Tugas Metil 2 Pembahasan

8/9/2019 Tugas Metil 2 Pembahasan

1/8

TUGAS METODOLOGI PENELITIAN & STATISTIK LANJUT

Prof. Dr. Ir. Simon Bambang Widjanaro

MENENTUKAN TEKNIK MEMBAHAS DATA

“An efficient Agrobacterium-mediated transformation method for the

edible mushroom Hypizgus marmoreus”

O!"# $

ARDHIA DEASY R.D

136100112011001

PASCASARJANA TEKNOLOGI PERTANIAN

BIOTEKNOLOGI PANGAN

UNIVERSITAS BRAWIJAYA

201


2/8

An efficient Agrobacterium-mediated transformation method for the

edible mushroom Hypizgus marmoreus

RESULT

Analysis of the GPD gene and its 5 flaning region

Following the degenerate PCR amplification, fragments of thesame size (550 bp)

were obtained from genomic DN and total RNof !" marmore#s" $#bse%#entl&, the

entire gpd genomic se%#encewas obtained b& 5and '$F PCR (ang et al" *00+),

which incl#des an #pstream region of *+0+ bp and a downstream fragment of -50

bp" .he coding se%#ence of /0/+ bp corresponding to'' amino acids is disr#pted

b& si1 introns" .he intron positionsof gpd gene between fo#r basidiom&cetes and fo#r

ascom&ceteswere anal&zed and the res#lt was shown in Fig" *" 2esides, the protein

was highl& conser3ed compared to 4nown PD proteins withan amino acid identit&

of 67"08 to PD se%#ences of other basidiom&cetes and the ph&logen& of the PD

proteins was shown inFig" /" 9n the gpd promoter, a potential transcription start site

waslocated at 5- bp #pstream of the start codon in a C.rich region, infront of which

a t&pical ..bo1 and fo#r C.bo1es were fo#nd(Fig" ')"

Teni !embahasan data tersebut sesuai dengan teori nomer " #adanya

teori !ustaa dan data !eneliti$% Pustaa dari Wang et al. 2007 memberian

cara untu mem!eroleh data hasil !enelitian !ada !aragra!h tersebut% &adi

setelah teori !ustaa ditulisan' !enulis mulai mema!aran data hasil

!enelitian yang diinter!retasian dengan gambar se!erti !ada gambar ('"' dan

) yang mem!erlihatan !ohon filogenetic' !erbandingan !osisi intron

basidomycetes dan ascomycetes dan !eta seuen D*A H.marmerous.

inter!retasi dengan gambar agar !embaca leboh memahami data hasil

!enelitian yang disa+ian !eneliti%

Agrobacterium-mediated transformation


3/8

9n attempt to de3elop a simple, highl& efficient transformation s&stem for H.

marmoreus, we tested the applicabilit& of the.:. method" .he protoplast, fr#it

bodies and f#ngal m&celi#mwere tested #sing the same 3ector p!mPD" .his

e1periment wascarried o#t in a similar wa& as described b& $hi et al", *0/*" s#spension mi1t#re of recipient materials and bacteria was spread ata ratio of /;/ on

sterile nitrocell#lose membranes on 9: agar plateswith 0"* m: $, and inc#bated at

*5


4/8

>27707 and ?'/0/, and no positi3e colonies were obtainedfor >/ strain (Fig"

5D)" .he optimal concentration of $ for coc#lti3ation was fo#nd to be 0"' m: (Fig"

5)" few transformantswere obtained when coc#lti3ation was cond#cted with 0"05

and0" m: $, b#t no positi3e colonies were obtained in coc#lt#reswitho#t $"comparati3e st#d& was performed b& coc#lti3ation of !"marmore#s with !/05

carr&ing one of the following plasmids;p!mgpd, plPD or p>ePD (Fig" 5F)"

.hese plasmids containa cassette in which hph is #nder the control of different

f#ngal gpd promoters" .he res#lts showed that the highest transformation efficienc&

was obtained #sing plasmid P!mgpd, in which hphis dri3en b& gpd promoter from

!" marmore#s" few transformantswere obtained #sing !/05 carr&ing the other

two plasmids, withsimilar efficiencies" .he hph gene in plPD and p>ePD is

dri3enb& promoters from " l#cid#m and >" edodes, respecti3el&"

2ara membahas data yang disa+ian !eneliti sesuai dengan teori nomer 5

yaitu !eneliti hanya menam!ilan data !enelitiannya sa+a yang di!eruat

dengan gambar dan grafi hasil data !enelitian% Paragra!h ini men+elasan

suhu dan atu o!timal co-ulti3asi' strain A. tumefaciens yang !aling

o!timal untu transformasi serta !lasmid yang digunaan untu membaa

gen insert !ada H.marmerous yang !aling efisien untu transformasi%

2haracteri0ation of re!orter gene integration and e4!ression

.o in3estigate the pattern of integration of the foreign DN fragment, three p#tati3e

transformants were randoml& selected for$o#thern blot anal&sis" s shown in Fig"

