TUGAS METODOLOGI PENELITIAN & STATISTIK LANJUT

Prof. Dr. Ir. Simon Bambang Widjanarko

MENENTUKAN TEKNIK MEMBAHAS DATA
An efficient Agrobacterium-mediated transformation method for the
edible mushroom Hypizgus marmoreus

Oleh :
ARDHIA DEASY R.D
136100112011001
PASCASARJANA TEKNOLOGI PERTANIAN
BIOTEKNOLOGI PANGAN
UNIVERSITAS BRAWIJAYA
2014

An efficient Agrobacterium-mediated transformation method for the

edible mushroom Hypizgus marmoreus

RESULT
Analysis of the GPD gene and its 5 flanking region
Following the degenerate PCR amplification, fragments of thesame size (550 bp)
were obtained from genomic DNA and total RNAof H. marmoreus. Subsequently, the
entire gpd genomic sequencewas obtained by 5and 3SEFA PCR (Wang et al. 2007),
which includes an upstream region of 2707 bp and a downstream frag-ment of 650
bp. The coding sequence of 1017 bp corresponding to338 amino acids is disrupted
by six introns. The intron positionsof gpd gene between four basidiomycetes and four
ascomyceteswere analyzed and the result was shown in Fig. 2. Besides, the pro-tein
was highly conserved compared to known GPD proteins withan amino acid identity
of 94.0% to GPD sequences of other basid-iomycetes and the phylogeny of the GPD
proteins was shown inFig. 1. In the gpd promoter, a potential transcription start site
waslocated at 56 bp upstream of the start codon in a CT-rich region, infront of which
a typical TATA-box and four CAAT-boxes were found(Fig. 3).
Teknik pembahasan data tersebut sesuai dengan teori nomer 2 (adanya
teori pustaka dan data peneliti). Pustaka dari Wang et al. 2007 memberikan
cara untuk memperoleh data hasil penelitian pada paragraph tersebut. Jadi
setelah teori pustaka dituliskan, penulis mulai memaparkan data hasil
penelitian yang diinterpretasikan dengan gambar seperti pada gambar 1,2, dan
3 yang memperlihatkan pohon filogenetic, perbandingan posisi intron
basidomycetes dan ascomycetes dan peta sekuen DNA H.marmerous.
interpretasi dengan gambar agar pembaca leboh memahami data hasil
penelitian yang disajikan peneliti.

Agrobacterium-mediated transformation

In attempt to develop a simple, highly efficient transforma-tion system for H.

marmoreus, we tested the applicability of theATMT method. The protoplast, fruit
bodies and fungal myceliumwere tested using the same vector pHm-GPD. This
experiment wascarried out in a similar way as described by Shi et al., 2012. A suspension mixture of recipient materials and bacteria was spread ata ratio of 1:1 on
sterile nitrocellulose membranes on IM agar plateswith 0.2 mM AS, and incubated at
25C. After 36 h, the membranes were transferred to PDA agar plates containing 200
g/mL cefo-taxime and 20 g/mL hygromycin B and incubated at 25C for twoweeks. A
few fungal colonies were obtained on culture plates. Thetransformation efficiency
was higher with protoplast than othermaterials, demonstrating that protoplast was
better for H. mar-moreus transformation using the ATMT method.
Teknik pembahasan data pada paragraph tersebut sesuai dengan teori nomer
6 (adanya data peneliti dengan data orang lain). Paragraph ini menjelaskan
bahwa metode transformasi dengan ATMT method (Agrobacterium-mediated
transformation) telah lebih dulu dilakukan Shi et al., 2012 pada jamur dan
terbukti berhasil. Oleh karena itu peneliti melakukan transfer gen dengan
ATMT method sesuai tahapan penelitian yang lebih dulu dilakukan oleh Shi et
al., 2012
Optimization of conditions for ATMT of H. marmoreus
Based on previous studies, 22, 24, 26 and 28C were chosen tooptimize the cocultivation temperature. The number of colonieswas found to be the highest at 26C,
which is the optimal culti-vation temperature for both EHA105 and H. marmoreus
(Fig. 5A).When the duration of co-cultivation was elongated, the transfor-mants were
decreased (Fig. 5B). This may be due to the over-growthof the bacteria after a longer
duration of co-cultivation inhibitedthe fungal growth. Therefore 24 h was chosen as
the

duration

of

co-cultivation

for

subsequent

assays.

