)+ unpaired t test (Welch) >°"ĝ* (*) & (**) +;; p <0.05 & p < 0.01 >Ɔč:unpaired t test (Welch) )+`[Õǌ^̍Excel
AbC̎>ȚTukey-Kramer ǰ& Dunnett’s ǖŃ)+w]Mɗʙʗǌ ver.
2 (ESUMI, Japan) >Ț

-57-

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ŀȕśŀˑ Ș®ʡɇŀȰȼŅ õɈ ƫƚ&Ķțɺ òİ)Ž79ô1$C. elegans ıȣ®>ơ²$ȳȞ1 Caenorhabditis

Genetic Center (CGC)National Bioresource Project (Japan) ʥƹţ ƫƚThe
Rockefeller University Cori Bargmann ƫƚpVxb>ơ²$ćĀŖĴŀ ǕːƂ ƫƚıȣ®)ã$pVxb>ơ²$ Center for Integrative Brain Research Aguan D. Wei òİ)ô11ƵǈŀʆƘɷ¨ ȏÙȰȼĒ DC )ƝȚ

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(A, B) kqt-25M9 kb_fbhZhMLkqt-2::gfpS/ DF_dX
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J.M7KQB0PRF>: (A)=, (B)>(C) KQT-2::GFPSVXI# CE=# '+S2DF>VXM6Me
UM1]chfgJ^TgLA?H= KQT-2::GFPM# B0PRF>(D) KQT-2M'+ >8!LA?H=kqt-2 cDNA::gfp Skqt-25
M9W\gM9 kb_fbhZh;S"?H# 4DF&(>KQT2::GFP NM]chfg I $L# B0PR=L1%J'+
IB0PRF>

77

A
1 kb
9 kb

4.6 kb

2.3 kbS\W^K^-

kqt-2) ;GFP
12?12
;EFJ].*;%PX^\];"(

B
30,000***25,000**20,00015,00010,000
5,000
0

1 kb

2.3 kb

4.6 kb

9 kb

S\W^K^10akqt-2) ;S\W^K^- ;GHE[V]N&(A, B) 1 kb2?9 kb;kqt-2) ;S\W^K^;:/kqt-2;bE
FJ]2?12;EFJ]/548gfp) B#46SYHUOB+:4/3$?A6.*;%PX^\];!;"(B ,460KQT-2::GFP<=1S\W^K^_1 kb`9[TZ32760
CMID ≥ 70EY^R^<SEM0LQMN :>@ '&0 *p < 0.05;**p < 0.010

78

B

OJRUb (AWA)
1
0.8
0.6
0.4
0.2
0
-0.2

**


-

)
)
32
10
y
7
(k
ok
9
(
2
tm
kq
os

C



Chemotaxis Index (%)

Chemotaxis Index (%)

A_eQJbX^Z (AWC)
0.8
0.6
0.4
0.2

-0.2
2)

3
7

ok
(
2
tkq

8)
7
(p6
4
tax

#%I$6




A




C

60.0$



3+1NaN3

**0

LS\fb1NaN3


A 




C



Chemotaxis index =gA@3F - C@3Fh / gA@3F + C@3Fh11ikqt-2 A ,WPY(A) kqt-2 AOJRUb@9F 3,2AWA=OJRUbA @/H
Fosm-9 B,&I 7?4;:2kqt-2 BOJRUb@9F,
&@B'EG?4;:2JVRK ≥ 92Laf]fBSEM2T[VY@DF")(2 *p < 0.05; **p < 0.012(B) kqt-2 A_eQJbX^Z@9F 3,2AWC=_eQJbX^ZA
@/HFtax-4A B,&I 7?4;:2kqt-2 B_eQJb
X^Z@9F,&@B'EG?4;:2JVRK ≥ 92Laf]fB
SEM2T[VY@DF")(2 *p < 0.05; **p < 0.012(C) N`fcI=281A@ 3+>NaN3I1C@BMeYdfb>8<LS\fb>NaN3I$3:2(@3F#%I@$5160.0$8:2-B 3+@8<*I 81AC!8:2
79

25 °C→15 °C (3 hr) →2 °C**

 (%)

80
60
40
20
0

)
)
L
x
x
K

3
; E (A S; E (A D))7"
32 t-2]32 t-2]
ok
(
7
7
t-2
ok ::kq
ok ::kq((qk-2 7 p-2 1 ptt
k q ra k q re [s[s2)

