A

Ahmed
JournalMansour
of Cell and Molecular Biology 6(2): 163-174, 2007
Hali University, Printed in Turkey.
http://jcmb.halic.edu.tr

Research Article

163

Expression of silk gene in response to P-soyatose (hydrolyzed

soy bean protein) supplementation in the fifth instar male
larvae of Bombyx mori
Chandrasekar Raman, Suganthi L. Manohar, Nirmala Xavier and Muthukalingan Krishnan *
Insect Molecular Biology Lab, Department of Environmental Biotechnology, School of Environmental
sciences, Bharathidasan University, Tiruchirappalli, 620 024, South India
(*author for correspondence: krish14156@yahoo.com)
Received: 17 August 2007; Accepted 04 December 2007
_______________________________________________________________________________________
Abstract
The silk glands of Bombyx mori L. have been used as a model system in the production of large amount of
silk proteins prior to cocoon spinning. The amount of silk proteins synthesized could be augmented by improving the nutritional status of silkworm larvae. The present study reports the changes in the silk gene expression by supplementing P-soyatose (hydrolyzed soy protein) during the fifth instar B. mori male larvae.
Western blot analysis of the silk gland protein in the soy protein supplemented group demonstrated higher
staining intensity of H and L chains. The rate of protein synthesis was increased in the male larva reared
with P-soyatose in comparison to control group. This was indicated by high incorporation of 3H leucine in
the posterior silk glands of supplemented larvae reared with P-soyatose. Northern blot analysis unequivocally prove that supplementation of soy protein increases the synthesis of silk fibroin, as it increases the levels
of fibroin mRNA, which reflects fortification in the economic traits of B. mori.
Key Words: Bombyx mori, silk fibroin, P-soyatose, nutrition supplementation, Northern blotting

Bombyx mori beinci evre erkek larvasnda P-soyatoz (hidrolize edilmi soya
proteini) takviyesine cevap olarak ipek geni ekspresyonu
zet
Bombyx mori L. ipek bezleri, kozaya dnmeden nceki dnemde fazla miktarda ipek proteinlerin retiminin modellenmesinde kullanlmtr. Sentezlenen ipek proteinlerinin miktar, ipek larvalarn beslenmesi
gelitirilerek arttrlabilir. Bu alma B. mori erkek larvasnn beinci instar dneminde P-soyatoz (hidrolize
edilmi soya proteini) takviyesi ile ipek gen ekspresyonundaki deiimleri sunmaktadr. Soya protein takviyeli gruptaki ipek bezi proteinlerinin Western blot analizi, H ve L zincirleri iin youn boyanma gstermitir. P-soyatozla beslenmi erkek larvada protein sentezi, kontrol grubuna gre artmtr. Bu durum, Psoyatozla besili larvadaki arka ipek bezlerindeki 3H leucine iaretlemesi ile kendini gstermektedir. Northern
blot analizi de ayrca soya protein takviyesinin ipek fibroin sentezini, dolaysyla fibroin mRNA seviyesini
arttrdn kantlamtr. Bu sonu, B.morinin ekonomik adan faydal olacak zelliklerinin glendirilmesini yanstmaktadr.
Anahtar Szckler: Bombyx mori , ipek fibroin, P-soyatoz, besin takviyesi, Northern blotlama
_______________________________________________________________________________________