-, all three transformantshad the .DN insertion" .he 3ario#s sized DN bands in

the $o#thern blot indicated that the .DN into the genome of !"

marmore#srandoml& was obtained".he transformants obtained from !" marmore#s

were anal&zedfor the presence of egfp=hph transgenes b& PCR and FP

e1pressionb& fl#orescence microscop&" .he presence of f#sion fragment containing

the gpd promoteregfp=hph in the transformants was alsoconfirmed b& PCR anal&sis

(Fig" -2 and C)" .he green fl#orescenceco#ld onl& be detected in the transformants

(Fig" +), s#ggesting thatthe transformants had not onl& integrated egfp b#t also

e1pressedthis gene"

.itotic stability of transformants


5/8

:ost of the transformants of !" marmore#s did not show an&phenot&pic difference

compared to the wildt&pe strain #nder thesame ph&siological conditions" .o

determine whether the transformed DN was stabl& integrated in the genome of !"

marmore#s,70 transformants were grown on PD plates witho#t h&grom&cin2 andreplated for fi3e generations" '7 mitoticall& stable transformants were obtained o#t

of 70 colonies (58), confirming thegenetic stabilit& of the integrated DN in this

f#ng#s" .he res#ltss#ggest that this method can be #sed as a tool to introd#ce

foreignDN into H. marmoreus%

Paragra!h ini +uga sesuai dengan teori nomer 5' dimana !eneliti hanya

mema!aran data hasil !enelitiannya% Peneliti menganalisis hasil transformant

menggunaan Southern blot yang ditun+uan dengan gambar ,A' lalutransformant +uga dianalisis dengan P2R yang ditun+uan !ada gambar ,%

Transformant didetesi +uga dengan green flouresence yang ditun+uan

dengan gambar 6%

Paragra!h edua +uga membahas data !enelitian sa+a sesuai dengan teori

nomer 5 dimana !eneliti menun+uan dari 78 transformant' )7 transformant

stabil mengintegrasian D*A !ada +amur H.marmerous

D9S2USS9/*

9n this st#d&, we obtained the complete gpd gene se%#ence from!" marmore#s" .he

coding region was anal&zed b& comparing thegenomic DN se%#ence with the

cDN se%#ence" 9t matches perfectl& with the cDN clone, s#ggesting this gpd

encodes a f#nctionalprotein" 9n :#cor circinelloides and garic#s bispor#s, which

harbormore than one cop& of gpd se%#ence, onl& one gpd mRN is identified,

indicating that there is onl& one f#nctional gpd (!armsen et al",/66*@ olff and

rna#, *00*)" 9t has been reported that gpd genein higher basidiom&cetes has 5A/0

introns (Bilar# and Bes, *005)".his is also tr#e for the gpd gene in !" marmore#s%

Paragraf ini menganut teori nomer ( yaitu teni membahas data dengan

membandingan data !enelitian dengan data !ada !ustaa% Pustaa Kilaru

and Kes, 200! memberian ru+uan +umlah intron !ada gen g!d di


6/8

basidiomycetes yang aan ditransformasian e 1%marmerous sehingga

mem!eruat menga!a !enelitian menggunaan AT.T method dilauan !ada

+amur +enis ini'

:olec#lar genetic st#dies of !" marmore#s ha3e been limitedd#e to the lac4 of an

efficient gene transfer s&stem" 9n the lastdecade, .:. has been s#ccessf#ll&

applied to different f#ngalspecies, mainl& owing to its efficienc& and technical

simplicit&(odio et al", *007@ :ichielse et al", *005)" .he main obecti3e of thiswor4

was to establish optimal conditions of .:. for !" marmore#s".ransformation

res#lts showed that the choices of recipient materials as well as 3ectors affected

final efficiencies (:i4osch et al", *00/@Cho et al", *00-@ ang et al", *00@ $hi et al",

*0/*)" 9n this wor4, theprotoplast was fo#nd to prod#ce the best res#lts intransformatione1periments as compared to fr#it bodies and f#ngal m&celi#m" 9n

"bispor#s and F" 3el#tipes, transformation efficiencies of single gill tiss#es were

reported to be appro1imatel& 708 and /-8, respecti3el&(Chen et al", *000@ Cho et

al", *00-)" !owe3er, few transformantsco#ld be obtained from gill tiss#es of !"

marmore#s deri3ing fromsmall m#shroom caps that #st de3eloped from primordi#m"

9nthis st#d&, we obtained at least /50 stable !" marmore#s transformants per

/05protoplasts, which is a relati3e high transformationefficienc&"

Teni membahas data !ada !aragfraf ini sesuai dengan teori nomer , dimana

!eneliti membandingan efiseinesi transformasi AT.T method !ada +amur lain

dengan +amur +enis H.marmerous dan mrembutian baha estabilan

transformasi !ada H.marmerous menca!ai :5; dibandingan +amur lain yang

hanya 78-,8; !ada !enelitian Chen et al., 2000; Cho et al., 2006

?ario#s grobacteri#m strains ha3e been tested, and it wasfo#nd that the

grobacteri#m strains !/05 prod#ced the highest transformation fre%#enc& in !"

