Effect of RNAi on expression of ecdysis-related genes in silk glands of the silkworm, Bombyx mori

2011·2.1 MB·

by  Qing-Rong, Li

Fffect of RNAi on expression of ecdysis-related genes in
silk glands of the silkworm, Bombyx mori
1 Qing-Rong!??, DENG Xiaw-Juui?, YANG Wan-Ving?, HUANG Zhi-l
FEMG GEL? GAO Yang?*

1. yeti a Apt Reman nine, amg Aone of Asin tS
2, Les fl, Solent Biased Behar, Cle aa Sete, Sach Oa Ags avery.
aang 50612, ius Cuateg Pei her L Anal of Bota he Pl even,
Sell of Lie Sta, Seok Cb Sal Laie, Gast, SIMU.

Abweraet; Tr inst, bse signal Ieaalaton pathos catia I tale 9 so in
srl waters erp C8189, wal rr BIC} al ETS ae Udievl ue the ety
tos Tatars Tv sual tle Tine aeae ativan Facts Laker, ue
ficaded [Ae dels} of ose genoa 0 Boner mari were erlkcezed da site ad “aes jato We
seandseng inves. benctpo changes were abcd ond the exeeion ef cose nd aelated gouce ere
Saloed hy qunrctmire reletine UIC qHeH"», lagmahennees ppm an elonion, devant ng nd
Aesth a poe and wake nie siete tie inerleneron of Bask BeaB © Bo IH. Toe PCR
tro eee ths oun son af RinFaR sesame ihe RMA. Ieerk vf BuBPAA. R748,
BuAHEA wed BefsPTZED. Pac henna BasBP64 wane te rKNA lel of Hiubsdl Ban
DnB TAL. BIE sal BeyBeTZ-8T. walle patil eave o Hat sera a 6 sipoicact deeeaie
fn te exproson lvls of sl tested gence ewopt fr BaP hese rsuke opliod thet Bmt-C may act
ft the ener stage in ovdesens gon pathway and regulate the expiosoa of ther mirseiptian fete
frves te walle eshsns inenplor gone Bene fr tse loin
Rey words Braise ovsy BVA interns rng cata phy wesaneg tmp Cas

ar

fehdeal Aramis tor een Hoult? sul
EinE74A peosedce thet of the ehor cedssitclated
leonneiption factor gute BeaHR3 { Fystathing

1 INTRODUI

TON

Te nore s apaptoni a the ogre aed
iui ach a pas yal, alsa gla
fat body, st iniisted yp odveteeide nang
uaunvnphaie (Dai wal Gilbert, 1997; Aller
fe, DIM, Sacfawarn e201). The ane
relates tauscepton fentargonos B93. LR-C, E74
fan BFTZET rogeluts. the empwoesion of the
cgopleninaea epee gee ale sepa
lates genie (lege) in the aslirarg_ land i
Drosophila melanogaster {Dacbrecle:. 2003; Harti
sd Peli in, S004 J ev the Tak Ilya Dh
ruta oejeour iia sabes. Unvesh
PIR and Tor sigzal wancducien pathrays. an
coals roca sige neler Ths aut
inerse hy thisrgulling the PBK pthesy
(Tusen ef ., 2004: Seott e af., 2004).

Toth ei glad of sna expos 9° the
fala payor sete Hct alles ee

ean rem
bata ale a
RAL secre oP 1898. AGEL A Rapes A

(a Sech AOD HALT SAEUN AAR Fama RAL
PL, Ho EERE ARIEL

sh MOT}, Hatin ae aly
MaPTETL "(Som ot al. 19943
[Seki of KT Re
CSmesers aay 202) ificatig thal er
DraDR-C upd BEPEL mvpbt bo et the up-eteam a
The arse signal pbisy. Herecer, Taller
seein ue eel rennin Ue rai li
woug the culsnozeluted rseopters unt
Trans iption atin aml their ile aw the
retamapais in mori 14 ra. 2040)

Tn thie vue, offers of NAL of the” genes,
Pookie, BB 748 and BBA, a he wei
of the dysisawlated tameription feet genes.
wuchwivg BARS, ElR®9, BrPTZ Fl
FuKTHE oral ob I5R, were incligall onl the
pei atop tin the vaqateou of ss
jenes ad phenotype elunyge daring te
Fnspliss lar ov adaltxa dsse

sigue)
Bed creed aa can
ate

2. MATERIALS AND METHODS

2.1 tnwwete

Loew of 8 rset Cslesin 9852 gure fl wil
aruTbiay Tees and teares a 25°C anu 70% race
Thai inthe Taesatny esitions onl adults or
10 the stages use for experinent
2.2 RNA laterforence of BmECR. BmBR-C wu
BmET#A