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Introduction
Larval nutrition is of great importance, which
influences growth, development and silk gland
function in B. mori (Ito, 1980; Akai, 1982). The
knowledge of the role of nutritional impairment on
biological and biochemical characteristics of insects stems from studies related to the components
of diet and chemical delineation of essential, beneficial and deleterious constituents of food (Dadd,
1985). A certain mode of approach in which silkworm nutrition can be studied is either by fortification of mulberry leaves as natural foods or supplementing various nutritive substances with standard artificial diets.
Supplementation results in multifarious effects
including increase in the concentration of hemolymph proteins (Nagata and Kobayashi, 1990;
Krishnan et al., 1995), increase in body weight,
silk gland and cocoon weight (Sarkar et al., 1995;
Vanisree et al., 1996). It has been demonstrated
that the elevation of dietary proteins to optimal
level results in acceleration of growth and silk
production in B. mori (Horie et al., 1971; Kamioka
et al., 1971; Horie and Watanabe, 1983). However,
these effects vary with types and levels of dietary
proteins used. Among the supplementary proteins,
the extracts of soyabean protein have the most
attractive nutrition quality value.
This highly nutritive protein including all the
indispensable amino acids (Reinecke, 1985) promotes growth and improves the economic characteristics of the silkworm (Ito, 1981; Nirmala et al.,
2003). An increase in protein synthesis is suggestive of an alteration in the expression of silk protein genes. This is supported by Hesketh and Partridge (1996), who stated that the interaction between nutrition and genetic information, results in
precise variability in the cell function. Nutrition
transcriptionally regulates the expression of various proteins (Goldman et al., 1985; Noriega et al.,
1994; Iritani et al., 1996).
Thus, we hypothesized that P-soyatose supplementation influences the growth of silkworm
and consequently, the amount of silk synthesized.
There are many studies reporting an increase in
silk gland, cocoon and shell weight upon dietary
supplementation.
However, there is little information on regulation of silk gene expression in response to nutritionally enriched diets. This study shows an increase in the fibroin protein synthesis and its

mRNA transcript accumulation in the silk glands of

B. mori reared with respect to P-soyatose supplementation.
Materials and methods
Insects
Disease free eggs of B. mori (the crossbreed race,
Tamilnadu White X NB4D2) were provided from
the Government grainage center, Tiruchirappalli,
South India and maintained at a temperature of
272C and a relative humidity of 755% (Nirmala
et al, 1996). When compared between sexes, the
rate of protein synthesis in the posterior silk glands
(PSG) was higher in males than females. Also, the
uptake of higher amount of P-soyatose was observed in males rather than females (Nirmala,
1998). Thus, male larvae were used in the present
study. Newly hatched larvae were sexed based on
the external genitalia (Nirmala et al., 2003). Male
larvae were fed with leaves of mulberry leaves
(MR2 variety). Bed cleaning and spacing of larvae
in trays were done as described by Krishnaswami
et al., (1978).
Hydrolyzed soyprotein
Hydrolyzed soyprotein, P-soyatose (Warkem Company, Mumbai, India) is an enzymatic digestive
product of soyaprotein which consists of 8.8% total
nitrogen (neutral in pH, water soluble and pathogen
free). Since the hydrolyzed soyprotein is devoid of
trypsin inhibitors and is known to be highly nutritive compared to other protein supplements, it was
used as the supplementary nutrient.
Soyprotein supplementation
Newly molted fifth instar male larvae were used in
the experiments. Stock solutions of soyprotein were
initially prepared in distilled water at different
concentrations ranging from 5-50%. The control
group was fed on mulberry leaves. The experimental male larvae were divided into three groups,
which received 2, 4 and 6 mg of P-soyatose per
larva per day. Micropipettes were used for feeding
larvae with a stock solution of 20% soyprotein. All
larvae groups were first fed with soyprotein, before
feeding with mulberry leaves. In the supplementation experiments, fifth instar larvae of 2-6 days
were used.

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Extraction of silk proteins from the silk gland and

cocoon

Labeling of proteins in vivo in the posterior silk

gland

Silk glands and cocoon were daily dissected out

from both the control and the supplemented
groups. For further analysis, the posterior (PSG)
and middle silk glands (MSG) where the synthesis and storage of fibroin takes place were washed
to remove hemolymph. The membrane surrounding the proteins was carefully removed as far as
possible. Each part was then cut into small pieces
and immersed in 25 volumes of the dissolving
buffer (50mM Tris-Cl (pH 6.8), 1% SDS, 2mM
EDTA, 1% -mercaptoethanol and 10% glycerol)
for 5 hours (Ichimura et al., 1994). The protein in
buffer solution was filtered through cheese cotton
and the filtrate was used immediately for the
analysis of silk proteins. A cocoon was cut into
small pieces and desericinated by autoclaving
(Chiang et al., 1980). The fibroin fibers were
solubilized in 60% of lithium thiocyanate and
dialyzed against the dissolving buffer to remove
the excess lithium thiocyanate. The resulting
dialysate was used for the analysis of silk protein.

