Download (GRP78) gene in silkworm Bombyx mori

Biologia 68/3: 559—564, 2013
Section Cellular and Molecular Biology
DOI: 10.2478/s11756-013-0182-6
Molecular cloning and expression analysis of glucose-regulated
protein 78 (GRP78) gene in silkworm Bombyx mori
Xing-Zi Xi1 & Ke-Shi Ma2*
1
Department of Education Sciences, Xinxiang University, Xinxiang 453003, People’s Republic of China; e-mail:
[email protected]
2
College of Life Sciences, Zhoukou Normal University, Zhoukou 466001, People’s Republic of China
Abstract: GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein),
is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding,
ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the
full-length cDNA of GRP78 from silkworm Bombyx mori (BmGRP78). It is 2645 bp, including an open reading frame
(ORF) of 1977 bp encoding a polypeptide of 658 amino acids. We sequenced the ORF sequence from genomic DNA, and
found only one intron (104 bp) existed in Bmgrp78 gene structure. The deduced amino acid sequence of Bmgrp78 carries
the ER retention signal KDEL at its C-terminus and is highly homologous to GRP78 of Fenneropenaeus chinensis (83.59%)
and Homo sapiens (80.27%). RT-PCR analysis shows that Bmgrp78 is ubiquitously expressed in tissues of silkworm. Heat
shock over 35 ◦C notably enhanced the expression of Bmgrp78 in head tissues. In response to starvation, the transcript level
of Bmgrp78 in head tissues was 2.8-fold (at WS stage) and 3.2-fold (at SD1 stage) higher than the control level, but it
remained stable in the midgut tissues. After silkworms were challenged by bacteria, the relative expression level of Bmgrp78
in midgut was up-regulated and reached maximal level at 24 hour after injection, and remained higher level than that of
controls at about 48 hour post challenge. We infer that BmGRP78 may play important roles in chaperoning, protein folding
and immune function of silkworm.
Key words: silkworm; GRP78; heat shock; starvation; bacterial challenge.
Abbreviations: ER, endoplasmic reticulum; EST, expressed sequence tag; GRP78, glucose-regulated protein 78; HSP70,
heat shock protein 70; ORF, open reading frame; RACE, rapid amplification of cDNA ends.
Introduction
Endoplasmic reticulum (ER) is a multifunctional organelle which controls important cellular processes, including protein synthesis, protein trafficking, Ca2+ homoeostasis and apoptosis (Ma & Hendershot 2004;
Schröder & Kaufman 2005). Disruption of any of these
processes causes ER stress, and prolonged ER stress
leads to cell death. One characteristic of the ER
stress is the induction of the ER resident stress proteins, such as glucose-regulated proteins (GRPs). One
of the best characterized GRPs in mammal, GRP78
(78 kDa GRP), also known as BiP (immunoglobulin heavy-chain-binding protein), is thought to function in ER stress, oxidative damage and Ca2+ depletion (Rao et al. 2002; Qian et al. 2005; Kitamura & Hiramatsu 2010). Additionally, it is an antiapoptosis protein. Overexpression in vitro showed
that GRP78 could protect cells against cell death
caused by disturbances of ER homoeostasis (Rao et
al. 2002; Reddy et al. 2003). Interestingly, recent
works have manifested the role of GRP78 in the de-
fence system against foreign pathogens (Luan et al.
2009).
Currently, there is no more information of GRP78
in silkworm, compared with numerous works on mammalian GRP78s. In the present paper, we cloned the
full-length cDNA of GRP78 from silkworm Bombyx
mori (Bmgrp78), and studied the expression profiles
of Bmgrp78 in response to thermal stress, starvation
and bacterial challenge. The data could be very helpful to understand the physiological and immune roles
of GRP78 in silkworm.
