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/ 48 ƲˣɡC$ŧ0ȑĶȍɊ 3̌¢ŋ!$˚ȑǄ0ȑĶȍ1 0̌˚ȑǄ0˩,¢ŋ!*.Ɉĵĭț´ gsp-4 0ȑĶȍ1Ɋ 44̌%'$0/Ņ ˚ȑǄ0˩,0¢ŋ!$ gsp-4 ĭț´1Ɋ 17̌,¢ŋ!*. gsp-4
10

ĭț´0³ǰɣų0țŒ˓Úȡ/ėū$(Ě 6) C@0,@ Ɉĵ0țŒ/?'*³ǰɣų0țŒşʱCB,ȯđC$

Ɉĵ0 MSP ³ǰɣų/ţ˶FB ĭț´>Ɉĵ0ňÎĽ̅@
C$



*.

Ɉĵ³ǰɣų/ţ˶FB,ȯđɈĵ-0?/³ǰɣų/ţ˶FB01Ư@,.'"+Ɉĵ³ǰɣų/Bţ˶/(*ʣ6B$</

Ɉĵ+ǏɨB,Ư@,.'*Bː¯ĵ/ĭțF=( 22 ɉɒ0ĭț´0³
ǰɣųFǱļ$(Ě 7)(L'Hernault, 2006) "0ɑǂ20̀ɦŧ/ 2/ɡ

C$ spe-15 , spe-39 0ĭț´+³ǰɣų0țŒʓńC$̍Ě 7̎ spe-15
1ɈĵŢž0ȾǯƢÚʆ+Ɉĵɍɧ(spermatid)Ǖ¶ŉ´(residual body)
@ÙɸB˨/ Fibrous Body-Membranous Organelles (FB-MOs)>}jYkI,'$ɍɧŉĔĻFÿƋˏƂ*ʺˀBŤåFƆ(}P] VI FYk*B(Kelleher et al., 2000) spe-39 1 FB-MOs 0Ţž/ůʌ.ƧʏʒǜųdrUʮFYk*B(Zhu and L'Hernault, 2003) 0(0ː¯
ĵ1-&@=ɈĵŢž/˙ʌ.ŤåFƆ(ɍɧŉĔĻ FB-MOs /ˤB(Kelleher et al., 2000; Zhu and L'Hernault, 2003) FB-MOs 1ɈĵŢž/˙ʌ.drUʮįƢĈ9C*BɍɧŉĔĻ+ Z^´ȕƿ0ŉɧ+

B MO

˓²,ɑƴǍ0ɝɔ´+ B FB ˓²@.B FB ˓²1˳IUfų0ɍɧ̆ǆĘĵ MSP ®Ą$ɝɔ´+

A MO ˓²ȕƿ0ɰǌˆ+ʍEC*B

FB ˓²FǌžB MSP 1Ɉĵɍɧ@Ɉĵ5,žȃBˊȷ+śʕˬ*
ɍɧʮ/ƟÙCɈĵ0«ʳ0ɍɧ̆ǆ,*Ó˪ĄB(Italiano et al.,11

1996; Kelleher et al., 2000; Smith, 2006)Ɉĵˉë1 MSP 0˙Ą,ɫ˙Ą/?B«ʳ0°ɛ/?'*ʃEC MSP 0ʕˬ1ɈĵȇțȡxhKz_vH
db GSP-3/4 /?'*áŪCB(Wu et al., 2012)"+̀ɦǰś»Ķ

ȡ/ȟȎĭëB msp ː¯ĵFugUIgx vJiJV RNAi Ǣ+ogUeL$ eri-1; lin-15B ĭț´Â´0³ǰɣųFǱļ$̍Ě 8̎ eri-1;

lin-15B ĭț´1 RNAi /?Bː¯ĵogUeL/ŠźĀųF=(ɉɒ+B "0ɑǂ msp-19 > msp-38 .- 21 ȸ˾0 msp ː¯ĵ0ogUeLÂ´/* 20̀ɦŧ/ 2/ɡC$ŧ/nRhJwYj0 eri-1;

lin-15B ĭț´?A=ȑĶȍƭB³ǰɣų0țŒʓńC$̍Ě 8̎
20̀ɦŧ/ 2/ɡC$ŧ/Yjɉɒ?A=ȑĶȍƭBț
Œ1 Ɉĵĭț´+=ʓńC$̍Ě 5, 7̎ C@0ɑǂ@/ˤEBÚĵǏǌ³ǰɣų/ˤBāɨųɢ@C$Ɉĵ0 MSPC@0?.

20̀ɦŧ0³ǰâǺ+ȑĶȍƭBțŒ1 ǰśĀŁm ASJ 0˛´ G drUʮɐʵ>

ɭ0K_ĀŁ´0ĭț´+=ʓńC*

B(Ohta, Ujisawa et al., 2014)³ǰɣų/*Ɉĵ0 gsp-4 1K_ɐʵ0ǧ+ǏɨB 9+0ĭț´Fȓ$³ǰɣų0ʕǁ@,ȯđC$"+Ɉĵ³ǰɣų/ˤB³ǰɣų/ˤEB,ȯđC*Bɏɟ+

BȲɐɉ>ɭ,Ɉĵ,0ˤ¾Fː¯ķȡ/ʣ6B,,$(Ohta, Ujisawa etal., 2014) C9+/ Ȳɐɉ>ɭ/BK_ŷĥ¯ˋɐʵ ³ǰɣų/ů˺+B,ĥĊC*B 9$ ƻȬȺ0 DNA |KUIKF
12ȓ$ɑǂ@K_ŷĥ¯ˋɐʵį0Ɉĵː¯ĵ0ȟȎFáŪB,ȯđC$ (Ě 3)C@0ŷĥ/Ĥ)

ĵ0ˤ¾-0?.=0ʣ6B$</³ǰɣų/Bɭ,Ɉ

³ǰɣų/ˤB"C#C0ɏɟ0ː¯ĵĭțFàȓ*ː¯ķȡʕǁFʃ'$ (Ě 9, 10, 11)C9+0Ȭ

Ⱥ@ ɭ1Ȳɐɉ@ÚǡC$K_FK_ĀŁ´ DAF-2 +ĀŁB,+ː¯ĵȟȎFĭî!³ǰ/ŅBˍűųFĭî!B,Ú'*B(Ě 2) (Kimura et al., 1997; Ohta, Ujisawa et al., 2014) "+ ɭ+1$@ː¯ĵ,*K_ĀŁ´FYkB daf-2 ː¯ĵ Ɉĵ+1$@ː¯ĵ,*ɈĵȇțȡxhKz_vHdb PP1 FYkB

gsp-4 ː¯ĵFȓ* "C@0˙ĭț´F·ʈ ³ǰɣųFǱļ$ 20̀ɦŧ/ 2/ɡ$, ˚ȑǄ07,G-ǔǶB0/Ņ Ɉĵ0 gsp-4

ĭț´,ɭ0 daf-2 ĭț´1̇ȑĶȍFȯ$ "* gsp-4; daf-2 ˙ĭț´1"C#C0õȊ0ĭț´,ąȷś0țŒFȯ$(Ě 9)0,@

gsp-4 , daf-2 1ː¯ķȡ/ąɐʵ+ǏɨBāɨųȯđC$ @/ļ˛ȡ PCR Fȓ* ˚ȑǄ,K_ĀŁ´ daf-2 ĭț´/BɈĵȇțȡxhKz_vHdb gsp-3 , gsp-4 0 mRNA 0ȟȎ˛Fǘʹ$"0ɑǂ K_ĀŁ´ daf-2 0ĭț´/* gsp-3 , gsp-4 0ȟȎyƭ$(Ě 10, 11)
¯ˋɐʵ
$

(9A

ɭ0K_ĀŁ´ DAF-2 F¦$ŷĥɈĵ0 gsp-3 , gsp-4 0ȟȎ/ţ˶F*B,ȯđC

C@0ɑǂ@ ː¯ķȡ/ DAF-2 0ǧ+Ɉĵ0 GSP-4 ǏɨBāɨųȯđC$0ȑîķýű/*ː¯ķȡ/ǧ9$1ǧ,1BȎʩ/ŅBˇţ˶FBː¯ĵ0˹ȚFȯˤ¾0,+
13B ʅȎŢ1ǧ0ː¯ĵ0ţ˶ÊÌCBɈĵ0 gsp-4 ĭț1 ASJ m0ǰś¯ˋɐʵ0țŒ+ƁáCB Ǒ/ Ɉĵ,³ǰɣų/ˤB,Ú'*B ASJ ǰśĀŁm,0ː¯ķȡ.ˤ¾Fʣ6B$</³ǰɣų/ˤB"C#C0ɏɟ0ː¯ĵĭțFàȓ*ː¯ķȡʕǁFʃ'$(Ě 12, 13)