TRINA oa isola wing the THzal Reagent

Ue meter” =

Towiayn, Gala, Califia, USA} ard wee
Si spite the ist steal DRA ang the
HDNA Synthesis Rit CTOVORO Ca. Os,
eye} Ta price sbKNA Tor TENA; PCIE
Tesermuls ene ample I reverse Iams
rirg the eomasponding LIA. ae tanplate. the
primera containing tho "T? promiter eaaonce of
Gindek, DmDR and BnETAA, aes ahown in Table
Le aaRVAs of DoufeR, DDR ond BoE 7A wore

shesived using T? TINA polyenerase fo the Ainbnon
MRIeAverigt RNAI Ri Aiton USA) eng
Ube rmasucturer» nseation,

Laas st 1 d alter weaudering ess ned for
AARNE ier tine. hur snr ash yo
injected ito tho 7 segment of ae abdoucn of the
Tareas uring teint Vase wn ape
rink the Inge ery Mi Fan npr gps
Feludig ioe ACD, endl? ON. Wed
AARNE wl RNA elainn laos Cueva rtd
vwee pefonnod, 20 Teast 108 Ina were fected for
feich aca. Aller AANA init
rman dod iets con vol goepe rary
ral rls PC svt (40 Fare pe
Zang) ond ese a alerts £8695 lame
bee subse
2.3. Realtime PCR detection
Fr ouch injection {Bou daRNA. Rou C
AGRNA, RAZ SANA srt BA ni fll
Fay La
gis ie sping.
ul rons leg al Ui pte ik
alace woe disetelw qPCTE a1 pa pupa
Tree drIIKA injection repeace ote conrad fo
sah of the deta
2.4. Morpbolagcal observation
Fox cach RNAi menmert { DoufeR dsRNA,
BoBR LRN, BvE?49 URW uel ROA elution

te la ee

baer? , 60 28 lyme eo wally dive nts
thee nil gouge tempt olagal beri
Deceagenental almuewlitine. ievlaing Tile tn

pees avenge, secant wing sul lh

2.8 Analyse of quantitative real-time Pt
(aper’

Thal RNA as ional ung Teoh Reagent
vecomiug to the mumfiuror "5 mstcton

Une, Conlebul. Calinana, USA} anal wwe
to sytbene the Sst stancleTINA aoeurding to the
eDSA Senthoris Kit (TOVOBO Co., Oaska,
Iau. De pines una ir GPCI we ses ra
Ineal onthe saysemes of Ure voresen ding beget

or Basic, the primer nae ign! al
{or he wong ai thy Us ies RR
F, BnEeR-2-1 oud BrBeR-2-2. Foe BBR €, te
Phunere were desimcd based on the conserved tez0n
ofthe toe infers BBR-C-2!. HiE-€-22 and
wie 74, vow the. mancerptin Tater sone
BohF4B, BoPTEAPE, HolKI9, Bb 75H,
Biola, “oe prinars worm slesignel baw on the
sped rion al thee gens Ravi? ur el

as tho gent, Phin stncucos ane shown in Tol
2. qPCR war perlomed weconding te SYER-Cieen
Euocescont tclaure queuttatse approaches ; Livak
sant Echnittgon, 2001) "nar AMET appara

Tlie ialependhal reyes wr ronda ar el

1 Ue dpi

2.6 Data analyte
The sha analysis he ste ein a
sing SPSS statin! anal atbenny Significance

test including the pheuotspe ehunges asl ebouges ua
cspuseson of acted gens bervcea injostion ea
ee

fal BNA cltion tf rap Covering cml
roi), cela il Sl

“Table 1_Paimersemnenes wed n PCR

sree gg mee

3 RESULTS

3.1 fect of RIVAL on expresilon of the target
ees BuER, BuaPT4A and BuBieL

Temrreson of Umer, fex744 al Pet
syppeared cure hum thate othe trnecriptioa fiero
ies A FIRGA, FooPRS9, Roysh TZ FY e748
HBTSH sl BouRPS(0 an Mose ies gees igh
Ie at the pnamean of the seve. aigel
‘wonsdneticn pathway tor progoumed ell. ceuth
CPCIV} etation iv the sill gland C15 a? al
BOK. Tin asuisine pletler tie iubekereof
BoE, BETA aud ErlRAC inverse the
expression of tho above truecrption titer genes
ning metomarphxis, dBA of Fe genes me
rrtvel inerind ak tvewealerng aro Te
eanka fubrated” that the econesion lorale of
BoB. DnE74l aud BDAC wine xgubcunls
CSM} sesso ler BNAT Irulnents at
soon ly 1K 26, 110% waa 16.46 uf the mark
souuel, ecaposvely (Fig. 1)
2.2. Merpholopleal changes in respuuse to RNA
‘of mbh, RMPTAS and HRB

he hua inert ill ULNA of Bit,
BrsBR-C ox Liab sppeatel to bo ampacbeons i
poporion aud culosion, as compare! tw the coud
imu Anong the 6478 lane injec, 4 =a