Material and methods
Animals and treatments
Silkworms of the normal strain (DaZao) were reared routinely on mulberry leaves at 25±0.5 ◦C under a 12L:12D
photoperiod and 70% relative humidity. After animals had
ecdysed to the last larval stage (5th instar), they were
staged and synchronized according to previously established
method (Kiguchi & Agui 1981). For starvation treatment,
starting from L5D3 (5th instar, Day 3), silkworm larvae were
* Corresponding author
c
2013
Institute of Molecular Biology, Slovak Academy of Sciences
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subjected to complete food withdrawal. Six larvae were randomly selected at five time points, corresponding to L5D3,
L5D4, L5D5, WS (wandering stage), and SD1 (Spinning
phase, Day 1) (Casati et al. 2012). The head and midgut
of larvae were dissected out and stored in liquid nitrogen
for RNA extractions. For thermal stress, larvae (L5D3, 20
animals/group) were cultured at 30 ◦C for 24 hours, followed
by 35 ◦C for 24 hours, and 40 ◦C for 24 hours. Six larvae were
randomly selected from each group, and their heads were
preserved in liquid nitrogen immediately for RNA extraction. For the bacterial challenge experiment, the larval (5th
instar, Day 3) hemocoels were injected with 10 µL/larva
of live E. coli DH5α (resuspended in phosphate buffered
saline, OD600=0.8). The non-injected larvae and larvae received injections of 10 µL autoclaved phosphate buffered
saline were used as blank group and control group, respectively. The midgut tissues were collected at 4, 8, 12, 16, 24
and 48 hours post-injection.
RNA extraction and Bmgrp78 cDNA cloning
Three or four of silkworm larvae (5th instar, Day 3) were
collected and grinded in liquid nitrogen to powders and
stored in Trizol reagent (Invitrogen, USA). Total RNA was
extracted and treated with DNase I (Takara, Japan) for
removal of DNA contamination. The RNA was quantified
by the ultra-violet spectrophotometer, and then 2 µg RNA
were used for reverse transcription. A 640 bp expressed sequence tag (EST) fragment for grp78 gene was previously
obtained by differential-display RT-PCR in our laboratory.
Based on the known EST of Bmgrp78 cDNA, Bmgrp78 3’RACE specific primer (5’-GAT GAC CAA ACT CAT TCC
TCG TAA CAC-3’) and Bmgrp78 5’-RACE specific primer
(5’-GCA CGG TTG TCT TTT CTG ATG TCC TTG3’) were designed for rapid amplification of cDNA ends
(RACE). Both 5’-RACE and 3’-RACE were carried out using a TaKaRa RACE cDNA amplification kit according to
the manufacturer’s instructions. The PCR products were
gel-purified, ligated into the pUCm-T vector, and submitted for sequencing. The resulting sequences were verified
and subjected to cluster analysis.
Amplification of Bmgrp78 ORF sequences from genomic
DNA
Genomic DNA was extracted using the Takara genomic
DNA extraction kit Ver.3.0 according to the manufacturer’s
instructions. Based on Bmgrp78 cDNA sequence, we designed a pair of specific primers (forward: 5’-ATG GTC
AAA ATG CGG TGG AGT ATG TTC-3’; reverse: 5’-GAT
TCA GTC TGA TTA CAA CTC GT CC-3’) to amplify
Bmgrp78 open reading frame (ORF) sequences from genomic DNA. The PCR program was carried out at 94 ◦C
for 5 min, followed by 30 cycles of 94 ◦C for 30 s, 62 ◦C for
30 s, 72 ◦C for 3 min and a final extension step at 72 ◦C for
10 min. The PCR products were sequenced.
Homology analysis
The similarity analysis of nucleotide and amino acid sequences was carried out using BLASTn and BLASTp
tools at the National Center of Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The
deduced amino acid sequence and protein motif features
were analyzed with the Expert Protein Analysis System (http://www.expasy.org/). Multiple alignments of the
GRP78 protein sequences were performed using the software
Dnaman 6.0 (http://www.lynnon.com/pc/framepc.html).
A phylogenetic tree was constructed using the programs
Clustal X 1.83 (http://www.clustal.org/) and Mega 3.1
(http://www.megasoftware.net/) based on the amino acid
sequences of BmGRP78 and its homologues. Bootstrap
analysis was used with 1000 replicates to test the relative support for the branches produced by neighbour-joining
analysis.