C9+0ȬȺ@

ASJ ǰśĀŁm1ƹąļ0ĀŁ´+ǰśFĀŁ ˛´ G drU
ʮ[wmgj(G)+B GPA-3 >VIm˗]Ub(GCY)+B

ODR-1 F¦*ǰśŷĥF¯ˋ ASJ 0]lx_˓²@0K_0Úǡ F á Ū  B  ,  Ú  ' *  B ( Ě 2) (L'Etoile and Bargmann, 2000;
Lochrie et al., 1991; Ohta, Ujisawa et al., 2014)
,0ː¯ķȡ.ˤ¾Fʣ6B$</
G> GCY 0ĭț,Ɉĵ, ASJ ǰśĀŁm

ASJ ǰśĀŁm+1$@

Ɉĵ+1$@ː¯ĵ0ĭț,0˙ĭț´F·ʈ"0³ǰɣųFǱļ$(Ě 12, 13) 0ʕǁ+1 ASJ m+ǏɨBG,* GPA-3 F GCY ,* ODR-1 Fȓ$ Ɉĵ0ĭț,* ³ǰɣųțŒFşʱ ɈĵȇțȡxhKz_vHdb PP1 FYk
B gsp-4 ː¯ĵ0ĭțFȓ$ (Ě 12, 13) 20̀ɦŧ/ 2/ɡ$, ˚ȑǄ07,G-ǔǶB0/Ņ ASJ

ǰśĀŁm0ǰśŷĥ¯ˋɐʵ/țŒF=( gpa-3 ĭț´, odr-1 ĭț´1-&@=˚ȑǄ?A=>>̇ȑĶȍFȯ$(Ohta, Ujisawa et al.,2014) ąǍ/ Ɉĵ/țŒF=( gsp-4 ĭț´=̇ȑĶȍFȯ$(Ě 12, 13)

gsp-4; gpa-3 ˙ĭț´, gsp-4; odr-1 ˙ĭț´1 ŸĮ.,/Ɉĵ0
14

gsp-4 ĭț´0țŒƁáC odr-1 ĭț´9$1 gpa-3 ĭț´,ąȷś0ȑĶȍFȯ$(Ě 12, 13)@/ļ˛ȡ PCR ʕǁ0ɑǂɈĵ/țŒF=(

gsp-4 ĭț´+1 ASJ ǰśĀŁm+ȟȎ*B gpa-3 > odr-1 ː¯
ĵ0ȟȎyĭî*$(Ě 14, 15) C@0ɑǂ@ ASJ ǰśĀŁm
0 GPA-3 , ODR-1 1ː¯ķȡ/Ɉĵ0 GSP-4 0ǧ+ǏɨBāɨųȯđC$Ɉĵźʑm0ȲɐǦë/ţ˶FB ˙ĭț´Fȓ$ː¯ķȡʕǁ,ļ˛ȡ PCR ʕǁ0ɑǂ@ ³ǰɣų/
* ASJ ǰśĀŁm0˛´ G drUʮɐʵɈĵ0ǧ+ǏɨBāɨųȯđC$"+Bţ˶/(*ʣ6B$</

Ɉĵ0țŒ ASJ ǰśĀŁm/
Q]L~K^VFȓ*Ɉĵĭț´

0 ASJ ǰśĀŁm0ȲɐǦëFǱļ$(Ě 16) Q]L~KiJ
Wd,*QPdrUʮF¸ȓ$(Miyawaki et al., 1997) QPdrUʮ1ː¯ĵ/?'*YkC N ƺ/ CFP C ƺ/ YFP F=(

@/ĳ/Q]L~ɑĄdrUʮ+BQ^0˓+

BQ]L~KPɑĄ˓²(EF pkkK),Q^,ɑĄ

B}P]ʸ˟Slb(M13)0Q^ɑĄ˓²F=( QP
1 440 nm 0éʱÍFȂŇ$˨/ ɍɧÒ0Q]L~Ǽś³,/1
CFP 0˱ɷ(480 nm)0ʀÍFȟB1ƨɍɧÒQ]L~Ǽś̇,

Q]L~KPQPÒ0 EF pkkK/ɑĄB,+

M13 ,ɑĄB "C/?'* CFP , YFP ˣ0ʴˬʽ) 0˨/ 44015

nm 0éʱÍFš*B, CFP 0˱ɷ(480 nm)0ʀÍNnT YFP /ȶë* YFP 0̋ɷ(535 nm)0ʀÍFȟB FRET(ʀÍÐ̉NnTȶë),
č2CBȎʩʱB 0˨0 YFP , CFP 0ʀÍ0ǘFÿB,+ɍɧÒ
Q]L~Ǽś0ĭîFļ˛îB,+B

ɶȡ/Ȳɐɍɧ1Q]L~ǼśƭB,"0Ǧų=ƭB,ɢ@C*B$<QPFȓ*ȲɐǦëFǱļB,+B
 QPː¯ĵFɈĵ/țŒF=( gsp-4 ĭț´0 ASJ ǰśĀŁmȇțȡ/ȟȎ!$ː¯ĵňÎɉɒF·ʈǰśâǺF$˨0 ASJ

ǰśĀŁmÒ0Q]L~Ǽś0ĭîFǱļ$(Ě 16)
ʃEC$Q]L~K^Vʕǁ@C9+/˚ȑġ0ǰśĀŁm ASJ

0ȲɐǦë1 ǰśâǺ/?'*ĭîB,ĥĊC*B(Ohta, Ujisawa

et al., 2014) ƻȬȺ+= ASJ ǰśĀŁmȇțȡ/QPFȟȎ!$˚ȑġɉɒ/ǰśâǺF$ĦĄ/
L~Ǽś0ĭîʓńC$(Ě 16)

ǰśâǺ/ű$ɍɧÒQ]"C/Ņ*

ɈĵȇțȡxhKz_vHdb PP1 /țŒF=( gsp-4 0ĭțɉɒ+1

˚ȑġɉɒ,ąǰśâǺF*= ASJ ǰśĀŁm0űɁų1³*$(Ě 16) @/

gsp-4 ĭț´ȯ$ ASJ ǰśĀŁm0ǰśűɁų0³1

spe-11 xdFȓ*Ɉĵȇțȡ/ gsp-4 ː¯ĵFȟȎ!$ gsp-4 ĭț´+ėū$(Ě 16) (9A gsp-4 ĭț´0Ɉĵ0țŒFėū!B,+
ASJ ǰśĀŁm0ǰśűɁų0³ėū$ɈĵȇțȡxhKz_vHdb PP1 +C@0ɑǂ@

B GSP-4 1

ASJ ǰśĀŁm

0ǰś/ŅBűɁų/ţ˶FB,ȯđC$16 ː¯ķȡʕǁ,ȑȏķȡʕǁ@ ³ǰɣų/*Ɉĵ ASJ ǰśĀŁm
0ȲɐǦë/ţ˶FB,ȯđC$§0ȐĨűɁ/ţ˶FBāɨųFɢ
Į0ȲɐǦë

(/Ɉĵ0ȈŻ

Ɉĵ ASJ ǰśĀŁmª$,2ēʑm0űɁų.-/=ţ˶FBFʣ

6$ Ñ´ȡ/1 Ɉĵ/țŒF=( gsp-4 ĭț´ AWA ēʑm+ĀŁCB^Iaf> AWC ēʑm+ĀŁCBKcI}IY/ŅBîķʰų0țŒFȯFǱļ$(Ě 17) (Bargmann et al.,
1993; Kuhara et al., 2002)îķʰųh_jFʃ'$ɑǂ

˚ȑġ1^Iaf

,KcI}IY/ŠʟşC$0/Ņ gsp-4 ĭț´1-&@/Ņ*=7,G-ʟşC.'$(Ě 17)
hKz_vHdb PP1 +C@0,@ɈĵȇțȡxB GSP-4 1 AWA ēʑm, AWC ēʑm0ēʑűɁ/=ţ˶FBāɨųȯđC$³ǰɣų/ˤEBɏɟˣFɞ_hKkzĀŁ´0ƐɌ 9+0ː¯ķȡʕǁ>ļ˛ȡ PCR ʕǁ Q]L~K^VFȓ$ʕǁ@ ɭɈĵ/ţ˶F