The

number

of

coloniesincreased with the increase of bacteria-to-protoplast ratio andreached the

peak at a ratio of 1000:1 (Fig. 5C). The number ofcolonies obtained at this ratio was
twofold higher than the numberof colonies obtained at a bacteria-to-protoplast ratio
of

1:1.

Four

A.tumefaciens

strains

were

evaluated

and

the

highest

transformantswere obtained by using the A. tumefaciens strain EHA105, followedby

LBA4404 and GV3101, and no positive colonies were obtainedfor AGL-1 strain (Fig.
5D). The optimal concentration of AS for co-cultivation was found to be 0.3 mM (Fig.
5E). A few transformantswere obtained when co-cultivation was conducted with 0.05
and0.8 mM AS, but no positive colonies were obtained in co-cultureswithout AS.A
comparative study was performed by co-cultivation of H.marmoreus with EHA105
carrying one of the following plasmids:pHm-gpd, pGl-GPD or pLe-GPD (Fig. 5F).
These plasmids containa cassette in which hph is under the control of different
fungal gpd promoters. The results showed that the highest transforma-tion efficiency
was obtained using plasmid PHm-gpd, in which hphis driven by gpd promoter from
H. marmoreus. A few transformantswere obtained using EHA105 carrying the other
two plasmids, withsimilar efficiencies. The hph gene in pGl-GPD and pLe-GPD is
drivenby promoters from G. lucidum and L. edodes, respectively.
Cara membahas

data yang disajikan peneliti sesuai dengan teori nomer 5

yaitu peneliti hanya menampilkan data penelitiannya saja yang diperkuat

dengan gambar dan grafik hasil data penelitian. Paragraph ini menjelaskan
suhu dan waktu optimal co-kultivasi, strain

A. tumefaciens yang paling

optimal untuk transformasi serta plasmid yang digunakan untuk membawa

gen insert pada H.marmerous yang paling efisien untuk transformasi.
Characterization of reporter gene integration and expression
To investigate the pattern of integration of the foreign DNA frag-ment, three putative
transformants were randomly selected forSouthern blot analysis. As shown in Fig.
6A, all three transformantshad the T-DNA insertion. The various sized DNA bands in
the

South-ern

blot

indicated

that

the

T-DNA

into

the

genome

of

H.

marmoreusrandomly was obtained.The transformants obtained from H. marmoreus

were analyzedfor the presence of egfp/hph transgenes by PCR and EGFP
expressionby fluorescence microscopy. The presence of fusion fragment con-taining
the gpd promoter-egfp/hph in the transformants was alsoconfirmed by PCR analysis
(Fig. 6B and C). The green fluorescencecould only be detected in the transformants
(Fig. 7), suggesting thatthe transformants had not only integrated egfp but also
expressedthis gene.
Mitotic stability of transformants