Detecting the tissue distribution of Bmgrp78 gene by RTPCR
For spatial expression analysis of Bmgrp78 gene, 7 main tissues, head, muscle, midgut, silk gland, fat body, Malpighian
tubule and gonads, were dissected from Day 3 of the 5th instar larvae, and also immediately frozen in liquid nitrogen,
respectively. Every tissue sample was collected from three
or four larvae. cDNA was synthesized as described above. A
pair of specific primers (forward: 5’-TGC TGT TGT TAC
TGT GCC TGC TT-3’; reverse: 5’-ATC AAA GGT TCC
ACC ACC CAA AT-3’) were designed to amplify Bmgrp78
cDNA fragment of 193 bp. Two specific primers, BmActinF
(5’-ACC CAT CTA CGA AGG TTA CGC-3’) and BmActinR (5’-ACG AAC GAT TTC CCT CTC AGC-3’) were used
for controls. The PCR program was carried out at 94 ◦C for
5 min, followed by 24 cycles of 94 ◦C for 30 s, 58 ◦C for 30 s,
72 ◦C for 30 s and a final extension step at 72 ◦C for 5 min.
Changes of expression pattern of Bmgrp78 in response to
different stressors
In order to detect the relatively accurate expression pattern
of Bmgrp78 in response to different stressors, the quantitative RT-PCR was performed using Real-Time PCR Detection System with a SYBR Premix Ex Taq kit (Takara,
Japan). The PCR was carried out as follows: 30 s at 95 ◦C,
followed by 40 cycles of 5 s at 95 ◦C, 1 min at 60 ◦C. The
primers of the Bmgrp78 gene and the actin gene for realtime PCR analysis were the same as those in the RT-PCR
analysis. The determination of the expression ratios was
carried out using the method 2−∆∆CT , described by Livak
& Schmittgen (2001). The data obtained from quantitative
RT-PCR analysis for the expression of Djgrp78 were subjected to one-way analysis of variance (one-way ANOVA).
Differences were considered significant at P < 0.05.
Results
cDNA cloning and gene structure of silkworm Bmgrp78
gene
Based on the Bmgrp78 EST sequence, we designed
two specific primers to clone the full-length cDNA sequence of Bmgrp78 gene. A cDNA fragment of approximately 1200 bp was amplified using a Bmgrp78 3’RACE specific primer and a TaKaRa 3’-RACE inner
primer, which contains stop codon TAA and canonical polyadenylation signal sequence AATAAA. A cDNA
fragment of approximately 1000 bp containing a start
codon was amplified using a Bmgrp78 5’-RACE specific primer and a TaKaRa 5’-RACE inner primer. A
2645 bp nucleotide sequence representing the complete
cDNA sequence of Bmgrp78 gene was obtained by cluster analysis of the two above fragments. In addition, we
sequenced the ORF sequences from genomic DNA, and
found only one intron (104 bp) existed in the Bmgrp78
gene structure. The intron interrupts the codon AAG
at the position of 587 and the codon GTT at the position of 588. The 5’- and 3’-putative splicing site follows
the typical “GT-AG” rule (data not shown).
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Fig. 1. Complete BmGRP78 nucleotide and deduced amino acid sequence from silkworm Bombyx mori. The signal peptide is signified
in bold letters and the C-terminal KDEL in bold and italics represents the ER retention sequence. Three HSP70 protein family
signatures are underlined. The ATP/GTP-binding site motif A (P-loop) is double-underlined. The start codon, stop codon and the
polyadenylation signal (AATAAA) are boxed.
Characterization of BmGRP78
The cDNA sequence of the Bmgrp78 gene was deposited in GenBank (Benson et al. 2012) under the
accession No. JX948747. The full-length cDNA of Bmgrp78 was of 2645 bp, including a 5’-terminal untranslated region of 134 bp, a 3’-terminal untranslated region of 534 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail (Fig. 1),
and an ORF of 1977 bp encoding a polypeptide of 658
amino acids with a predicted molecular mass of 73.13
kDa and theoretical isoelectric point of 5.03. The Signal
P software analysis indicated that BmGRP78 contains
a signal peptide of 20 amino acids (MVKMRWSMFALVLVVLAVCA). SMART analysis showed that BmGRP78 displays three conserved heat shock proteins
70 (HSP70) family signatures: IDLGTTYS at the position of 36-43, VFDLGGGTFDVSLL at 224-237, and
IVLVGGSTRIPKVQQ at 361-375. The ATP/GTPbinding site motif A (P-loop), AEAYLGKK, was located at amino acid residues 159-166 (Techel et al.
1998; Luan et al. 2009). The C terminal of BmGRP78
contains the putative ER targeting tetrapeptide KDEL
(residues 655-658) (Fig. 1).