12Jkqt-24H@>FI $kqt-24#'+8 5ADL03kqt-2 cDNA:),8(136./ *-%ASK05 5792&.-%;BA< ≥ 18%=GI
DI5SEM%CFI>I?G<EI 368! % *p < 0.05; **p < 0.01%

80

A
Avoidance index

1-octanol (ADL, ASH, AWB)

B

1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2

*
**
**

8)
0)
2)

7
1
3

k7 L)
(ky SH) -4(p6 B)o,
9(t-2 (AD osm L, A tax L, AW
q
k
D
D
(A
(A

!9 cmTZ[Q

2
1

3

4 5

6 7 8








9

10
11
12

2

12 -.&

%'D&3

1

3

4 5

6 7 8

9

10
11

 12






1-octanol
Avoidance index = ΣWiNi/total(Wi: -2.5 (#1)/-1.5 (#2)/-0.5 (#3)/+0.5 (#4)/+1.5 (#5)/+2.5 (#6) )(Ni: 7C8C> =1B)
13\kqt-2>+( PKQ(A) ADL/ASH/AWB; 4CB1-octanol=6B+( PKQ0ADL<
ASH;6Bosm-9</ADL<AWB;6Btax-4?1octanol=6B+( =D"590ADL;6BIXGUNVRYkqt-2
?1-octanol=6B+( =D"590EOLF ≥ 90HW[S[?SEM0MROQ=AB$*)0 *p < 0.05; **p < 0.010(B) JV[ZD125/3  >%=1-octanolD&190)=1B%'
D=&2/12-.&590,?1-octanol=5:+( D"5/
@# 59081& (%)

A

15
10

( (25 ℃*)5

( (15 ℃*)

0

23 °C
15 °C

0

50

B

100 150 200 250 300
) (sec)
& (%)



-5

**

10
8
6
4
2
0



(

5
(2

)

*
℃


(

5
(1

)

*
℃

14SADL!HLPNO>(A) 25 ℃:*37(?15 ℃:*37(=@9- =
4B1,087. JFPO?EMK=$%Q17 ℃→23 ℃→17 ℃R.
(B) EMKA>160-170 sec>10)> &> .EMK>?EMKA>; .n ≥ 16.DMPIP?SEM.t (Welch) =AB#". *p <0.05; **p < 0.01.25 ℃*>(>GPF?15-17-20-23-24-2526-30;'.15 ℃*>(>GPF?16;'.5C6C> +?
=/2<87.

82*0 (%)

A

25 °C 4(

15

2 
10
kqt-2(ok732)
5
kqt-2(ok732);
Ex[sre-1p::kqt-2 cDNA]

0



-5
23 °C
15 °C

0

*0 (%)

B

50

100 150 200
3 (sec)

12
10
8
6
4
2
0

**


2

250

300

**

)
x A]
32
E
7
k
); cDNo2(
73 t-2
-2
t
k
kq
(o kq
-2 1p::tkq re[s15c25 ℃4(Hkqt-2 !HADL+W\`^_H&(A) kqt-2 !IADLH G=L&9=L!N$<6:H!IADLD!#Gkqt-2 cDNAN";>L:EGKBC<A7=JC 25 ℃4(7 YT`_IS]ZG./7
a17 ℃→23 ℃→17 ℃b7(B) S]ZAH170-180 secH11%3H*0H 7S]ZH)IS]ZAH)E7n ≥ 187P]`X`ISEM7U\`Q`R]O[` GK
L'-,7 *p < 0.05; **p < 0.0172 HV`TI 146176206236
24625626630E17kqt-2 !HV`TI 20626630E17?M@MH 5IG8:FBA783

15 °C -!