"0ŧɈĵ ASJ ǰśĀŁm/vJkqgUB,+ȲɐǦë/ţ˶FB,ȯđC$ (9AȲɐ@ɭ5

ɭ@Ɉĵ5ǰɣų/ˤB,ɢ@C$Ɉĵ@Ȳɐ5,ɏɟˣngjU³
C9+/ ASJ ǰśĀŁm@ɭ50ŷĥ¯ˋ1K_F¦*ʃECB,Ư@/.'*B(Ohta,
Ujisawa et al., 2014)"C/Ņ*

ɭ@Ɉĵ50ŷĥ¯ˋ>Ɉĵ@˼˓0 ASJ źʑm50ŷĥ¯ˋ/ˤEBÚĵ1ąļC*.
17C.

elegans 0´Ò/* Ɉĵ, ASJ mˣ0ʴˬ1ȦŅȡ/İȆȏȡ/ȥƑ0YdUjFƆ(,1+.$<Bŷĥ¯ˋFƄÚĵ,*C@0ɏɟˣ/

Úǡų]Vl0āɨųFɢ$C9+0ʕǁ@ _hKkzĄž/ˤEBskS]_hKk 17isk
XlbFYkB dhs-4 ː¯ĵ0ĭțĥĊC*B(Ohta, Ujisawa et al., 2014)³ǰɣų/ţ˶FB,"+_hKkzĀŁ´+ B Nuclear Hormone Receptor (NHR)0ː¯ĵ³ǰɣų/B

ţ˶Fʕǁ$(Ě 18, 19)
 DNA |KUIKʕǁFȓ* 250ǰśâǺF$˨/ȟȎĭëBː¯ĵ(Ě 3, 4)F˚ȑǄ,K_ĀŁ´ daf-2 0ĭț´+ǘʹB,+
_jî "0@ NHR FYkBː¯ĵFugUIgx$ ugU
IgxC$ː¯ĵ0ǐƖĭț´

B1 RNAi /?BogUeLÂ´0³

ǰɣųFǱļ$(Ě 18, 19) "0ɑǂ 20̀ɦŧ0 nhr-88 , nhr-114 0ĭț´/* 2/ɡC$ŧ=̇ȑĶȍʓńC$(Ě 18)

0(0nhr ĭț´0& nhr-88 ĭț´+1 AWA ēʑm+ĀŁCB^
Iaf, AWC ēʑm+ĀŁCBKcI}IY/ŅBîķʰų³*$ (Ě 17) NHR-88 1ɩʮʫɽ/ˤB,ĥĊ
C*B(Ashrafi et al., 2003)

NHR-114 1

̃0ȸ˾+ĭîBɭ0©ʥĭî/?Bȑǖɍɧ50ʪɺFɚďBŤåĥĊC*B(Gracida andEckmann, 2013) nhr-88 ː¯ĵ, nhr-114 ː¯ĵ0ȟȎɍɧFʣ6B$</
ɗɷʀÍdrUʮ GFP F|Q,*ʕǁFʃ'$ nhr-114 ː¯ĵ/ˤ*1

xdDNA ˔Ü,ː¯ĵÏˡ/ GFP ː¯ĵFʁĄ!$x
18

_}kF·ʈ nhr-88 ː¯ĵ/ˤ*10˓/ GFP ː¯ĵFʁĄ!$x

xdDNA ˔Ü,ː¯ĵ_}kF·ʈ$ C@0x_}kF˚ȑǄ/ňÎ GFP ȟȎ*BɍɧFʣ6$,D -&@=ɭ/* GFP 0ȟȎʓńC$(Ě 20)
 nhr-88 ː¯ĵ, nhr-114 ː¯ĵ ³ǰɣų/ˤEBȲɐ,ɭ,ɈĵF¦$ɏɟngjU00-0ɏɟˣ0ŷĥ¯ˋFƄ'*BFʣ6B$</³ǰɣų/ˤB"C#C0ɏɟ0ː¯ĵĭțFàȓ*ː¯ķȡ.Nu_d]_ʕǁFʃ'$(Ě 21, 23-26)

9Ɉĵ+1$@ɈĵȇțȡxhKz_vHdb PP1 FYkB gsp-4 ː¯ĵ0ĭț,

nhr-88 

nhr-114 ː¯ĵ0ĭțFȓ*˙ĭț´F·ž "C@0³ǰɣųFǱļ
$(Ě 21) nhr-88 ĭț´, nhr-114 ĭț´,Ɉĵ/țŒF=( gsp-4 ĭț´
1 20̀ɦŧ/ 2/ɡC$ŧ

C=˚ȑǄ?ȦȑĶȍFȯ$"C/Ņ gsp-4; nhr-88 ˙ĭț´, gsp-4; nhr-114 ˙ĭț´1-&@=

gsp-4 ĭț´,ąȷś0țŒFȯ$(Ě 21) nhr ĭț1 gsp-4 ĭț/?'*Ɓ
áC$,@ gsp-4 ĭț1ː¯ķȡ/

nhr-88 ĭț, nhr-114 ĭț0ǧ/²ɡB,ɢ@CB @/ ļ˛ȡ PCR ʕǁ@ Ɉĵ+ȟȎ*B gsp-4 ː¯ĵ0ȟȎy nhr-88 ĭț´, nhr-114 ĭț´0ƨ+ĭî*$(Ě 22) C@0ɑǂ@ ɈĵȇțȡxhKz_vHdb PP1+

B GSP-4 1 ɭ+ȟȎB_hKkzĀŁ´ NHR-88 , NHR-114

0ǧ+Ǐɨ*B,ɢ@CBǑ/

ASJ ǰśĀŁm0ǰśĀŁŷĥ¯ˋ/ˤEB GCY FYkB odr-1 ː¯ĵ0ĭț,> nhr-114 ː¯ĵ0ĭțFȓ*˙ĭț´F·ʈ19nhr-88 ː¯ĵ

"C@0³ǰɣųFǱļ$(Ě 23, 24)

ASJ ǰśĀŁm0ǰśŷĥ¯ˋɐʵ/țŒF=(odr-1 ĭț´120̀ɦŧ/˚ȑǄ?A=>>̇ȑĶȍFȯ$(Ě 24)(Ohta, Ujisawa et al., 2014) ¥ėʃ'$ nhr-88; odr-1 ˙ĭț´0Ľ̅+1

˄Œ0 2/ 48 Ʋˣ˲ɡBƾ¬+1 odr-1 ĭț´0İòǔǶ*9

˚ȑġ,ǘ6*İ.țŒ1ʎ@C.'$$<ɡBƾ¬+ʕǁFʃ'$"0ɑǂ

2/ 12 Ʋˣ˲

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0 2+ 48 ƲˣɡC$ŧ nhr-114 ĭț´,ąǍ0³ǰɣųțŒFȯ$(Ě24) "C#C0˙ĭț´ nhr ː¯ĵ0ĭț´,ąǍ0³ǰɣųțŒFȯ$,@ ː¯ķȡ/ɭ+ȟȎB NHR-88 , NHR-114 1 ASJ ǰśĀŁm
0ǰśŷĥ¯ˋɐʵ+ǏɨB GCY +B ODR-1 0ǧ+ǏɨB,ɢ@CB(Ě 23, 24) C@0ɑǂ@ ³ǰɣų/*ɭ+ȟȎB_hKkzĀŁ´ NHR-88 , NHR-114 1_hKkzF¦*ǰśĀŁm ASJ @µ@0]VlFĀÿB,ȯđC$
C9+0ȬȺ@

ASJ ǰśĀŁm@ɭ50ǰśŷĥ¯ˋ1K_F¦*ʃECB,Ư@,.'*B(Ě 2)(Ohta, Ujisawa et al.,2014) "+

ɭ0K_ĀŁ´ DAF-2 ,(0_hKkzĀŁ´ NHR-88 > NHR-114 ,0ː¯ķȡˤ¾Fʣ6B$</ daf-2 ː¯ĵ,

nhr-88 ː¯ĵ> nhr-114 ː¯ĵ0ĭțFȓ*˙ĭț´F·ʈ "0³ǰɣųFǱļ$(Ě 25, 26) daf-2 ĭț´,"C#C0 nhr ĭț´1 20̀ɦ
ŧ/ 2/ɡC$ŧ˚ȑǄ?A=̇ȑĶȍFȯ$ƨ

nhr-88; daf-2

˙ĭț´, daf-2; nhr-114 ˙ĭț´1"C#C0õȊ0ĭț´?A=̇
20

ȑĶȍFȯ$(Ě 25, 26)

nhr-88; daf-2 , daf-2; nhr-114 0ƨ0˙ĭț´+³ǰɣų0țŒĩŠB,@ K_ĀŁ´ DAF-2 ,"C#C
0ǅÒĀŁ´ĀŁ´ NHR 1 ßɐʵ+³ǰɣų/ţ˶F*B,ȯđC$Ɉĵĭț´0ɩʮɏž əɿ C. elegans FĈ;į0ȑȆ/*´FǌžBǍ

