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Oleh :
ARDHIA DEASY R.D
136100112011001
PASCASARJANA TEKNOLOGI PERTANIAN
BIOTEKNOLOGI PANGAN
UNIVERSITAS BRAWIJAYA
2014
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
duration
of
co-cultivation
for
subsequent
assays.
The
number
of
1:1.
Four
A.tumefaciens
strains
were
evaluated
and
the
highest
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
South-ern
blot
indicated
that
the
T-DNA
into
the
genome
of
H.
gene
expres-sion)
resulted
cerevisiae,Monascus
in
higher
purpureus,
transformation
and
the
frequencies
Oomycete
in
S.
Phytophthora
efficient
Agrobacterium
in
transferringT-DNA to
strainLBA4404
(Park
Cryphonectria
and
Kim
2004).
parasitica
than
However,
the
systemic
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
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.