الفهرس 
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Abstract
This study aimed to isolate and characterize the induced
antibacterial genes from the bacterial-challenged third larval instar of
house fly, M. domestica using differential display technique. The larvae
were injected with gram (+) bacteria, S. sanguinis; gram (-) bacteria, P.
vulgaris and mix. Whole body and haemolymph challenged larvae were
collected at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h postinfection as
the challenged insects died after 24 h postinfection. Then total haemocyte
counts, quantitative protein analysis were estimated. Antibacterial assay
and protein banding patterns (Native and SDS PAGE) were investigated.
The differential display technique was employed to screen the genetic
variation (at RNA level) between bacterial-challenged and control M.
domestica third instar larvae. More than forty reproducible bands were
eluted and sequenced to characterize the full length cDNA of the induced
genes. In addition, sequence and phylogenetic analyses of resultant genes
were studied.
1- Total haemocyte counts:
Significant gradual increases in THCs were observed within the
control group as well as within each treatment group. Such increases
reached plateau phase at 12 h postinfection. Significant increase in THCs
was observed in the case of treated larvae as compared with controls.
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Mixed infection created significantly higher THCs than separate bacterial
infection and control, as well.
2-Quantitative protein analysis:
Significant gradual increases in total soluble protein concentration
were observed within the control group as well as within each treatment
group. Such increases reached plateau phase at 12 h postinfection.
Significant increase in total soluble protein was observed in the case of
treated larvae as compared with controls. Mixed infection showed
significantly higher protein concentration than separate bacterial infection
and control, as well, over the first 12 h postinfection.
3- Antibacterial assay:
The bactericidal activity of whole body homogenate and
haemolymph of M. domestica third instar larvae (control and treated)
using agar well-diffusion technique was estimated. The bacterialchallenged
larvae was tested for their antibacterial activity against three
gram (+) bacteriam; and three gram (-) bacteria based on the microbial
growth inhibition zone (in mm). In control insects, the antibacterial
activity of third instar larvae showed no inhibition effect against any of
the tested bacteria at all time intervals. Meanwhile, significant
antibacterial activities of the immunized whole body and haemolymph
were observed against the tested gram (+) and gram (-) bacteria. Notably
the antibacterial activity of the challenged samples 48 h postinfection was
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more than 24 h postinfection for all the tested bacteria and the bacterialchallenged
haemolymph samples showed a significantly higher
bactericidal activity than those of whole body.
4- Native and SDS PAGE:
Whole body and haemolymph proteins of control and infected
larvae were separated using Native and SDS-PAGE at at 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22 and 24 h postinfection. The results of native-PAGE
demonstrated that there were differences in the overall protein banding
pattern in the infected larvae as compared to control. The results of the
SDS-PAGE clarified that the molecular weight of the separated proteins
ranged from 116 to 10 KDa and bacterial infection had led to the
induction of different proteins as compared to control. The molecular
weight of these bands was 18, 10 and 8 KDa.
5- Differential display results:
Whole body and haemolymph samples were differentially
displayed using 11 decameric arbitrary primers at 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22 and 24 h postinfection with S. sanguinis, P. vulgaris and
combination of both strains. The results indicated the presence of
differentially displayed bands in the bacterial-challenged larvae and not
observed in controls. More than forty reproducible bands were eluted and
sequenced. The resulting sequences were blasted to defensins, diptericins
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and attacins. As for defensins, single orf that could encode a polypeptide
of 97 and 99 amino acids was detected for MdDefWB and MdDefHL,
respectively. One stop codon was found at the 3 end of both sequences.
The length of 3’ untranslated regions were 69 and 58 bp before the poly
(A) track for MdDefWB and MdDefHL, respectively. Two putative
polyadenylation sequences AATAAA were located 37 and 49 bp
downstream from the stop codon of MdDefWB and one polyadenylation
sequence AATAAA was located 33 bp downstream from the stop codon
of MdDefHL. The identified defensin orfs included signal peptides of 26
and 29 a.a., propeptides of 30 and 30 a.a. and mature peptides of 41 and
40 a.a for MdDefWB and MdDefHL, respectively.
As for diptericins, single orf that could encode a polypeptide of 99
and 80 amino acids was detected for MdDipWB and MdDipHL,
respectively. One stop codon was found at the 3’ end of both sequences.
The flanking region of the initiation codon ATG was AAAATGCAA for
MdDipWB sequence and was CCGATGATA for MdDipHL sequence. The
lengths of 3’ untranslated regions were 74 and 62 bp before the poly (A)
track for MdDipWB and MdDipHL, respectively. One polyadenylation
sequence AATAAA was located 40 bp downstream from the stop codon
of MdDipWB. Meanwhile, two putative polyadenylation sequences
AATAAA were located 3 and 32 bp downstream from the stop codon of
MdDipHL. Signal peptide sequence was predicted for MdDipWB but not
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predicted for MdDipHL. Meanwhile, prosequence was predicted for
MdDipHL and not predicted for MdDipWB.
Single orf that could encode a polypeptide of 67 and 67 amino
acids was detected for MdAttWB and MdAttHL, respectively. One stop
codon was found at the 3’ end of both sequences. The flanking region of
the initiation codon ATG was (TTAATGTTT) for MdAttWB and
(AAAATGGCT) for MdAttHL sequences. The length of 5’ untranslated
regions were 64 and 91 bp for MdAttWB and MdAttHL, respectively. The
length of 3’ untranslated regions were 88 bp before the poly (A) track for
both MdAttWB and MdAttHL. One putative polyadenylation sequence
AATAAA was located 54 bp downstream from the stop codon of both
MdAttWB and MdAttHL. The identified attacin orfs does not include signal
but have mature peptides of 41 and 38 a.a for MdAttWB and MdAttHL,
respectively.