.ɩɥ˗0ǘ

ȍ1³ǰɣų/İţ˶B,ĥĊC*B(Murray et al., 2007; Ohta
et al., 2014) C9+0ȬȺ+ ɭ+³ǰɣųFáŪB,Ư@,.'*BK_ĀŁ´ DAF-2 0ǏɨFǐƖB daf-2 ĭț´/* Â´FǌžBɩɥ˗0ǘȍĭîB,ĥĊC*B(Ohta, Ujisawa et al.,2014) "+ ³ǰɣų/ˤB,ȯđC$ɈĵȇțȡxhKz_vHdb PP1 /țŒF=( gsp-4 ĭț´/*=ɩɥ˗0ɩʮåĄFǱļ$ ˚ȑǄ, gsp-4 ĭț´0ɖɩʮFǘʹ$ɑǂ 16:1n-7 > 18:1n-9 .
- 11 ȸ˾0ɩɥ˗+ƷŹ.ŏFȯ$(Ě 27)



SFA̍́ďɩɥ˗̎

MUFA̍õ́ďɩɥ˗̎ PUFA̍į¼́ďɩɥ˗̎/İß$ĦĄƷŹŏ
1ʎ@C.'$+C9+0ȬȺ+1

B9%Ú'*.$<B$</1ăÂ

-0?.ɩɥ˗³ǰɣų/˙ʌ
0ɩɥ˗,³ǰɣų,0ˤˇųFąļ0©ʥɐʵ0ĭț´0³ǰɣųFʕǁBůʌ

@CB

21

B,ɢ

ɢń
³ǰɣų/Ɉĵ0 MSP ˤB ɈĵŢž>Ǐɨ0ĭț´>Ɉĵ/țŒF=( gsp-4 ĭț´,˚ȑǄ0˩,

0ƎĄE!/?BɈĵňÎĽ̅@Ɉĵ³ǰɣų/ţ˶FB,ȯđC$(Ě 5, 6) @/ Ɉĵː¯ĵ0ĭț´, RNAi /?BogUeLÂ´0³ǰɣų0ʕǁ@ Ɉĵ0 MSP ³ǰɣų/ˤB,ȯđC$(Ě 7, 8)

MSP 1Ɉĵȇțȡ.˳IUfų0ɍɧ̆ǆĘĵ+ɈĵˉëFʃ«ʳ0ɍɧ̆ǆ,*ǏɨB(Italiano et al., 1996; Kelleher et al., 2000;
Smith, 2006)

MSP 1ɈĵŢž0ÝƸ@ǉÙC ǑųɍɧƸ@ǑųɍɧƸ1Z^´ȕƿ0ŉɧ+

B MO /˪Ą*ɑƴǍ0ɝɔ´ FB FŢž MO ȕƿ0ɰǌˆ/ʍECB,+ FB-MO ,č2CBɈĵȇțȡɍɧŉĔĻ,.B ɈĵɍɧǕ¶ŉ´@ÙɸBƲ FB-MO 1 SPE-15 /?'*
Ɉĵɍɧ/ʺˀC "0ŧ FB ɫ˙Ą* MSP 1ɍɧʮ5,ƟÙC «ʳ0ɍɧ̆ǆ,*Ó˙ĄB «ʳ0ɍɧ̆ǆ+

B MSP 1«ʳÌȽ˓+˙Ą «ʳĤ˓+ɫ˙Ą*°ɛ Ɉĵ0ˉëFáŪB(Ě 28)(Smith, 2006)³ǰɣų/ţ˶FB,ȯđC$ SPE-39 , SPE-15 1 FB-MO 0Ţž,ʺˀ/ˤB,@ MSP 0ǓŒ.Ǐɨ³ǰɣų/ţ˶FBāɨųɢ@CB(Kelleher et al., 2000; Zhu and L'Hernault, 2003) 9$«ʳFŢžB MSP 0ɫ˙Ą1ɈĵȇțȡxhKz_vHdb GSP-3 ,GSP-4 /?'*áŪCB(Ě 28) (Wu et al., 2012)0ɈĵȇțȡxhK

z_vHdb GSP-3 , GSP-4 1z˾0ɈĵȇțȡxhKz_vH
db PP10PcV+Az˾/*1Ɉĵ0˵Ǚ/?Bˉëų
22

,žȃFáŪB(Varmuza et al., 1999)ɈĵȇțȡxhKz_vHd

bəɿ,z˾+ąǍ0ŤåFB,,ˊú/ʃEC$ȬȺ+sj,ə

ɿ0ˣ+Ð˄BQm`~įʎ('*B,@sj0ǰśˍű/=ɈĵˤBÐ˄0Qm`~ĶĞB,FƸŦ*B

9$

MSP 1ɈĵˉëFáŪB«ʳ0ɍɧ̆ǆªĮ/ Ɉĵ@ÚǡCB,+ŷĥ¯ˋȆʮ,*ǏɨB,ĥĊC*B(Kosinski et al., 2005)
MSP /?Bŷĥ¯ˋ1Ɉĵɍɧ>žȃɈĵ@ÚǡC ÷ɍɧ+ǯƢÚʆ0
žȃ>ȑǖɮ˴ɍɧ+÷ɍɧFʫɈĕ5ƍ÷F¿ˈBː¯ķȡʕǁ>ļ˛ȡ PCR ʕǁ Q]L~K^VFȓ$ʕǁ.-@ ³ǰɣų1ɈĵFĈ;ʉƢ0ɏɟȦ·ȓ*áŪ*B,ȯđC*B(Ě 9-16)

C. elegans 0Ɉĵ1ʫɈĕ/ĶĞ Ȳɐɉ,ʴˬ B$<Ȇȏȡ/ȥƑ0YdUjFƆ(,1+. "0$< MSP 0?.Úǡz/?
'*ċʼɏɟ,ŷĥ¯ˋFBāɨųɢ@CBC9+0ʕǁ@_hKkzĄž/ˤEBː¯ĵ0ĭț³ǰɣų/ţ˶FB,ĥĊC*A(Ohta, Ujisawa et al., 2014),@ʫɈĕFʲ$ŷĥ¯ˋFʃɈĵ,§0ɏɟ0ŷĥ¯ˋ/1_hKkzˤ*Bāɨųɢ@CB ¥ė0ȬȺ+Ɉĵ³ǰɣų/ˤB,ȯđC$

"+

Ɉĵ

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Ɉĵ0ƖÇ1ʀÍ0ǯŊFʓńB,+Ȯʞ$(Ě 29)\ȂŇŧ/ 20+̀ɦ*@ 2+ 48 Ʋˣ0³ǰâǺFĦĄ ̊˕0:0gUØȏFʃ'$YjÂ´1Ɋ 15̌0ȑĶȍFȯ$0/ŅɈĵ/\ȂŇFʃ'$Â´+1Ɋ 42̌0ȑĶȍFȯ$(Ě 29) (9A\ȂŇ/?BɈĵ0Ȇȏȡ.ƖÇ20̀ɦŧ0³ǰɣų/BȑĶȍFƭ!B,Fȯđ$əɿ C. elegans 1Ɋ 300 Â0ɈĵFŢžB0+\ȂŇ+Ï*0Ɉĵ/ƖÇFB,1˭'$(Ě 29)

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L3 ŘɿƸ0³ǰɣų/(*=Ǳļ$(Ě 30)

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0ȑĶȍ1žɿ?A³B,ʓńC$(Ě 30) .@ L3 Řɿ@žɿ51ɫȣFɐ*´ǌˆİĭîB$<ɈĵŢžªĮ0ţ˶=İ,ɢ@CB 9$ žɿ, L3 Řɿ+1´0[K`=İˌ,@ ȐĨǰś/?Bţ˶F L3 Řɿ0ƨİĀBāɨųɢ@C$ 
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ʌɋİ

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ȇ/əɿ0?.ņĎȫĵĸFįǕ?.ȑǦǍŞ024

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ĶĞB

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C. elegans 0˫˩ą´0Ɉĵ1 L4 ŘɿƸ/·@C"078Ï*ĀɈB0+ĀɈ÷0ɖƢFǓŒ.Ɉĵ0Ƣ,*ɢB

,+B x^MmQLj0ɑǂ ˚ȑǄ0ĀɈ÷Ɋ 300 Â/Ņ

spe-6 ĭț´ªĮ0Ɉĵĭț´+ĀɈ÷Ƣ1ǯŊ$  20̀ɦŧ/ 2
/ 48 ƲˣɡC$,0ȑĶȍ, ɖȒ÷Ƣ?3ĀɈ÷Ƣ/Ȧˤˤ¾1ʎ@
C.'$ Ñ´ȡ.¹+1 20̀ɦŧ/ 2/ɡC$ gsp-4 ĭț´1Ɋ
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78ƹĀɈ÷+'$(Ě 31)ĭț´ʉƢ

'$,@

ąǍ/Ɉĵ0țŒ/?BȒ÷Ƣ0ǯŊ,³ǰɣųțŒ/ȥƑȡ.ˤ¾1.,ɢ@C$ˇų1ʎ@C.'$BĥĊ1Ȓ÷ƢŊ.*=³ǰɣųǓŒ.