#) (%)

A
15
10

+

5

kqt-2(ok732)

0

23 °C
15 °C

B

0

#) (%)



-5

50

100 150 200 250 300
, (sec)

10
8
6
4
2
0

NS

2)

3

k7
o
+
(
-2
t
kq

16Q15 ℃-!<kqt-2 <ADL$FJNLM<(A) 15 ℃8-!26kqt-2 =+9<A3/
HDNM=CKI;'(O17 ℃→23 ℃→17 ℃P/(B) CKIA<160-170 sec<11,<#)< /CKI<"=CKIA<"9 /n ≥ 16/BKNGN=SEM/t (Welch) ;>? &%/ *p <0.05; **p < 0.01/15 ℃-!<+<END=149*/4@5@< .=;01:76/

84

25 °C 4&(. (%)

A
30
20

2!
10

N2; Ex[sre-1p::kqt-2 cDNA]

23 °C
15 °C

0

50 100 150 200 250 300
3 (sec)B(. (%)



0

30
25
20
15
10
5
0

**

A]

N
!
cD
2
2
tq
k
::
p
1
er
s
x[
E
;
N2
17akqt-21 0" GADL)TY^\]G$(A) kqt-2 cDNAL2!GADLC0" ;>JD6ADLG F=JO[
QMX9<A725 ℃4&7 VR^]HPZWF,_17 ℃→23 ℃→17 ℃`7(B) PZWAG170-180 secG11#3G(.G 7PZWG'HPZWAG'D7n ≥ 257NZ^U^HSEM7t (Welch) FIJ%+*7 *p < 0.05;**p < 0.01725 ℃4&G2!GS^RH 14615620623624625626630D
/7?K@KG5H F8:EBA7

85

B

80

80

 (%)100

 (%)

100

60
40
20



D


k

(
qt-1

13
o k4

25 ℃→15 ℃ (8 hr) →2 ℃
100

60
40

0

)

15 ℃→25 ℃ (0 hr) →2 ℃E
 (%)

80
60
40

3

k4 1
o
(

1
kq t


k

1 (o
kqt-

)
413

60
40

0

)

15 ℃→25 ℃ (3 hr) →2 ℃



F



k
-1 (o
kq t

413

)

15 ℃→25 ℃ (8 hr) →2 ℃
100

80
60
40

80
60
40
20

20

20

80

20

100

100

0

C

20

0

 (%)

25 ℃→15 ℃ (3 hr) →2 ℃

 (%)

25 ℃→15 ℃ (0 hr) →2 ℃

 (%)

A

0




k
1 (o
kqt-

)
413

0


3)

k4 1
o
(
1
kqt-

18I:F8C=D@Gkqt-1.
(A) 25 ℃ -15 ℃-;B?('-2 ℃.5$) .!
(B, C) 25 ℃+ 15 ℃-3  "3#/8 ;B?&).!#'4
0 /24,%*)!
(A, B, C) 6><7 ≥ 11!9EHAH/SEM!t (Welch) -13! *p <0.05; **p < 0.01!
(D) 15 ℃ 25 ℃-;B?('-2 ℃.5$).!
(E, F) 15 ℃+ 25 ℃-3  "3#/8 ;B?&).!#'4
0 /24,%*)!
(D, E, F) 6><7 ≥ 7!9EHAH/SEM!t (Welch) -13! *p <0.05; **p < 0.01!

8625 °C→15 °C (3 hr) →2 °C

*



 (%)

80

*

60
40
20
0

)
)
)
2
1
2
3
w
7
a
73
k
(
k
o
(
#
(o
t-3
2
2
q
t
k
t
q
k
kq
;
1)
w
(a
3
t
kq


19Hkqt-3 2'
25 ℃/&15 ℃13$=CA,.%2')kqt-3 3'*-5 7,.)kqt-3; kqt-2" /3kqt-2 2'2 *+6(kqt-3 02 7,.)8?>9 ≥ 12):FGBG3SEM)@EG;G<F9DG 145! ) *p < 0.05; **p <
0.01)

87

"

,3! (%)

A

25 °C8*25
20

6$
kqt-2(ok732)

15
10

kqt-3(aw1)

5
0
-5

23 °C
15 °C

kqt-3(aw1); kqt-2(ok732)

0

50 100 150 200 250 300
7 :>=<;,3! (%)

B
20
16
12
8
4
0

**

**

**

2)
1)
2)

3
3
w
7
7
$
(ok
3(a
ok
6
(
2
t
2
tkq
qt
k
kq
;
1)
w
a
(
t-3
q
k

20jkqt-3%PADL.^cgefP"((A) kqt-3%QADLP"(BFS%U&EI@kqt-3; kqt-2
5%LQkqt-2%P"(PBkqt-3%ORJK DT?
kqt-3%M
P-# U&EI@25 ℃8*@"`[gfQZda
O12h17 ℃→23 ℃→17 ℃i@(B) ZdaAP160-170 secP11'7P,3!P @ZdaP+QZdaA
P+M@n ≥ 16@Wdg_gQSEM@\cgXgYdVbgORS)0/@*p < 0.05; **p < 0.01@6$P]g[Q 14?15?17?23?24?25?26?30M
4@kqt-2%P]g[Q 15?26?30M4@GTHTP9Q OA
CNJI@
88