marmore#s" .ransformants werealso obtained b& #sing the grobacteri#m strains

>27707 and?'/0/, b#t no transformants were fo#nd with >/" Different

grobacteri#m strains ha3e different transformation efficienc&according to different

f#ngal strains (:ichielse et al" *005)" .hreest#dies showed that the #sage of

grobacteri#m strains deri3edfrom the s#per3ir#lent */ strain (high le3el of 3ir


7/8

gene e1pression) res#lted in higher transformation fre%#encies in $"

cere3isiae,:onasc#s p#rp#re#s, and the Eom&cete Ph&tophthora

infestans,compared with .:. #sing grobacteri#m strain >2//00 (Campo&et al"

*00'@ Piers et al" /66-@ ?in and o3ers *00')" 9n anotherst#d&, it was also fo#ndthat the s#per3ir#lent grobacteri#m strain*/ and its deri3ati3e (>/) were

more efficient in transferring.DN to Cr&phonectria parasitica than the

grobacteri#m strain>27707 (Par4 and Bim *007)" !owe3er, s&stemic

comparisons ofthese strains in relation to transformation fre%#encies ha3e notbeen

performed, ma4ing it diffic#lt to determine which strain is thebest to #se (:ichielse et

al", *005)" From these res#lts, we clearl&4now that the choice of grobacteri#m

strain for .:. of f#ngi canha3e an effect on transformation efficienc&%

Paragra!h ini sesuai dengan teori membahas data !ada nomer ( yang

menyataan baha Agrobacterium strain "HA10! !ada hasil !enelitian

memilii efisiensi tranformasi !aling tinggi !ada +amur dibanding strain lain%

1al ini diduung oleh !ustaa Michielse et al. 2005

$e3eral st#dies ha3e shown that each f#ng#s has a #ni%#e combination of

temperat#re and coc#lti3ation d#ration to obtain ama1im#m n#mber of

transformants (Combier et al", *00'@ :e&eret al", *00'@ Rolland et al", *00'@

ardiner and !owlett, *007@:ichielse et al", *007b@ $hi et al", *0/*)" 9n this st#d&,

the mosts#itable temperat#re is *-


8/8

!usataa #om$ier et al., 200%& 'eyeret al., 200%& (olland et al., 200%& )ardiner

and Ho*lett, 200+&'ichielse et al., 200+$& Shi et al., 2012. Data !enelitian telah

ditun+uan dengan grafi !ada gambar ,%

.his is the first time the egfp gene is e1pressed in f#sion withhph in the

basidiom&ceto#s m#shroom !" marmore#s, #nder thecontrol of !" marmore#s gpd

promoter" 9n " l#cid#m, the constr#ct carr&ing the promoterof itself res#lted in the

highest transformation efficienc& ($hi et al",*0/*)" 9n o#r st#d&, promoter from !"

marmore#s had the highest transformation efficienc& and promoters from two

homologo#sspecies had lower transformation efficienc& than !" marmore#s" :itotic

stabilit& anal&sis showed that o3er 58 of the transformants tested remained

mitoticall& stable e3en after s#bc#lt#re inthe absence of h&grom&cin 2" .he antibioticresistance conferred b&the hph gene was still maintained" $o#thern blot and PCR

anal&sisres#lts demonstrated that .DN was integrated into the chromosome of !"

marmore#s"9n o#r another st#d&, fo#r de3elopmental stages transcriptomesof !"

marmore#s ha3e been done and the res#lts s#ggested thatsome genes pla&ed

important roles in the fr#it bod& formation of!" marmore#s, s#ch as laccase genes,

NDP! o1idase genes and thegenes in3ol3ed in :PB signaling pathwa& (Cano

Doming#ez et al",*00@ De Pa#la et al", *00@ >angfelder et al", /66@ :# et al",

*0/')"s the transformation method was constr#cted, these genes ma&be as o#r

target genes and their f#nctions in the de3elopmentalprocess of !" marmore#s co#ld

be st#died b& this method in f#t#rest#d&"

Paragraph tera4hir ini memberi4an bahasan data mengan#t teori nomer / dimana

data &ang dihasil4an diper4#at dengan p#sta4a &ang mend#4#ng" :en#r#t ($hi et

al",*0/* !"marmero#s memili4i promotor paling 4#at sehingga 4eberhasilan

transformasi mencapai 58" $elain it# CanoDoming#ez et al",*00@ De Pa#la et al",

*00@ >angfelder et al", /66@ :# et al", *0/' men&ata4an bn&a4 fa4tor &ang

mempengar#hi 4eberhasilan transformasi dari segi 4ondisi mole4#leram#r"

Tugas Metil 2 Pembahasan

Documents

Transcript of Tugas Metil 2 Pembahasan