¥ė0ȬȺ+1ȑǖɨæ,³ǰɣų/ˤãʿ0?/ȑǖɨæ0³+_j_ɣųƭƻƿȑǖ/¸ECBNnT0c_Â´0ȑĶ/˔ÚCB,+ȑBȎʩ%,ɢ@CBɈĵː¯ĵ0ĭț/?AɈĵŢž/ț25ŒȑC2"0ŧ0žˡ1ɈĵțŒ0ţ˶FĀB0+NnTc_FÂ´0ȑĶ0$</˔ÚBāɨųFɢ@C=.G@0ţ˶FBāɨųɢ@CB³ǰ_j_50űɁ/ɈĵŢžĺ*

B gsp-4 ĭț´0˫˩ą´0žɿ/˚ȑġ0˩F¢ŋ!*ɈĵFňÎB
,+³ǰɣų

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DNA |KUIK0ǘʹį0Ɉĵː¯ĵ0ȟȎK_ɐʵ/?'*ţ˶FĀ

B,Fȯđ$(Ě 3)ː¯ķȡʕǁ,ļ˛ȡ PCR ʕǁ@

ɭ0K_

ɐʵ0ǧ+Ɉĵ³ǰɣų/ţ˶FB,ȯđC$(Ě 9, 10, 11)ļ˛ȡ PCR ʕǁ+1

9$

K_ĀŁ´ daf-2 ĭț´/*Ɉĵ0

gsp-4 ː¯ĵ, gsp-3 ː¯ĵ0 mRNA ȟȎ˛ĩç$̍Ě 10, 11̎ ³ǰɣųh_j/ȓ$ daf-2 ː¯ĵ> gsp-3 ː¯ĵ gsp-4 ː¯ĵ0ĭț´1

C=ǏɨǯŊġ0ĭț+ ³ǰâǺŧ0ȑĶȍƭ$ 0,@daf-2 ĭț´+ gsp-3 ː¯ĵ, gsp-4 ː¯ĵ0ȟȎĩç*=³ǰɣų+ȑĶȍƭB,,1ʎȩȧB?/=ʎ@CB daf-2 ĭț´ş26ʱțŒFµ@0Ţ+ȦǗB$</ȟȎĩç*Bāɨųɢ@CB

0?.ː¯ĵ0ǐƖ/?'*ȑBțŒFȦǗB$<0ȟȎĩç1§0Úĵɐʵ/*=ʎ@CB,

B(Sergina et al., 2007)Ɉĵźʑm/Bţ˶ ː¯ķȡʕǁ ļ˛ȡ PCR ʕǁ Q]L~K^VFȓ$ʕǁ/?
AɈĵ@ǰśĀŁm ASJ 50vJkqgUĶĞB,ȯđC$(Ě 12-16) əɿ C. elegans /* ȑǖɨæ1ȐĨŷĥFĀŁ$Ȳɐėʵ/?'*YjCB,1ĥĊC*$(McKnight et al.,2014) ǰśˍű/*ȑǖɨæ0țŒȲɐɉ/ţ˶FB,ĥĊ1Ý<*0W_+'$vJkqgU]_h~1*ȐĨ/ˍÛ/ˍűB$<0ȑ´Qm`~+

B$<ɲʶ0ȈŻFýƱǰśˍűªĮ+=Ɉĵ0ȈŻ§0ȐĨűɁ/ţ˶FBāɨųɢ@C$ɈĵȇțȡxhKz_vHdb PP1 FYkB gsp-4 0ĭț´0ēʑűɁ0ʕǁ@ Ɉĵ0 GSP-4 0țŒ ASJ ǰśĀŁm%+. AWA ēʑm, AWC ēʑm/?'*áŪCBēʑűɁ/=ţ˶FB
āɨųȯđC$(Ě 17)¥ė0ēʑűɁĽ̅+ʎ@C$ gsp-4 ĭț´0^Iaf,KcI}IY/Ņ*ʟşC..B,îķʰų0
țŒ/=µ@0ŹČ B,ɢ@CBəɿ C. elegans 0˫˩ą´1

ɈĵF L4 ŘɿƸ/0:·B0+˚ȑġ1Ɋ 300 Â,ƢǞ9'*B žɿ+÷ɍɧF·Aɓ*B 9$ gsp-4 ĭț´1x^MmQLj@ĀɈ÷Ƣ (9AɈĵǯŊ*B,Ú'$(Ě 31) (9A gsp-4 ĭț´
27

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0ȲɐǦëFQ]L~K^V+Ǳļ$(Ě 32) Ɉĵ0 gsp-4
ĭț´,ąǍ/ 300 ȵ* 17@ 230ǰśĭîF$ɑǂ daf-2 ĭț´0 ASJ ǰśĀŁm0ȲɐǦë1˚ȑǄ?A=³űɁųFȯ$(Ě 32)

űɁų³$ȏȕ,*ɭ0K_ɐʵɈĵ/ţ˶F$,+Ɉĵ@Ȳɐ50vJkqgU/?AȲɐǦëĭî$ĦĄ,K_ɐʵ@vJkqgUʃEC$ĦĄ/ţ˶F$āɨųɢ@CB

B1"0ƨȲɐ¥ė0ȬȺ+1 daf-2 ĭț´+

ASJ ǰśĀŁm0ǰśˍű³$ȏȕ1ȇļ/ɳ@.'$ɑʠǰśˍű0ɏɟngjU/BɈĵ,Ȳɐˣ0vJkqgU]_h~ ǰś1ȑ´ýű/ȥƑţ˶Fü8$<

ëȆ1ȐĨǰś0ĭî/ŅBˍű0$</įƢ0ɏɟ/?BɒĄȡ.ǰśˍűQm`~FƷ*B,ɢ@CBəɿ C. elegans 0³ǰɣų/*¥ė0ȬȺ+128Ɉĵ³ǰɣų/ţ˶FB,, ɈĵȇțȡxhKz_vHdb GSP-4 0ț
Œ ASJ ǰśĀŁm0ȲɐǦë/ţ˶FB,Ư@,.'$(Ě 5-8, 16) C9+0ȬȺ,¥ė0ː¯ķȡʕǁ>ȑȏķȡʕǁ@ũ@

C$ȪʎFɏ:ĄE!B,+³ǰɣų/*ª/ʙʿB?.v

JkqgU]_h~FĈ;ɏɟngjUiɢ@C$ĀŁmȐĨǰśFĀŁ

ASJ ǰś

K_,_hKkzF¦*ɭ/ŷĥ¯ˋC ɭɈĵ/ţ˶F "0ŧ Ɉĵ ASJ ǰśĀŁm0ǰśŷĥ¯ˋ/ţ˶FB(Ě 33)

C. elegans 0Ɉĵ1ʫɈĕ/ĶĞ ˼˓0 ASJ ǰśĀŁm,Ȇȏȡ/ʴˬ

B$</Ȇȏȡ/ȥƑ0YdUjFƆ(,1+.,ɢ@CB



ƨ+

¥

ė³ǰɣų/ˤEB,ʎ('$ MSP 1 ÷ɍɧ0žȃ,ƍ÷/ˤEBɀɤˉë0áŪ/ˤ*Úǡų0]VlÚĵ,*È

÷ɍɧ>ƍ÷Ƀ0

ɀɤ/ȥƑ·ȓB,ĥĊC*B(Kosinski et al., 2005)

C@0,@ MSP /?BÚǡų0]VlȲɐɉ50vJkqgU/ˤE'*Bāɨų=ƈ@CB 9$ Ɉĵ@ ASJ ǰśĀŁm50vJ
kqgU/1Ɉĵ§0ɏɟF¦*ˣƑȡ/ ASJ ǰśĀŁm0ȲɐǦëFʣɄBāɨų=ɢ@CBˬC$ɏɟˣ0ŷĥ¯ˋůʌ+qgUáŪ/1
"+