Intra-coelomic injection of 10Ci 3H leucine

(BARC, Mumbai, India) (7000mCi/Mmole/g larva
was given from day 1~7 of fifth instar larva for
control and soy protein supplemented larvae to
study the alteration in the synthesis rate of silk
proteins due to supplementation. After 6 hours of
labeling period, PSGs were collected from five
larvae and the silk proteins were extracted. The
total rate of protein synthesis was evaluated by
determining the rate of fibroin synthesis. The fibroin polypeptides are exclusively synthesized by
the PSG. Furthermore, 80% of the protein synthesized by PSG in fifth instars is only fibroin (Prudhomme, 1976). Thus, the study on the rate of fibroin synthesis was restricted only to the PSG
region of the fifth instar larvae. Fibroin, the core
protein of the silk fiber, is composed of two polypeptides, as H and L chains, 350 kDa and 25 kDa,
respectively (Gamo et al., 1977).
Quantitation of 3H leucine incorporation into the
posterior silk gland proteins

Protein electrophoresis and Western blot analysis

Protein concentration in the samples (silk gland
and cocoon) was determined according to Bradford (1976), using bovine serum albumin as the
standard. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a 4-15% gradient gel under denaturing
conditions (Laemmli, 1970). The polypeptide
markers as standard proteins ranging from 7.3 to
204 kDa, were run simultaneously to identify the
fibroin polypeptides. The protein bands on gels
were stained with silver nitrate (a solution of 50%
ethanol, 12% acetic acid and 0.1% formaldehyde)
(Merril et al., 1984). Quantitative differences in
specific polypeptides resolved in the gel were
measured and scanned using a densitometer
(Hoefer, GS 300). Western blot analysis was
carried out according to Towbin et al., (1979)
with the primary antibody and probed with an
anti-rabbit IgG (1:100 dilution). Polyclonal antibodies were kindly gifted by Prof. Dr. S. Mizuno,
Tohoku University, Sendai, Japan. The purple
colored bands were visualized and photographed.

To determine the amount of radioactivity incorporated into the silk glands, an aliquot (5l) from
the silk protein extract was spotted on Whatmann
filter paper (3mm) discs. It was washed with icecold 5% TCA and subsequently with ethanol for 3
times. The discs were dried and the radioactivity
was determined by immersing the filters in 5 ml of
scintillation cocktail including 0.6 g of 2- pphenylene bis 5-phenyloxazole (POPOP) and 4 g of
2, 5-diphenyloxazole (PPO) in 1000 ml Toluene
and counted in a liquid scintillation counter (LSC,
Beckman Co. Inc., USA) with 96% efficiency for
internal standard. To determine the specific incorporation of 3H leucine into silk proteins, equal
counts (5000 cpm) were loaded on a 4-15% gradient SDS-PAGE. The gels were stained in 0.5%
coomassie blue R-250 and the polypeptide bands
corresponding to H and L chains of fibroin were
excised. They were immersed in 5ml of scintillation cocktail and counted in an LSC counter (Bosquet and Calvez, 1985).
Preparation of probes and Northern blotting
The recombinant plasmid clones pJ9HPE1 for fibroin H chain (Waga and Mizuno, 1993) and pFL 18