Homology analysis of BmGRP78
A sequence homology search using BLASTn and
BLASTp tools revealed that BmGRP78 is highly homologous with other GRP78s from invertebrates and
vertebrates. It displayed a high degree of sequence similarity to GRP78 of Fenneropenaeus chinensis (83.59%,
EF032561), Dugesia japonica (75.45%, JN968463),
Danio rerio (79.21%, NP 998223) and Homo sapiens
(80.27%, NP 005338). In addition, many GRP78 homologues from different species of insects were collected
from the GenBank (Benson et al. 2012). The complete
alignment was done upon these sequences, and showed
that all of these homologues contain the ER retrieval
signal KDEL or RDEL. These tetrapeptides are responsible for the retention of proteins in the ER lumen
(Munro & Pelham 1987; Persson et al. 2005; Byun et
al. 2007), which suggested that BmGRP78 is an ERlocalized protein.
Based on the sequences of BmGRP78 and its homologues in insects, a phylogenetic tree was constructed
(Fig. 2). The phylogenetic tree shows that animals from
Lepidoptera, Hymenoptera, Diptera and Orthoptera
are clustered together, respectively. Interestingly, all of
the complete metamorphosis insects are clustered together, and form a sister branch with that of incomplete metamorphosis insects. This phenomenon suggests that GRP78s in insects display highly species
specificity.
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Fig. 2. A phylogenetic tree of GRP78 family members in insects constructed with the neighbour-joining method. The GenBank
accession numbers are in brackets. Numbers at each branch indicate the percentage of times a node is supported in 1000 bootstraps
pseudoreplication by neighbour joining.
Fig. 3. RT-PCR analysis of the tissue distribution of Bmgrp78
mRNA in Bombyx mori. Lanes 1-7, head, fat body, muscle,
silk gland, midgut, gonads and Malpighian tubule, respectively.
Bmactin was used as the control to normalize the amount of templates.
Analysis of the tissue distribution of Bmgrp78 gene in
silkworm
RT-PCR showed that the transcript of Bmgrp78 gene
could be detected in all seven tissues as described above
(Fig. 3). High expression was observed in head, midgut
and gonads, in contrast to the weak signals in muscle,
fat body, silk gland and Malpighian tubule.
Expression pattern of Bmgrp78 in response to different
stressors
Expression profile of Bmgrp78 after heat shock treatment is shown in Figure 4A. Silkworms were transferred
from 25 ◦C culture condition to 30 ◦C for 24 hours, Bmgrp78 expression level had no notable change in contrast
to the normal level. After elevating the culture temperature to 35 ◦C for 24 hours, Bmgrp78 transcription level
was 1.7-fold higher than the control point. After elevating the culture temperature to 40 ◦C for 24 hours, silkworms could still survive, with Bmgrp78 mRNA level
2.2-fold higher than the control level.
Previous studies have revealed that GRP78 could
be induced by starvation (Lee et al. 2002; Mamady
& Storey 2006). Therefore, we were greatly interested
in understanding whether starvation influences GRP78
expression in silkworms. We examined the Bmgrp78
mRNA in head and midgut at different time points
after the initiation of starvation. In the head of silkworms, the transcriptional level of Bmgrp78 remained
unchanged after 24 hours starvation (L5D4), increased
slightly (1.3-fold) after 48 hours starvation (L5D5), and
reached a higher level at WS (2.8-fold) and SD1 (3.2fold) stage (Fig. 4B). Unexpectedly, we did not observe
the up-regulation of Bmgrp78 expression in the midgut
of the starved larvae during the period of starvation
(Fig. 4B).
In addition, we investigated the bacterial challenge
on the effects of Bmgrp78 expression. At 8 hours postinjection, the mRNA level of Bmgrp78 began to rise
(1.2-fold higher than that of blank), and reached the
maximum (2.5-fold higher than blank group) at 24
hours post-injection (Fig. 4C). At 48 hours post injection, the transcriptional level of Bmgrp78 in midgut of
bacteria-challenged group remained higher than that
of controls. The phosphate buffered saline challenged
group was used as the control, and there was no significant difference from 0 to 48 hours (Fig. 4C).