B,@

C/ˤ*=

Ȇȏȡ/ʴˬ0Ɉĵ@Ȳɐɉ50vJkÚǡų0ŷĥ¯ˋĘĵ¦Ğ*Bāɨųɢ@CBŷĥ¯ˋ/_hKkzˤ*B0+1.,ɢ$ŷĥ¯ˋĘĵ0ąļ/1ɳ@.'$ ǰśɣų/*Ɉĵ@Ȳɐ50vJkqgUȯCB01Ý<*0
29

W_+

B

+ɢ@C$ɏɟngjUi1

ɐ/vJkqgUB,+~+1.,ɢ@CBɈĵ0ȈŻȲȐĨ/ŅBɜǖʃëFƶˍîBQm`

Ĥƻȡ.ȑȏȡ(ÚĵȑȆķȡQm`~0į

1əɿ C. elegans @sjFĈ;̇ȿëȆ/ɳB9+ÀĶC*B$<̇ȿëȆ/*=ąǍ0ǏǌĶĞBāɨųƸŦCB

ƻȬȺ@ʎ

('$ǰśˍű/ˤBɏɟngjU,vJkqgUǏǌFi/ʕǁFˈ<B,+ëȆ0ǰśˍűQm`~>/àȓ+Bāɨųɢ@CB(Ě 33)

30

ǰśˤEBȝŶ0ʕǁƼƥ,ƨǢ

¸ȓ$ C. elegans ɉɒ
wild-type

N2

Bristol,BA17

fem-1(hc17),

CB4108fog-2(q71),

FX01505

gsp-3(tm1505), FX05415 gsp-4(tm5415), gsp-4(y418), SS712 ife-1(bn127), RB2191
msd-4(ok2971), BA819 spe-11(hc77), DR1572 daf-2(e1368), IK038 daf-2(e1370),
NL335 gpa-3(pk35), CX2336 odr-1(n1936), VC1290 nhr-125(gk578), FX00886
nhr-19(tm886),

MH1955nhr-25(ku217),

FX01033

nhr-88(tm1033),

VC1759nhr-95(gk836), FX016661 nhr-121(tm6661), VC1554 nhr-155(ok2026), FX01819
nhr-156(tm1819), VC2567 nhr-209(gk1135), RB1407 nhr-60(ok1600), RB1425
nhr-60(ok1622), FX02174 nhr-65(tm2174), VC1760 nhr-114(gk849), VC1565
nhr-116(gk728), VC1591 nhr-116(gk746), FX01185 nhr-205(tm1185), VC2761
nhr-243(gk1213),

VC1095

nhr-62(gk478),

RB1592nhr-64(ok1957),

RB1578nhr-69(ok1926), VC1140 nhr-132(gk523), FX01698 nhr-136(tm1698), FX00699
nhr-152(tm699),

VC1364nhr-206(gk603),

VC1751

nhr-246(gk856),

puf-8(q725),

SL754

spe-39(eb9)/nT1[unc-?(n754)

let-?],

JK3231SL753

spe-39(tx12)/nT1[unc-?(n754) let-?], spe-6(hc163), BA714 sDf5/spe-4(hc78), BA763
spe-5(hc93); sDp2, VC2253 spe-17(ok2631), BA744 spe-10(hc104), BA6 fer-6(hc6),
BA15 fer-15(hc15), BA840 spe-26(hc139), SL3 spe-15(hc75), BA1 fer-1(hc1), BA785
spe-8(hc40), BA783 spe-12(hc76), BA963 spe-27(it132), BA962 spe-29(it127), PS4330
spe-41(sy693); him-5(e1490), BA708 spe-9(hc52), AD271 spe-38(eb44); him-5(e1490);
asEx78[spe-38p::spe-38(cDNA)::spe-38 3’UTR + myo-3p::GFP], BA14 fer-14(hc14),31SL1138

spe-42(tn1231)/

nT1[unc-?(n754)

let-?

qIs50],

oxIs318[spe-11p::mCherry::histone + unc-119(+)]; unc-119(ed3).
unc-?(n754)! , let-?1ʅȎġȮļ*Bƹąļ0ĭț!

¸ȓ$x_}k!

pPD95.75! !
pPD49.26!
pBluescript! SK+!
pSND01![spe%11p::gsp%4cDNA]!
nhr%88(anterior8half)%GFP8
nhr%114(full)%GFP!
!
·ʈ$ɉɒ!
gsp%4(tm5415);8daf%2(e1368)!
gsp%4(tm5415);!gpa%3(pk35)!
gsp%4(tm5415);!odr%1(n1936)!
gsp%4(tm5415);!nhr%88(tm1033)!
gsp%4(tm5415);!nhr%114(gk849)!
nhr%88(tm1033);!odr%1(n1936)!
nhr%114(gk849);!odr%1(n1936)!
nhr%88(tm1033);!daf%2(e1368)!
daf%2(e1368);8nhr%114(gk849)!
32

EG4883

gsp%4(tm5415);8Ex[rx-1p::yc3.60]8
gsp%4(tm5415);8Ex[spe%11p::gsp%4cDNA,8trx-1p::yc3.60]8
N2;8Ex[nhr%88(anterior8half)%GFP,8trx%1p::dsRed,8ges%1p::tagRFP(30ng/Ol),8pRF48
rol%6(gf)]8
N2;8 Ex[nhr%114(full)%GFP,8 trx%1p::dsRed,8 ges%1p::tagRFP(30ng/Ol),8 pRF48
rol%6(gf)]8!
¸ȓ$İɭɻ!
OP<50! Ǆ! :! əɿ0̃,*! NGM! xj/ħŐ$!

!
¸ȓ$˽Ŭˠ!
˂ˊŞĽ´˽Ŭˠ! (Leica:!MZ16)!
Olympus IX81 ˽Ŭˠ!
ÐȀǽ\˽Ŭˠ(FV1000-IX81 with GaAsP PMT, Olympus)!
!
¸ȓ$ģğ!
̍1̎Nematode!Growth!Medium!(NGM)!Plate!(1000!ml)!
3!g 0 NaCl 20!g 0 agar(­˒Ńı) 2.5!g 0 Bacto!Peptone(BD!Falcon)
975! ml 0 H2O Fʔv

_Y.-ɣȄŁĔ/ÎCƙƅĵ+ǬĄ 40

ÚPjUw+çȄǶɻ$ "0ŧƙƅ.@Ɋ 50! /Õ9*@ 1!ml 0 MgSO4!(1!M) 1!ml 0 CaCl2!(1!M) 1!ml 0 Cholesterol!(5!
mg/ml!in!EtOH)

25!ml 0 Potassium!Phosphate!(1!M,!pH!6.0)Fç$
33CFȥť 6!cm 0]/ 14!ml(ɲëÚǤǏ+ÚǤ*ě<$B1 3.5!cm 0]/ 6!ml!

!
̍2̎ODR!Plate!(1000!ml)!
20! g 0 Difco! Bacto! Agar

1000! ml 0 H2O Fʔv

_Y.-ɣȄŁĔ

/ÎCƙƅĵ+ǬĄ 40 ÚPjUw+çȄǶɻ$"0ŧƙ

ƅ.@Ɋ 50! /Õ9*@ 1!ml 0 MgSO4!(1!M) 1!ml 0 CaCl2!(1!
M)! 25!ml 0 Potassium!Phosphate!(1!M,!pH!6.0)Fç$

9!cm 0]/ 12!ml(ɲëÚǤǏ+ÚǤ*ě<$

CFȥť
!

!
!
¸ȓ$xK|!

̍1̎į˙ĭț´F·ʈB˨/ȓ$ȇļ0ĭțFǉÙB$<0xxK|0Ćã KHR220_gpa<3_new_fwd_primer! !

DNA 0˔Üŷĥ GCAGAGGACAAAGAACTGAC!
!
x

K|0Ćã KHR221_gpa<3_new_rev_primer! !

DNA 0˔Üŷĥ TACAAACCGCATCCTTGC!
!
x

K|0Ćã KHR155_gpa3(pk35)_internal_fwd! !

DNA 0˔Üŷĥ TGGTTCATCAATACTTCAATGAT!
!
34

K|!

x

K|0Ćã KHR132_gsp<4tm5415_fwd1! !

DNA 0˔Üŷĥ AGCAGGCAGGATTTTATGC!
!
x

K|0Ćã KHR133_gsp<4tm5415_rev1! !

DNA 0˔Üŷĥ TGGCAAACGGAAAAACGC!
!
x

K|0Ćã KHR134_gsp<4tm5415_int_fwd2! !

DNA 0˔Üŷĥ TACTGTCCGTGGATGTCTCC!
!
x

K|0Ćã KHR137_odr<1n1936_RsaI_fwd! !

DNA 0˔Üŷĥ TCCTACGCTATCAACGCAG!
!
x

K|0Ćã KHR138_odr<1n1936_RsaI_rev!