2 8

Trae ile pup snialy (Figs 22 3

TT Ginna fe Tas oF IVA ennai

Fae | op arte alts concn af BR, RPL
HBR os yensend y TE w Revre ns
vs ger SIG tr ce wi Sd Sis he eo

sed 3 ~$ ls appeal to Le wieght (or devia
Teng) afr emerges Farm the eons Fig 2
DL PY i wih toate Tlie 3 shows the reine
Ve fit the, BonFaR, BBR ol nT
FNAL oc ts deelopacat lhe wens Boe. “he
tans se type tinge in Bk oo
Cad Unset RNA Ueabene wee 12.256, 8.84
sed 9.9 wenpively CP 20084 al ve ater of

reaglot wee 6.756, 10,08. and 10.7%. exportiely
£20.08 2; the ter of wenaliy wane 28. 0%

13.38 nd 22, 75 _ mopeds {720.05 1, The
sce of abwnncalily in Rnd, Bel aod
RNAI oman weve 48.25%, 20.405 aud
elag sgt bier haa

4.76, ‘sepecdray
Me sons Pet
3.3 Ufoet of RNAI of Bie, Bmat744 and
BinH-C on exprowion of other transcription
Factor genes

lls al Rift, MocP4A aml Attic
RAAF an the espminn af e prlane rere
eww Bakidt, e lher Unio Caron seven

a eee
BoB ETE PE, at 25H sd Bad, al the
sinptenisiaiiue praise Shalt wore
Samined ig, 2. While njeoton of Aaa

TRHINA anal Sittin shew tee BoA
HINA Devel (Figs 1) te earn Ps the
Transeiption factor gona nA7AL, Se 74i,
Hast an High TZAEL rns snificatly
Asasated 1221, DU, wheres He eapacaton of
HOt, HowB S80 UD sty at slaely
stable Towle (Wg. 3. A}. This vert indicated that
‘eked nt beat tho eet up-to of ae a8
iad 741, Sk TAB, Hout and Eph Tz-+7 and
seas csitcal for the initiation of the ontysone signal
cascade. Ursie24 IEMA sietficantiy devveaced the
1 "ENTE to Bone San

saat someon of AueRT#S (ig. 1) alle gia
feypreio’ the exenwion of TinFak, Ba,
BoEF4B, BeR34 sd Ligh TEES CP 0.01)
hw the essing af ReiR7SB, AimeRA nad Rll?
nas slips inevaed (Fg. 3-'). IESAG of Bt
Famed sign ese i Ube meson af al
testo taveripton facta genes except AML (ig
350) (Pe.
oak PIA ie on oor tameription fitor of
cedvsone sigaaling pathway (Eee ea, 1996)
fet i oe nga the tvsertinn ety
cedsaone rooptor Uinall. lowcrex the tonscrpion
sevivily af BinBc ess sigiicanlyssererulted
thor NWA‘ of HHA Cig 3 1). Ths roel
ena I are sie

4 DISCUSSION

4.1 finch, ak-748 and Bra might be
the upstream fuctors of the ‘cedysone signal
(transduction pauhivay in the silk plana

Dmel, EmE74A and LaBare the cance
esprcesion genes 2 the elk gland dung the larval ts
papel tansition in mori (Li ota, 2010) , and it
2 suposed that dey night eat the upstream ofthe
relying saat iting, DCD sing,
the degen silk glans. In this Hy, NAG
ft oly styyresea the eyiesinn'the Lage gene
Fook, Hub T#8 sd BB, la al suppl
the omens te lle sion eo ene

Bink 74, BllR3A, Bll and Doak 75B,

Fig 2 Pl af RNA a Bo EA, Bs, Bk

‘Tale 3 tellunoe of UNAE Mf Ye, AC aed DTA eames a Boe mar

pte and sabe (8)~72 oe een eae" Sg ees Po CS Renee ese vn he ais em
=

ii

Bho RA Serco
ef eedpameaal sa e Kae

indicating tbat theso tho goucszaayfomation at the

tiles lage suing le» PCD tigger by arose. 3
oe are ow, fae pata lene of hone ee go
Jey RAG od Gv th Ll a asin Ca, val
peal sig oro Inve all omg nga tos
Sinile seule wow sojecd i Laatuen
‘Morphy vunges of the aero lk ells
mort wie seoeupanicd ‘by the loeelization an