Most of the transformants of H. marmoreus did not show anyphenotypic difference

compared to the wild-type strain under thesame physiological conditions. To
determine whether the trans-formed DNA was stably integrated in the genome of H.
marmoreus,40 transformants were grown on PDA plates without hygromycinB and
replated for five generations. 34 mitotically stable trans-formants were obtained out
of 40 colonies (85%), confirming thegenetic stability of the integrated DNA in this
fungus. The resultssuggest that this method can be used as a tool to introduce
foreignDNA into H. marmoreus.
Paragraph ini juga sesuai dengan teori nomer 5, dimana peneliti hanya
memaparkan data hasil penelitiannya. Peneliti menganalisis hasil transformant
menggunakan Southern blot yang ditunjukkan dengan gambar 6A, lalu
transformant juga dianalisis dengan PCR yang ditunjukkan pada gambar 6B.
Transformant dideteksi juga dengan green flouresence yang ditunjukkan
dengan gambar 7.
Paragraph kedua juga membahas data penelitian saja sesuai dengan teori
nomer 5 dimana peneliti menunjukkan dari 40 transformant, 34 transformant
stabil mengintegrasikan DNA pada jamur H.marmerous
DISCUSSION
In this study, we obtained the complete gpd gene sequence fromH. marmoreus. The
coding region was analyzed by comparing thegenomic DNA sequence with the
cDNA sequence. It matches per-fectly with the cDNA clone, suggesting this gpd
encodes a functionalprotein. In Mucor circinelloides and Agaricus bisporus, which
harbormore than one copy of gpd sequence, only one gpd mRNA is identi-fied,
indicating that there is only one functional gpd (Harmsen et al.,1992; Wolff and
Arnau, 2002). It has been reported that gpd genein higher basidiomycetes has 510
introns (Kilaru and Kes, 2005).This is also true for the gpd gene in H. marmoreus.
Paragraf ini menganut teori nomer 1 yaitu teknik membahas data dengan
membandingkan data penelitian dengan data pada pustaka. Pustaka Kilaru
and Kes, 2005 memberikan rujukan jumlah intron pada gen gpd di

basidiomycetes yang akan ditransformasikan ke H.marmerous sehingga

memperkuat mengapa penelitian menggunakan ATMT method dilakukan pada
jamur jenis ini,
Molecular genetic studies of H. marmoreus have been limiteddue to the lack of an
efficient gene transfer system. In the lastdecade, ATMT has been successfully
applied to different fungalspecies, mainly owing to its efficiency and technical
simplicity(Godio et al., 2004; Michielse et al., 2005). The main objective of thiswork
was to establish optimal conditions of ATMT for H. marmoreus.Transformation
results showed that the choices of recipient materi-als as well as vectors affected
final efficiencies (Mikosch et al., 2001;Cho et al., 2006; Wang et al., 2008; Shi et al.,
2012). In this work, theprotoplast was found to produce the best results in
transformationexperiments as compared to fruit bodies and fungal mycelium. In
A.bisporus and F. velutipes, transformation efficiencies of single gill tis-sues were
reported to be approximately 40% and 16%, respectively(Chen et al., 2000; Cho et
al., 2006). However, few transformantscould be obtained from gill tissues of H.
marmoreus deriving fromsmall mushroom caps that just developed from primordium.
Inthis study, we obtained at least 150 stable H. marmoreus transfor-mants per
105protoplasts, which is a relative high transformationefficiency.
Teknik membahas data pada paragfraf ini sesuai dengan teori nomer 6 dimana
peneliti membandingkan efiseinesi transformasi ATMT method pada jamur lain
dengan jamur jenis H.marmerous dan mrembuktikan bahwa kestabilan
transformasi pada H.marmerous mencapai 85% dibandingkan jamur lain yang
hanya 40-60% pada penelitian Chen et al., 2000; Cho et al., 2006
Various Agrobacterium strains have been tested, and it wasfound that the
Agrobacterium strains EHA105 produced the high-est transformation frequency in H.
marmoreus. Transformants werealso obtained by using the Agrobacterium strains
LBA4404 andGV3101, but no transformants were found with AGL-1. Differ-ent
Agrobacterium strains have different transformation efficiencyaccording to different
fungal strains (Michielse et al. 2005). Threestudies showed that the usage of
Agrobacterium strains derivedfrom the supervirulent A281 strain (high level of vir

gene

expres-sion)

resulted

cerevisiae,Monascus

in

higher

purpureus,

transformation

and

the

frequencies

Oomycete

in

S.

Phytophthora

infestans,compared with ATMT using Agrobacterium strain LBA1100 (Campoyet al.