DNA 0˔Üŷĥ !
TCCGATTTTTTTTTTTGTCTAAAACTTTCATTTTGAAGTA!
!
x

K|0Ćã KHR011_daf2e1368_AclI_fwd! !

DNA 0˔Üŷĥ TAAAGAAGACCGAGTCCGC!
!
x

K|0Ćã KHR012_daf2e1368_AclI_rev!

DNA 0˔Üŷĥ !
TTGAACATGTTCCGGAATTTACGACGTATTGAGGCAACGT!
35

!
x

K|0Ćã KHR320_nhr<114gk849_fwd1!

DNA 0˔Üŷĥ ACCTTGGGTTCCGGAATATC!
!
x

K|0Ćã KHR321_nhr<114gk849_rev1!

DNA 0˔Üŷĥ GAACCAGTCTCTCTCGTGCC!
!
x

K|0Ćã KHR322_nhr<114gk849_int_fwd2!

DNA 0˔Üŷĥ AAGTGACGAAGGAGCAAGCACC!
!
x

K|0Ćã KHR431_nhr<88_tm1033_int_fwd4!

DNA 0˔Üŷĥ TGAGCCAAATCCTAAGCCTACGC!
!
x

K|0Ćã KHR432_nhr<88_tm1033_fwd2!

DNA 0˔Üŷĥ CTCCTTCGACGGTTTATACCTATGG!
!
x

K|0Ćã KHR433_nhr<88_tm1033_rev2!

DNA 0˔Üŷĥ GAAGCTCCCTTCAAGTTTACCTAGG!
!
!
!
!
36

̍2̎ȇļː¯ĵ0ȟȎ˻Ģ/ GFP |QFȟȎ!B$<0x

K|!

nhr-114 0 3’UTR ˻Ģ 1 kbp
x

K|0Ćã KHR316_nhr-114_1K_fwd

DNA 0˔Üŷĥ TCGTAGAATTCCTGCTCACAAATTGTGAAGTG

x

K|0Ćã KHR317_nhr-114_1K_rev

DNA 0˔Üŷĥ CGAAAGGGCCCGAATCCTAAAATTGGCCTG

nhr-114 0ǧ 4 kbp FĈ; 6.6 kbp 0 PCR v
x

Vj

K|0Ćã KHR318_4K+nhr-114_fwd

DNA 0˔Üŷĥ !
ATGCCTGCAGGTCGACCAGATAAATCAAGTGATTTCCC

x

K|0Ćã KHR319_4K+nhr-114_rev

DNA 0˔Üŷĥ CCGGGGATCCTCAATTTGTGAGCAACAACGCTCG

nhr-88 0 3’UTR ˻Ģ 1 kbp
x

K|0Ćã KHR364_nhr-88_UTR_EcoRI_fwd primer

DNA 0˔Üŷĥ !
AGCATTCGTAGAATTCTCATCGTTTTTTTTGTTGTTGTG

37x

K|0Ćã KHR365_nhr-88_UTR_SpeI_rev primer

DNA 0˔Üŷĥ CGTACGGCCGACTAGTACAAGAGAAACGGGG

nhr-88 0ǧ 4 kbp FĈ; 6.7 kbp v
xVj

K|0Ćã KHR414_nhr88a_Inf_fwd

DNA 0˔Üŷĥ !
ATAAGCTTGCATGCCTGCAGGTTTCCAGGTTCCAACGCCC)x

K|0Ćã KHR415_nhr88a_Inf_rev

DNA 0˔Üŷĥ !
TTTGGCCAATCCCGGGGATCCTACGCGTTTCAGTGTCCGAAAGGG!
!
̍3̎Q]L~K^V/ȓBɈĵȇțȡ gsp%4 _Sɉɒ0·ʈ
0$<0x K|!
spe-11 0xd˻ĢFĈ; 294 bp v
xVj

K|0Ćã KHR848_spe-11promoter_SbfI_fwd

DNA 0˔Üŷĥ CCCCCCTGCAGGGTCGACAGAACATTTTTCCGx

K|0Ćã KHR849_spe-11promoter_XmaI_rev

DNA 0˔Üŷĥ TCCCCCCGGGTTTATCTAGTCGGTTTGCG

38

gsp-4 0 cDNA ˻ĢFĈ; 936 bp v
xVj

K|0Ćã KHR852_gsp-4cDNA_XmaI_fwd2

DNA 0˔Üŷĥ TCCCCCCGGGATGACAGCGACAATCGATGTCGx

K|0Ćã KHR853_gsp-4cDNA_KpnI_rev2

DNA 0˔Üŷĥ GGGGTACCCTATCCTTTACGAAGAGACTTCGG!
̍4̎ļ˛ȡ PCR ʕǁ/ȓBȇļː¯ĵǉÙȓx
gpa%3 ǉÙȓx
x

K|!

K|!

K|0Ćã KHR634_gpa-3_fwd_RT-PCR

DNA 0˔Üŷĥ GCCAATCTGCAGAGGACAAA

x

K|0Ćã KHR635_gpa-3_rev_RT-PCR

DNA 0˔Üŷĥ GCAGTAAATCCGTGATCGTGA

gsp%4 ǉÙȓxxK|!

K|0Ćã KHR638_gsp-4_fwd_RT-PCR

DNA 0˔Üŷĥ GCAACCGTCGTTACAAGTCG

x

K|0Ćã KHR639_gsp-4_rev_RT-PCR

DNA 0˔Üŷĥ GTTTGGAGGTGTGGGGAGAG

39

odr%1 ǉÙȓxxK|!

K|0Ćã KHR724_odr-1_fwd2_Q-PCR

DNA 0˔Üŷĥ GCGAAGACCCCTACCATTTA

x

K|0Ćã KHR725_odr-1_rev3_Q-PCR

DNA 0˔Üŷĥ CGCTGGCAACATTTCATTTA
KHR724 , KHR725 1ʤƤ?Aşȓ(Juang et al., 2013)!
!
!
PCR!solition ,[|[KU0ǰśxV~!

(1) ȬȺľ+Ɉʈ$ r!taq {bFȓ$! PCR!

1!]l 0 10r!taq!buffer 0.6!]l 0 MgCl2!(25!mM) 0.5!]l 0 dNTPs!(2.5!mM)
K|Fă 10!]M + 0.5!]lȤȡ0ː¯ĵFǉÙB$<0x( 0.25!]l

0 r!taq 5.65!]l! 0 dH2O F 200!]l fw+?ǬĄ hxj,.B DNA F 1!]l ç$ Ȥȡ0ː¯ĵ˙ʉʷȶF*BȮʞFʃ
ĦĄ1vOkx

K|,q_x

K|0˛ȿ.B?

/ 0.5!]l 0.25!]l 0.25!]l ,$ [|[KU9

94 F 2 Ú"0ŧ 94 F 15 ȵ

+F 1 [KU, 42 ėɠAʾ
!
!
40

0ǰśxV

58 F 30 ȵ

15+ÀĶ$~1

72 F 1 Ú9

(2) ! Ex!taq! Fȓ$ PCR!
1!]l 0 10Ex!taq!buffer 0.5!]l 0 dNTPs!(2.5!mM) Ȥȡ0ː¯ĵFǉÙ
B$<0vOkx

K|,q_x

K|Fă 10! ]M + 0.5! ]l( 0.1!]l 0 Ex!taq 6.4!]l! 0 dH2O F 200!]l fw+?ǬĄ hxj,.B DNA F 1!]l ç$ [|[KU~1 9

0ǰśxV

94 F 2 Ú "0ŧ 94 F 15 ȵ 58 F 30 ȵ 72 F 1

Ú9+F 1 [KU, 42 ėɠAʾ

15+ÀĶ$!
(3) KOD<plus<Neo Fȓ$ PCR!
1!]l 0 10KOD<plus<Neo!bufferFǉÙB$<0vOkx+ 0.3!]l0.1!]l 0 dNTPs!(2!mM)
K|,q_x

Ȥȡ0ː¯ĵ

K|Fă 10! ]M( 0.2!]l 0 KOD<plus<Neo 6.2!]l! 0 dH2O F 200!]l fw+?ǬĄ hxj,.B DNA F 1!]l ç$ [|[KU0ǰśxV~1 9

94 F 2 Ú "0ŧ 98 F 10 ȵ 62 F

30 ȵ 68 F 30 ȵ9+F 1 [KU, 42 ėɠAʾ

15+ÀĶ$!
(4) LA!taq!PCR!
1!]l 0 10LA!buffer 0.8!]l 0 dNTPs!(2.5!mM) 0.7!]l 0 MgCl2!(25!mM)Ȥȡ0ː¯ĵFǉÙB$<0vOkx

K|,q_xFă 10!]M + 0.5!]l

5.9!]l! 0 dH2O F 200!]l f( 0.2!]l 0 LA!taq

K|w+?ǬĄ hxj,.B DNA F 1!]l ç$ [|[41KU0ǰśxV

60 F 30 ȵ

~1

9

94 F 1 Ú"0ŧ 94 F 30 ȵ

72 F 1 Ú9+F 1 [KU, 42 ėɠAʾ@/72 + 20 Úɡ$ŧ 15+ÀĶ$!
!
á˥˖ɋ(RE)Øȏ!
¸ȓBá˥˖ɋ/ˍ$ 1!]l 0 NEB!buffer 9$1 Cut!Smart 0.2!]l 0á˥˖ɋ 3.8!]l 0 dH2O F 200!]l fw+?ǬĄ 5!]l 0 PCR ȒȆF
ç$ "0ŧ 37! + 12 Ʋˣª˲ɡ$

!