Discussion
In this paper, the complete cDNA sequence of GRP78
gene was successfully cloned from silkworm Bombyx
mori. Conserved sequences and characteristic motifs,
such as HSP70 family signatures, as well as ATP/GTPbinding motif, were found in the deduced BmGRP78
amino acid sequence. The signal peptide at the Nterminus and the tetrapeptide KDEL at the extreme
C-terminus of the BmGRP78, ensure its residency in
ER. KDEL is a retrieval motif essential for the preUnauthenticated
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Glucose-regulated protein 78 in silkworm
Fig. 4. Expression of Bmgrp78 in response to different stressors by
real-time PCR. (A) Heat shock treatment. 25 ◦C indicates animals
cultured in 25 ◦C as control; 30 ◦C indicates animals cultured in
30 ◦C for 24 hours; 35 ◦C indicates animals cultured in 30 ◦C for
24 hours, followed by 35 ◦C for 24 hours; 40 ◦C indicates animals
cultured in 30 ◦C for 24 hours, followed by 35 ◦C for 24 hours and
40 ◦C for 24 hours. (B) Starvation treatment starting from L5D3
up to SD1 stage; L5D3 as control group. (C) The larvae were
challenged by bacteria. Asterisk indicates significant differences
comparing to that of control samples (*P < 0.05, **P < 0.01).
Values are expressed as mean ± S.D. (n = 6) of three independent
PCR amplification.
cise sorting of ER resident proteins along the secretory
pathway. The KDEL proteins, such as GRP78, calreticulin and protein disulfide isomerase, perform essential
functions in the ER related to protein folding as well as
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assembly (Munro & Pelham 1986). Our work revealed
that mRNA of Bmgrp78 is ubiquitously distributed in
all tested tissues in silkworm, especially in head, midgut
and gonads displaying intense signals. The molecular
characteristic of BmGRP78 and the non-induced level
of expression indicated that it might play multiple functions in silkworm as its homologues reported in other
living organisms.
GRP78, the ER member of the HSP70 family, is
regulated by heat and/or other stresses (Shiu et al.
1977; Sun et al. 2006; Quinones et al. 2008). In this
paper, we verified that thermal stress and starvation
can induce the up-regulation of Bmgrp78 expression in
silkworms, especially in head tissues. However, we have
not observed the notable changes of Bmgrp78 expression in silkworm midgut when deprived of food. This
phenomenon may due to the flexibility of the gastrointestinal tract of animals, which can conserve energy
through self-digestion in response to food deprivation
(Chediack et al. 2012; Zeng et al. 2012). Interestingly,
other studies reported that GRP78 protein level was
higher in brain than in other tissues during animal hibernation (Lee et al. 2002; Mamady & Storey 2006).
The head with the central nervous system is more sensitive to ER stress. GRP78 accumulation in head tissues may have a neuroprotective role in response to
ER stress. This hypothesis has been supported by the
findings that overexpression of GRP78/BiP diminishes
α-synuclein neurotoxicity and the induction of GRP78
expression protects hippocampal neurons against excitotoxicity and apoptosis (Yu et al. 1999; Nakka et al.
2010).
Recently, more and more evidences indicate that
GRP78 plays diverse roles beyond the ER (Quinones et
al. 2008; Morito & Nagata 2012). Studies have shown
that GRP78 is expressed on the cell surface in many
tissue types, and involved in transducing signals from
ligands as disparate as activated α2-macroglobulin and
antibodies (Gonzalez-Gronow et al. 2009; Zhang et al.
2010). In the present study, the mRNA transcripts in
midgut increased and reached the maximum at 24 hours
and maintained high level at 48 hours following the bacteria injection. Luan et al. (2009) reported that white
spot syndrome can induce GRP78 expression in Fenneropenaeus chinensis. Our results indicate that BmGRP78 is potentially involved in silkworm immune response to bacterial infection. These data would be helpful to understand the significance of GRP78 in silkworm
immune defence.
In this study, we successfully cloned a full-length
grp78 cDNA from silkworm Bombyx mori for the first
time, and analyzed its expression pattern in response to
thermal stress, starvation, and bacterial challenge. Our
data indicate that Bmgrp78 may be a multifunctional
gene in silkworm.
Acknowledgements
We thank Dr. Oliver Clarke and Youzhong Guo from
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564
Columbia University for critical reading of this manuscript.
This work was supported by the Young and Key Teacher
in Colleges and Universities of Henan Province (2011GGJS163), and the Natural Science Research Program of Education Department of Henan Province (13A180107).
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Received November 3, 2012
Accepted March 17, 2013
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