A

25°C→15°C (3 hr) →2°C

 (%)

80
60
40
20
0




oc

B

(o
1
r

k

2
13
o

)

2(
r
c

a

7
k4

)

25°C→15°C (5 hr) →2°C

 (%)

80
60
40
20
0




oc

r

k
o
(
-1

2
13
o

)

2(
r
c

a

7
k4

)

21:ocr-1%ocr-2((A) 25 ℃ 15 ℃'3 "(
(B) 25 ℃$ 15 ℃'5 "(!.*),.&#"
(A, B) /420 ≥ 1318979)SEM3645'+-  *p <0.05; **p < 0.01
89



 (%)

25 °C→15 °C (3 hr) →2 °C
100

*

**

80
60
40
20
0

)
32

)
32


1
7
k

k
k
o
o
o
(

(
(
1
2
-2
t
t
r
q
k
kq
oc
;
)
32
1
k
o
(
r -1
c
o

2
73

)

22>ocr-1; kqt-2,"
25 ℃! 15 ℃+3$( ,"#kqt-2,"%
'/ -ocr-1+.)* &0(#1762 ≥ 8#3<=9=-SEM#
8;=4=5<2:= +./# *p < 0.05; **p < 0.01#

90

A



%+ (%)

25
20
15
10
5
0
-5
23 ℃
15 ℃

25 ℃/#

B-
ocr-1(ok132)
ocr-2(ak47)

0

50 100 150 200 250 300
. (sec)%+ (%)

25

**

20
15
10
5
0

)
)

47
32

k
1
(a
ok
2
(
1
r
roc
oc

23Xocr-1 1ocr-2 ?ADL&LQUST?!(A) ocr-1 @ADL?!47BD6:225 ℃/#2
 OGUT@FRP>)*V17 ℃→23 ℃→17 ℃W2(B) FRPA?140-150 sec?11 .?%+? 2FRP?$@FRPA?$< 2 n ≥ 172ERUNU@SEM2HMIK >AB"('2 *p <0.05; **p < 0.012-?JUG@14115117120124125126130<,2
ocr-1 ?JUG@24126<,2ocr-2 ?JUG@25<,28
C9C? 0@>35=;:291

25 ℃2%25
20

0 
ocr-1(ok132)
osm-9(ky10); ocr-1(ok132)
ocr-2(ak47); ocr-1(ok132)

15
10
5

0
-5
23 ℃
15 ℃

0

50 100 150 200 250 300
1 (sec)

B

25
20
15

'- (%)

'- (%)

A

**
**

**

10
5
0

)
)
2)
32
3
32
1
1
1
0
ok
ok
(ok
(
(
1
1
1
rcr
cro
o
oc
;
;
)
7)
10
4
y
k
9(k
2(a
r
m
oc
os


24\osm-9; ocr-1/ !4ocr-2; ocr-1/ !CADL(PUYWXC
#(A) ocr-1 !CADLC#7:F!Dosm-9 !4ocr-2 !B
E>?;G<G 9G=525 ℃2%5 RMYXDLVSB+,
Z17 ℃→23 ℃→17 ℃[5(B) LVSAC140-150 secC11"1C'-C 5LVSC&DLVSAC&@5n ≥ 185IVYQYDSEM5NUYJYKVHTY BEF$*)5 *p <0.05; **p < 0.0150 COYMD 14415417420423425426430@.5ocr1 !COYMD 23426@.5;G<GC3DB68A>=592

A#) (%)

25 ℃-!
15+

10

osm-9(ky10)

5

ocr-2(ak47)0

ocr-2(ak47) osm-9(ky10)



-5
23 ℃
15 ℃
0

50

#) (%)

B

100 150 200 250 300
, (sec)
12
10
8
6
4
2
0

)
10
0)