!
!
ɒʗʕǁ
Ě0Ï*0ʡŏ1ǎǴʡŏ(SEM)FȯɈĵ/țŒF=( gsp-4 ĭț´,

˚ȑǄ0˩,0ƎĄE!/?BɈĵňÎĽ̅(Ě 6),ɷɋ\ȂŇ/?Bɍ ɧ ȭ Ī Ľ ̅ ( Ě 29) ª Į 0 Ě  0 Ï * 0 ɒ ʗ ʕ ǁ / 1  Ë ˔ ɡ Ú ơ Ú ǁ(ANOVA)Fʃ ʉƢǘʹ0$<engjǉļ+Þļ$ ¢ŋ/?BɈĵ0
Ȝ±ėūĽ̅,ɷɋ\/?BɍɧȭĪĽ̅1vJg]0ȥƑȮȍǉ
ļFʃ'$ Ě0]VI_d_U(*),ewI_d_U(**)1"C#C p Ä 0.05 ƹǳ, 0.01 ƹǳFȯ "C#C0Ľ̅/ȓ$ǉļ1ˊú0ʤƤFûɢ/ʃ'$(Ohta, Ujisawa et al., 2014)

42

GFP /?B nhr ː¯ĵ0ȟȎʕǁ nhr-114 0 3’UTR ˻Ģ 1 kbp 1˚ȑǄ N2 0Xo~@ KHR316 , KHR317x

K|+ĩœC$ nhr-114 0 3’UTR ˻Ģ 1 kbp 0 PCR v

1 EcoRI , BanII 0[KjF¸ pPD95.75 /ƊÎ$FĈ; 6.6 kbp 0 PCR v
KHR319 xVjnhr-114 0ǧ 4 kbp

Vj1˚ȑǄ N2 0Xo~@ KHR318 ,K|+ĩœ$

nhr-114 0xdFĈ; 6.6 kbp ,ː¯ĵ˻Ģ1 SbfI , BamHI [KjF¸ 3’UTR ˻Ģ 1 kbp FƊÎ$ pPD95.75/ƊÎ$ nhr-88 0 3’UTR ˻Ģ 1 kbp 1˚ȑǄ N2 0Xo~@ KHR364 , KHR365x

K|+ĩœ$

nhr-88 0 3’UTR ˻Ģ 1 kbp 0 PCR v

InFusion(Clontech)Ǣ/?A

Vj1

EcoRI , BanII +ÛƦC$ pPD95.75 /ƊÎ$ nhr-88 0ǧ 4 kbp FĈ; 6.7 kbp v

Vj, nhr-88 ː¯ĵ0Ⱦ 3 K

j9+0˓Ú˻Ģ1˚ȑǄ N2 0Xo~@ KHR414 , KHR415 x|+ĩœ$

nhr-88 x    d  F Ĉ ; 6.7 kbp v

K

Vj1

InFusion(Clontech)Ǣ/?A SbfI , XmaI +ÛƦC nhr-88 3’UTR ˻Ģ 1 kbp
FƊÎ$ pPD95.75 /ƊÎC$

³ǰɣųh_j ³ǰɣųh_j/1

÷FȒ:Ĵ<$˸0Ƨ̈.žɿ 70

150 ï7-0'$xjF¸'$ Ƨ̈.˫˩ą´0žɿ 2 ïF İɭɻ OP50 ħŐC$əɿģ̂ģğ(NGM)43

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/ 20 Ú "0ŧ 20ÕɽStngj/ 48 Ʋˣ˲ɡ$(CRB-41A Hitachi,
Japan)

žˡ˅ś0ț.Bĭț´F¸ĦĄ1* Yj+ʒ/÷FȒ9!BƲˣF@

B˚ȑǄ,ąƲ/ 2/˲ɡ$ daf-2 ĭț1eŢžFşʱ0+ Yj+B˚ȑǄ,ɘ/÷@ L4 Řɿ9+15+̀ɦ Ƴ 12 Ʋˣª 20=1 25+̀ɦ*@Yk]
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KSyjxjF 15/ȶƳȑĶÂ´Ƣ,ǔ¡Â´ƢFQLj$

nhr-88; odr-1 ˙ĭț´Fʕǁ$ƲƸ1 odr-1 ĭț´0ʅȎġ˳Œ/³'$ C/1ǲ
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"0$<

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Vj1˚ȑǄ N2 0Xo~@ KHR848 , KHR849 x

K|+ĩœ$spe-11 xd˻Ģ 294 bp 0 PCR v Vj1 SbfI , KpnI 0[KjF¸ pPD49.26 /ƊÎ$ gsp-4 0 cDNA ˻ĢFĈ; 936 bp 0 PCR v
44Vj1 ˚ȑǄ N2 @ƏÿC$ RNA FˁʷÔ$ cDNA @ KHR852, KHR853 x

K|+ĩœ$ gsp-4 0 cDNA ˻Ģ 936 bp 0 PCR v

Vj1 XmaI , KpnI 0[KjF¸ spe-11 xd˻Ģ 294 bp FƊÎ
$ pPD49.26 /ƊÎ$!

Q]L~K^V/?BɍɧÒQ]L~Ǽś0Ǳļ Q]L~K^V1ˊú/ʃEC$Ľ̅Fûɢ/*ʃ'$(Kuhara
et al., 2011; Ohta, Ujisawa et al., 2014)Ľ̅/1ǰśĀŁm+B

ASJ ȇțȡ/ȟȎ!B$<0xd0 trx-1p / Q]L~KiJ
Wd+

BKNQPFYkB˔ÜFɞ%ɏ:Ƶː¯ĵ

(trx-1p::yc3.60 (pTOM13))FňÎ$Â´F¸'$ QP1 N ƺ/ CFP
C ƺ/ YFP F=& ĳ/Q]L~ɑĄ[KjF=(S

drU+

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0éʱÍF

*B,

̋ɷ0ʀÍFȟB

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B ASJ ȇțȡ/QPFȟȎ$Â´1 2̌(w/v)IRrgk0/ƑȨä(ðȞȓIIvH A, ǀĄžǄŞ®ȱ)Fȓ*ěļ

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Ĉ;K^MU]ǵǪFȑǖɮ/ǤÎB,+ʃEC$(Mello et al.,
1991)

ƻȬȺ+1

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!

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ɭɻ>Řɿ.-Fǥǧ$

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100 ng 0ɖ RNA , Low Input Quick Amp Labeling Kit (Agilent; 5190-2305)
F¸

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elegans 0 RNA

Kw(Fraser et al., 2000)

ſ

Dr. Ahringer /?'*ȮȼC$ C.

F Source BioScience @ʯÎ
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B AKT SlbFYk*BÐȀǽ˽ŬˠʕǁFȓ$ʀÍ0ʓń _KkR _/·ž$ 2% (w/v)IR_rgk/ 10 ]l 0 100 mM

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nhr-114 ĭț´F"C#C 20+̀ɦ$Â´F¸ȓ$ăɉɒ0žɿF

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50

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51

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52

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80

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Sonoda S., Ohta, A., Maruo, A., Ujisawa T., Kuhara, A.
Sperm affects head sensory neuron in temperature tolerance of Caenorhabditis elegans
Cell Reports, 16, 1, 56–65, 2016

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Ohta A., Ujisawa T., Sonoda S., Kuhara A.
Light and pheromone-sensing neuron regulates cold through insulin signaling in C. elegans
Nature commun, 5: 4412, 1-12, 2014

Ujisawa T, Ohta A, Okahata M, Sonoda S, Kuhara A.
Cold tolerance assay for studying cultivation-temperature-dependent cold habituation in C. elegans
Protocol Exchange, doi:10.1038/protex.2014.032, 2014

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