Be A-BT, iia Be Beck 8
spree ie portato do PCL the anti ee
hands (Gon are Park. 2K 240. The
Segensmn of tho ore all glands Fr lar «0
rye: os sured by atupgey an apni shied
fee aia bo sidysnne signe a late sg
Subst oy watering sye (Li a
vor suggest tht. FicBR, BeoBR €2 aed Bi
fot ae the vpestrcam factors io the ondgsoae sigmal
alta pl ey are ay Ie olen ae ea
of either ent By aeiatig He atopangy-rele:
fetes fer) on sunpliin by adn te apne
Imad vompese game Tow and Tachssekn, 2
Barba, 2003; Bein wal Batoweke, 20045 Lin

TT Ginna fe Tas oF IVA on era a

va 2
Th fe audys che fnjorr wife BY ofan
lati wae we a Uy vob. att Uke ily seal
inypecom be uring KRINA af et ged or of
tuctavorphccie each a5. GH as adders) oontel
Hiner the ssa cbutive Fn ts susly vou
rire baste firmer for Farther wa
4.2 RmHR-C might he regalated nit only hy
the cedysone hut also other hormame or Factors
Hs paral eens oP sla lar NAG
sedwoul the expeseien levels of BmiBell. mE 74d,
Fh F1B, BrcHRAA vel BePTZ FY UFig, 35 C)
Thiel gain thal BoBC? wey Tr on a
age of the walgsiee stale el

the wine
reaulate th: exavarion af ame teeta ths
tale lind drag the tarsiton too lures to pore
ger ea 1983), Hoover, the pil alone
nc olla a th spor a Re
(ig 32 8). Tie we posi hae U1 the exe
sf cdaons and Javeule mann JUN dune ths
pts fv Day 6 Scant ealer ego of pup
Murray oof, C2008) myer thatthe ie ab
vee sud JH inemed vino luring the
fre: Tan D6 3 oa os Dig 9
fiom Day 9 Sth instar to prpation. ceaheone “ol
shure a sherp decline eficr tho titer of IM decreased
rail to pony le lwo, but eve incre
Healy. The Gn nf AG “nj and sanging
Fo aPUCIC atthe saan was Dar 7 Sth tar
sod 1 d post pupatin. reapectivels. whoa the tte
cf codons and JIL roomed Ineb selutves
Therelive, it amy se supped thot JH ould
antago ie the lone of arlzaen, bile ght
frntelie the art oh face SAG
‘spuceio er of Unb.
Zhen e ai. (4998) sepented that JIT rodoced
sspiuttion rasumghisi in
he AAMC eayrossion yamaha
ental bor JUL ia inet (Daleks. 2008) I
fwldition, Kasna vt of. £1993 panpoced at the
teansinption fuer: BRC pla «key role in te
stage speiity of the eile espe i
Disaaphite sal tle adysourieylor yee
romples dave wie, vol sulfcbnt Hee apna
ruction of the cally peimary-rospouco gence, the
por capresnioa cf TRC arocioe wos definitely
Fae Casi ithe emer sulin (Raia
real, N85, Murata at al 2008} om
evita dae see i Bin wiht be
feguated ma ony by the eedsaane but alee br ocr
Teme dato,

tan the

ARE

Momo sexe

ecw

Hossevcr, sume nesta soul oh ae the
soles et ve nn gli of le

q

fave, wey apne he vepnenion Bol7
i "Ra te

ad ORR E cai ae doeonse of the RFR Pet 2. Nae bs 2

fespirssinn, stil eal tbe fanler investigate

hbiTomec armed Peng fctahy te ge Se 4

07 28 adams epi:

init Es Stak 00 S08 Vind ces oe) yoke @ cial deh nthe ibe ae is a

beer -_ Het , abou 22, he ms as ewe
so, Dee A Se 2. teat, Feng Ube 0 Fe at oA

RNA Tit xt RS 2 ARG BE A iA Ae

srs ae te
gaat deumelastecilernsestoneren
Maeno, © smppcrrastere. F bRIoebaBEHTE Meme

WAR ACNE NT CART RTI CRM EAT EE Cee. TERA He
ERAGE, NLA LER lel ERMC ALLHAA TPRIRY GEE AHEM, OFTHE TE
RFRA RATE, ORAL AS MHA CA, HALEY DAMA PHO RAMER,
ewes WMAP, LY SATHANA PIERS INES IES TAR Ma, Be,
gph 4. S205 AM ATS FRA VOGUE PCIE, ENGR HA HLS CORLL HS
HRs aN, CIR. GAG NHL VETTE Ug BAL it) gh lt, 0, Mets
Wager AH Cae ARETE? 60.08, RS Sa AMR Mie a aM, oI AL
Dig ince REAR PDA, HORS or ESI RETA SEARR SE R HM CA
EY aN TATRA 8 15, EIA, BoE CTRAT RES 4 FAP ch ALL
SRANFARARE META.

Bin, RE OU, a ERA, OE

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