2003; Piers et al. 1996; Vijn and Govers 2003). In anotherstudy, it was also found
that the supervirulent Agrobacterium strainA281 and its derivative (AGL-1) were
more

efficient

Agrobacterium

in

transferringT-DNA to

strainLBA4404

(Park

Cryphonectria

and

Kim

2004).

parasitica

than

However,

the

systemic

comparisons ofthese strains in relation to transformation frequencies have notbeen

performed, making it difficult to determine which strain is thebest to use (Michielse et
al., 2005). From these results, we clearlyknow that the choice of Agrobacterium
strain for ATMT of fungi canhave an effect on transformation efficiency.
Paragraph ini sesuai dengan teori membahas data pada nomer 1 yang
menyatakan bahwa Agrobacterium strain EHA105 pada hasil penelitian
memiliki efisiensi tranformasi paling tinggi pada jamur dibanding strain lain.
Hal ini didukung oleh pustaka Michielse et al. 2005
Several studies have shown that each fungus has a unique com-bination of
temperature

and

co-cultivation

duration

to

obtain

amaximum

number

of

transformants (Combier et al., 2003; Meyeret al., 2003; Rolland et al., 2003;
Gardiner and Howlett, 2004;Michielse et al., 2004b; Shi et al., 2012). In this study,
the mostsuitable temperature is 26C, which is close to the normal growth
temperature

(25C)

for

H.

marmoreus.

At

all

temperatures

tested,fungal

transformants were obtained. This result suggests that theAgrobacterium vir gene is
expressed over a wide range of tem-peratures, allowing for effective T-DNA transfer
and integration.Similar to the co-culture temperature, co-cultivation for differenttime
periods also resulted in positive fungal transformants. Unlikein G. lucidum, the
prolonged incubation period let to irreproducibleresults, possibly due to the increased
fungal background growthduring co-cultivation (Michielse et al., 2004b).
Paragraph ini sesuai dengan teori membahas data pada nomer 1 yang
menyatakan

bahwa

banyak

faktor

yang

mempengaruhi

keberhasilan

transformasi untuk menghasilkan positif transformant yang merujuk pada

pusataka Combier et al., 2003; Meyeret al., 2003; Rolland et al., 2003; Gardiner
and Howlett, 2004;Michielse et al., 2004b; Shi et al., 2012. Data penelitian telah
ditunjukkan dengan grafik pada gambar 6.
This is the first time the egfp gene is expressed in fusion withhph in the
basidiomycetous mushroom H. marmoreus, under thecontrol of H. marmoreus gpd
promoter. In G. lucidum, the construct carrying the promoterof itself resulted in the
highest transformation efficiency (Shi et al.,2012). In our study, promoter from H.
marmoreus had the high-est transformation efficiency and promoters from two
homologousspecies had lower transformation efficiency than H. marmoreus. Mitotic
stability analysis showed that over 85% of the transfor-mants tested remained
mitotically stable even after subculture inthe absence of hygromycin B. The antibiotic
resistance conferred bythe hph gene was still maintained. Southern blot and PCR
analysisresults demonstrated that T-DNA was integrated into the chromo-some of H.
marmoreus.In our another study, four developmental stages transcriptomesof H.
marmoreus have been done and the results suggested thatsome genes played
important roles in the fruit body formation ofH. marmoreus, such as laccase genes,
NADPH oxidase genes and thegenes involved in MAPK signaling pathway (CanoDominguez et al.,2008; De Paula et al., 2008; Langfelder et al., 1998; Mu et al.,
2013).As the transformation method was constructed, these genes maybe as our
target genes and their functions in the developmentalprocess of H. marmoreus could
be studied by this method in futurestudy.
Paragraph terakhir ini memberikan bahasan data menganut teori nomer 1 dimana
data yang dihasilkan diperkuat dengan pustaka yang mendukung. Menurut (Shi et
al.,2012 H.marmerous memiliki promotor paling kuat sehingga keberhasilan
transformasi mencapai 85%. Selain itu Cano-Dominguez et al.,2008; De Paula et al.,
2008; Langfelder et al., 1998; Mu et al., 2013 menyatakan bnyak faktor yang
mempengaruhi keberhasilan transformasi dari segi kondisi molekulerjamur.