7)
y
1
4

ak
9(k
(ky
(
+
9
2
m
rm
os
oc
os
)
47
k
a
(
r-2
c
o

25Vocr-2 /osm-9 <ADL$JOSQR<(A) ocr-2 /osm-9 =ADL<; A4:287025 ℃!0 LGSR=FPM;'(T17 ℃→23 ℃→17 ℃U0(B) FPMA<140-150 sec<11,<#)<0FPM<"=FPMA<"90n ≥ 170CPSKS=SEM0HOSDSEPBNS ;>? &%0 *p < 0.05;**p < 0.010+<ISG=14/15/17/20/23/24/26/309*0ocr-2 
<ISG=2W9*05@6@< .=;13:870

93

A

25 ℃9,#.4! (%)

25
20
7$
ocr-1(ok132)

15
10

kqt-2(ok732)
ocr-1(ok132); kqt-2(ok732)

5
0

-5
23 ℃
15 ℃

0

50

.4! (%)

B

25

100 150 200 250 300
8 (sec)
**
*

**

20
15
10
5
0

)
)
)

32
32
32
7
7
1
$
7
(ok
(ok
(ok
2
2
1
t
r
qt
k
kq
oc
;
)
32
1
(ok
1
r
oc

26iocr-1; kqt-26%IADL/ZafdeI#((A) ocr-1; kqt-26%IADLI# H@LQcUO_N`fVeT0I*;kqt-2%I&)"IJocr-1%HKDE ?M;
H;ocr-1%I&)"IJkqt-2%HKDE ?MC<25 ℃9,<# \WfeJ
Tb]H23g17 ℃→23 ℃→17 ℃h<(B) Tb]AI140-150 secI11'8I.4!I <Tb]I-JTb]AI-F
<n ≥ 18<Pbf[fJSEM<XafRfSbN^fHKL+10< *p < 0.05;**p < 0.01<7$IYfWJ 14;15;17;20;23;24;25;30F5<kqt-2%
IYfWJ 15;20;30F5<ocr-1%IYfWJ 23;24F5<AMBMI:J H=>GDC<
94



 (%)

25 °C→15 °C (3 hr) →2 °C
100
80
60
40
20
0

*

NS

 732)# 2(ok
tkq

 32)
 7
# 2(oktkq

6 cm
3.5 cm'
2

27B3.5 cm/6 cm2
.',-kqt-2 2(25 ℃.'15 ℃13$:?=,-%2(*3.5 cm.',kqt-2 .3#/2(6,-+)6 cm2
.
',-kqt-2 3& 0(26,-*7<;8 ≥ 8*9@A>A3SEM* t (Welch) 145"!* *p < 0.05; **p < 0.01*95PreSens OXY-1
SMA system

5-/3<

% 08<;( ),+*126

28= &(PreSens OXY-1 SMA system,#( %"*126,08<;' 495.:7% !#08<;(()12
6', !$, !#

96

25 °C→15 °C (3 hr) →2 °C
*



 (%)

80

*

*

*

60
40
20
0
!



k



-2
t
q

g

(

7
k
o

y
c

gc

)
2
3

3
y-

(
5
3

o

5

k
o
(

6
7
k

7

;
9)

)
9
6
k

-2
t
q

k
o
(

7

)
2
3

29F  gcy-35 0%
25 ℃.$15 ℃/3";A?)+#0%&  GCY-350
.1%'*3 5)+&kqt-2 0%'*3 1gcy-35 /2,-(4+&6=<7 ≥ 11&8DE@E1SEM&>CE9E:D7BE /23& *p < 0.05; **p < 0.01&

97

A

25 ℃3&(. (%)

151kqt-2(ok732)
gcy-35(ok769)
gcy-35(ok769); kqt-2(ok732)

10
5
0



-5
23 ℃
15 ℃

0

100
200
2 (sec)(. (%)

B

14
12
10
8
6
4
2
0

300

**

9)
2)
2)

6
3
3
7
7
7

ok
ok
ok
1
(
(
(
5
2
2
tqty-3
k
kq
c
g
9);
6
7
(ok
5
y-3
c
g

30X0#4gcy-35 ?ADL)LQUST?"(A) kqt-2 ? >8B $?@0#<6Bgcy35 >A:; 7C9525 ℃3&5 NIUT@HRO>,V17 ℃→23 ℃→17 ℃W5(B) HROA?140-150 sec?11!2?(.? 5HRO?'@HROA?'=5n ≥ 185ERUMU@SEM5JQUFUGRDPU >AB%+*5*p < 0.05; **p < 0.0151?KUI@ 14415417420423424425426=
/5kqt-2 ?KUI@ 15420426=/598

25 ℃→15 ℃ (3 hr) →2 ℃
( 6 cm )  (%)

80

NS

60
*

40
20
0
%
20

O2

%
10

O2

5%

O2



31? ,"  *#320 %$10 %$5 % )"'(!,#%5 % )"'( -20 % )"'( +.$#,&02(%4975 ≥ 9%6=><>-SEM%8;9: +/1% *p < 0.05; **p <
0.01%

99

25 ℃→15 ℃ (3 hr) →2 ℃(%6 cm )

!

(%)

50
**
25

**

0
%
20

O2

%
10

O2

5%

O2

kqt-2(ok732)

32f kqt-2#K3*1&$I4
kqt-2#U20 %510 %55 %1&F3*?C2K4610 %G5 %1&F3*?Ckqt-2#J:8E54K;-QTC6V]ZW ≥ 96
Yce`eLSEM6[_]^JPS'/.6 *p < 0.05; **p < 0.016=K.FL51&U>ASCMJ"KaX\JTE(,U3*?C6a
X\JTE3*@S=GJPR520 % O2J:8ENkqt-2#K4;
?E8C6:BQ<5aX\JTS=GJPR bde^KOaX\K IH;03*?Cbde^GL#IDC=G; F7S +;)9QTS6100

25 ℃→15 ℃ (3 hr) →2 ℃
(6 cm)  (%)

80
NS

60
40

*

20
0
%
20

O2

%
10

O2

5%

O2

gcy-35(ok769)

33@ gcy-35 -#! +$
gcy-35 420 %%10 %%5 %!*#()"-$&5 %!
*#()gcy-35 .20 %!*#()gcy-35 ,/%
$-'13)& 5:86 ≥ 9&7>?=?.SEM&9<:; ,02 
& *p < 0.05; **p < 0.01&101

25 ℃→15 ℃ (3 hr) →2 ℃
(6 cm)  (%)

60
40

*
**

20
0
%
0
2

O2

%
0
1

O2

5%

O2

gcy-35(ok769); kqt-2(ok732)

34D gcy-35; kqt-2" 2$! 0%
gcy-35; kqt-2" 820 %&10 %&5 %!.$+,#2%'10 %/5 %!.$+,gcy-35; kqt-2" 1)(-&%2
*57,'9><: ≥ 9';BCAC3SEM'=@>? 146 ' *p <0.05; **p < 0.01'

102

KCNQ
+;*7093<

TRP 093<
OCR-1

KQT-2

OCR-2
OSM-9

KQT-3

ADL#
35@ADL#(& # 
TRP093<!&OCR-1$OCR-2"OSM-9#3,1)5=-:=?/?" '%#TRP093< $ADL#(&42#KCNQ093<#68>.!&KQT-2$KQT-3#3,1)5=-:=?/?" ADL#(&

103

Wild typeO2
GCY-35
URX

kqt-2
O2

GCY-35

RMG

ADL
KQT-2

!/

×

GCY-35URX!gcy-35; kqt-2

URX

&(
RMG

ADL
KQT-2

×
×

!/

&(
RMG

×

ADL
KQT-2

×
!/ ×

36\+'"%B!/ L ;J&()TPX-#C32?0NWMSOURXKQT-2EADL! QV[YZ@! C;J&( L9<J8ACGI0!/ 5 9KJ18D! QV[YZ
ADLD&( E D+' QV[YZURX4HD+'D.L 7J1
URX+' QV[YZ@+' A:? 6GCY-355+'L :0RMGQV[YZL:?0ADL! QV[YZD&( L 9<J1kqt-2$@E0ADLD&( 5;J=F0!/ 5;J1gcy-35; kqt-2,$@E0GCY-35CG>?+'5*9KB2=F0kqt-2$CGJADLD&( D59K0kqt-2$D!/ 5;J$5 9KJ1

104

1A3.5 cm26 cm4

4"" '%+! *$6 cm"& **+! ((3.5 cm"" #$+" !$6 cm"& *$+! (*3.5 cm"" "$+" "!

6 cm"& '*+! *%3.5 cm"" !%+" ""6 cm"& $*+! *%3.5 cm"! ((+" "*6 cm"& $)+" !"3.5 cm"! "&+" $!

6 cm"% )(+" #!

3.5 cm* (*+" %!

6 cm"% %"+" "*

*

64

3.5 cm*62

"' #$+! $$

**

60

6 cm

**

58

"% !(+! $%

**

56

3.5 cm**54 4 ±SEM (%)

**

52



**

24

>?@=4

4 

24.9646143.5 cm26 cm4
4 ,3.5 cm456 cm4
8:73- </0,n = 10,t (Welch) 38;, *p < 0.05; **p <
0.01,

105

2.   

PrimerMutaion check用プライマー
プライマー番号

配列(5'-3')

KHR854_kqt-2_inner_fwd
KHR855_kqt-2_inner_rev

GAG AAT GCC GGA AAA TTC AA
TGG CAA TAA AGT GAC GCT TG

KHR856_kqt-2_internal1_rev

GCG TAA ACG AGT TCT ATT TGC G

KHR1179_kqt-3_deletion_fwd

GCT CGA TAC CGT AAC ATA AAG GAG

KHR1180_kqt-3_deletion_rev
KHR1181_kqt-3_deletion_inter

AGG AAT CCG ATG TAG AGA GTT GTGCGA CAT TTA GCA GGA CCA G

KHR2022_ocr-1(ok132)_fwd

GAT GAA TTC GAT CAG GGA CAA GC

KHR2023_ocr-1(ok132)_rev
KHR2024_ocr-1(ok132)_inter_r

TTT CCC GAT CCA TTT CAT CCA GTCT TTC ATC AAC GCT CCA TAC C

KHR2025_ocr-2(ak47)_fwd

CAA GCT AGC ATT AGA ATG TGG AGC

KHR2026_ocr-2(ak47)_rev

CCT GTC GTT TAT ACT CCA GTT GTG

KHR2027_ocr-2(ak47)_inter_r
KHR2074_gcy-35_f

GGC AAA TGC TTC CCA TTT CG
TGG AAA ACT CCG CAA CAA CG

KHR2075_gcy-35_r

GTT GAG CTT CCC AGT CTC GC

KHR2076_gcy-35_inter_f

ATG AGA CAC TCT GAC ACC ACG TG

KHR132_gsp-4tm5415_fwd1
KHR133_gsp-4tm5415_rev1

AGCAGGCAGGATTTTATGCTGGCAAACGGAAAAACGC

KHR134_gsp-4tm5415_int_fwd2

TACTGTCCGTGGATGTCTCC

用途

kqt-2(ok732) 変異チェック用プライマー

kqt-3(aw1) 変異チェック用プライマー

ocr-1(ok132)変異チェック用プライマー

ocr-2(ak47) 変異チェック用プライマー

gcy-35(ok769) 変異チェック用プライマー

gsp-4(tm5415) 変異チェック用プライマー

レスキュー用 PCR断片
プライマー番号

配列(5'-3')

KHR1053_kqt-2_full_fwd
KHR1054_kqt-2_full_rev

GAT CAT GTC GAT GAC GCA ATG AG
CTA GCG ATC CAA TTG ATT TCC GTC

用途

kqt-2遺伝子上流3kbプロモーター領域とkqt-2遺伝子全長を増幅するためのプライマー

KHR1055_kqt-2_full_out_fwd

CCA GTG GAA TTT TAT GCT TTC CGG

KHR1053、KHR1054で増幅したPCR産物をTemplateにして kqt-2のプロモーターとKHR1056_kqt-2_full_out_rev

GTC AGG AGA TTT TTG GTT TCC TGG

Plasmid作成に用いたプライマー
プライマー番号

配列(5'-3')

KHR1112_kqt-2cDNA_primer1_rev

TCC GTT GCC TTT GAT TTC C

KHR1113_kqt-2cDNA_primer2_fwd

GAC GAT GCC GAA GAT TCA G

KHR1114_kqt-2cDNA_primer3_fwd
KHR1115_kqt-2cDNA_primer4_fwd

CAT TCT CAT CAT CCC AGC C
ATG GTG GTT CAC TGC TGT C

KHR1116_kqt-2cDNA_primer5_fwd

TCT ATG TTA CCG AGG AGC G

KHR1117_kqt-2cDNA_primer6_fwd

GAA GTA GAC ATC AGC CTG C

KHR1118_kqt-2cDNA_primer7_fwd
KHR1141_kqt-2_3UTR_infus_fwd

ACA AAG AAC CAT TGG ACC G
CAT TAT CAC CAC GTG CGT AGA ATT CCA ACT GAG CG

KHR1142_kqt-2_3UTR_infus_rev

CCC GCG GTG GCG GCC GAC AAG CTG TGA CCG TCT CCG

KHR1157_kqt-2_arti_infu_fwd
KHR1158_kqt-2_arti_infu_rev

GGG AAC AAA AGC TGG GTA CCA AGC TTC CTG CAG GGC ATG C
ATA TGG CGC TGA CGC TGC ATT CAA TGG TTT TTC AAG G

KHR1159_kqt-2cDNA_primer8_fwd

CAC CTC TAA CAA TGA AAT TGT G

KHR1160_kqt-2cDNA_primer9_fwd

CAA GTA TTG ATG AGC ACG ATG C

KHR1342_kqt-2_pro_4.5k_inv_f
KHR1343_kqt-2_pro_2.3k_inv_f

TGA CCT TCT GCT ATC ACC GAA CTC
TTC TCG AGT TCC TTC TTT CGA TCG

KHR1344_kqt-2_pro_1k_inv_f

GAC TTC GTT GCT CTT GAT CTC ACC

KHR1345_kqt-2_promoter_inv_r
KHR1947_pMSK027_kqt-2cDNA_Insert_f

GAC ACT CAT AGA TGA AAA CGA CTG GGACTCTAGAGGATCATGCCAAAATACGTTGCGTTKHR1948_pMSK027_kqt-2cDNA_Insert_r

TCATTTTTTCTACCGAGACTCTATCCAAGCTGACT

KHR1949_pMSK028_kqt-2promoter_infu_f

GAAATGAAATAAGCTTGCTCTAAGACACATATGAAA

KHR1950_pMSK028_kqt-2promoter_infu_r

CCTCTAGAGTCGACCTAATTCATTGATTTAATTTTCCAT

遺伝子全長を増幅するためのNested primer

用途

kqt-2cDNAのダイレクトシーケンス用

kqt-2cDNAの下流にunc-54 UTRを導入するためのプライマー
kqt-2cDNAの人工遺伝子を導入するためのInfusion用プライマー
ダイレクトシーケンス用

kqt-2遺伝子のプロモーター領域のシスエレメント解析用プラスミド作成に用いたプライマー

表1. 本研究で使用したプライマー一覧

106

KQT-2の局在解析用プラスミド作成に用いたプライマー（ kqt-2cDNA::GFP ）3.    

Plasmid
Rescue系統作製用
プラスミド番号
pMSK004
pMSK005
pMSK007
pMSK008
pMSK024

ges-1p::kqt-2cDNA::unc-54UTR
osm-6p::kqt-2cDNA::unc-54UTR
sra-7p::kqt-2cDNA::unc-54UTR
kqt-2cDNA::unc-54UTR
sre-1p::kqt-2cDNA::unc-54UTR

発現細胞解析用
pMSK028
pMSK029
pMSK030
pMSK031

kqt-2_9kb_promoter::kqt-2cDNA::GFP
kqt-2_1kb_promoter::kqt-2 genomic gene(1st-12th exon)::GFP
kqt-2_2.3kb_promoter::kqt-2 genomic gene(1st-12th exon)::GFP
kqt-2_4.5kb_promoter::kqt-2 genomic gene(1st-12th exon)::GFP

Calcium imaging系統作製用
プラスミド番号
pTOM63sre-1p::yc3.60

Injection marker用
プラスミド番号
pKDK66
pAK62
pRF4

ges-1p::NLS::GFP
AIYp::GFP
rol-6gf

表2. 本研究で使用したプラスミド一覧

107

1)

Okahata M., Wei A. D., Ohta A., Kuhara A.

Cold acclimation via the KQT-2 potassium channel is modulated by oxygen in Caenorhabditis elegans
Science Advances, 5, 2, eaav3631, 1-12, 20192) Okahata M., Ohta A., Mizutani H., Minakuchi Y., Toyoda A., Kuhara A.
Natural variations of cold tolerance and temperature acclimation in Caenorhabditis elegans
Journal of Comparative Physiology B, 186, 8, 985–998, 2016

1)

Ohnishi K., Takagaki N., Okahata M., Fujita M., Ohta A., Kuhara A.

Molecular and Cellular Network Systems Underlying Cold Tolerance of Caenorhabditis elegans
Cryobiology and Cryotechnology, 64, 2, 53–59, 2019