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for fibroin L chain (Yamaguchi et al., 1989) were

used as probes. These clones were kindly gifted by
Prof. Dr. S. Mizuno, Tohoku University, Sendai,
Japan. These plasmids were further propagated by
transformation into E. coli cells. Radio-labeling of
the probe was done using Random Primer Labeling kit (Genei Bangalore, India).
Northern blot analysis
Total RNA from PSG was isolated according to
Chomczynski and Sacchi (1987). Aliquots of 20
g of the total RNA was separated on a 1.2%
formaldehyde agarose gel and transferred to a
nylon membrane (Amersham Co., Chennai, India). The filter was hybridized with 106cpm/ml of
32
P labeled cDNA probe (denatured), washed and
exposed to the X-ray film (Sambrook et al.,
1989).
Results
Silk gland protein profiles and identification of
the fibroin polypeptides
Electrophoretic analysis separated 10-15 bands in
silk gland of fifth instar male larvae of B. mori
(Figure 1A). Western blot analysis confirmed L

chain as 25 kDa band and H chain as a streak of

multiple bands from 350 to150 kDa in cocoon
fibroin (Figure 1B).
Pattern of fibroin accumulation
Electrophoretic analysis showed that fibroin level
was increased in the soyprotein supplemented larvae in comparison to control. The staining intensity
of both the H and L chains gradually increased
from day 4 to 6 (Figure 2A). Western blot analysis
of the H and L chains of fibroin, confirmed the
results of SDS-PAGE (Figure 2B and C).
Pattern of protein synthesis in the silk glands
Since high amounts of the fibroin polypeptides
were obtained in all the supplemented groups, only
one group of male larvae that received 4mg soy
protein was used for this study. In controls, the
incorporation of 3H leucine by the acid precipitate
of the silk gland extract increased exponentially
during the development of fifth instar control larval
period (Figure 3). The rate of protein synthesis
during development was markedly increased until
day 3, while it showed a slight increase between
day 3 and 5. The maximum increase was obtained
on day 6, and then it decreased on day 7 in the male

Figure 1. 4-15% gradient SDS-PAGE pattern of silk gland protein profile from fifth instar male, B. mori (A), and immunoblot
analysis of heavy [H] and light [L] chain of fibroin(B). Lanes 1-6 indicate developmental days of fifth instar (Molecular weight
marker: M, Heavy chain: H and Light chain: L).

P-soyatose mediated gene expression in Bombyx mori

larvae. A similar trend in the incorporation of

radioactivity (3H) was observed in the PSG of
supplemented larvae. However, this increase was
higher than that of controls. The increase with
104% in the larvae was highest on day 6, followed by a decline of 22.68% on day 7.
Pattern of 3H Leucine incorporation into fibroin
polypeptides
The incorporation of 3H leucine into the H and L
chains of the soy protein supplemented groups
were higher, when compared with the controls.
The cpm/pair of PSG in the male control was

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15,007 cpm, while it was 16,140 cpm in the soy

protein- supplemented group, on day 3 for H chain.
It was 7,313 and 7,395 cpm in the control and supplemented group for the L chain respectively (Figure 4). The incorporation of radioactivity increased
markedly to a level of 12,779.1 cpm in the control
and 16,352.5 cpm in the soy protein supplemented
groups on day 6 for the L chain respectively.
Pattern of the fibroin transcription
Total RNA, extracted from larvae of control and
soyprotein supplemented groups was probed with
cDNA for the H and L chain of fibroin. Figure 5

Figure 2
SDS-PAGE (4-15%
gradient) of the
middle (MSG) and
posterior (PSG) silk
gland proteins in 6th
day of fifth instar
male, B. mori reared
with control diet and
different concentrations of P-soyatose
(A). Western blot of
fibroin H (B) and L
(C) chain in silk
glands of day 4
control (0.00) and
soyprotein supplemented group (2, 4
and 6 mg) in 4th day.

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and 6 explain the hybridization pattern for the H

and L chains of fibroin in control and soyprotein
supplemented groups. Hybridization analysis
showed that the levels of fibroin H and L chain
mRNA increased with the developmental period
from day 2 to 6 of the fifth instar. Thus, the addition of 4mg soyprotein into the normal diet of B.
mori male larvae increased the amounts of fibroin
H (Figure 5) and L (Figure 6) chain transcripts.
Discussion
Protein profiles of silk gland
The previous study showed that protease activity
was increased in the gut of B. mori larvae reared
with soyprotein (Nirmala et al., 1996). Similar
results were obtained by supplementation of
different nutrients including protein (Horie and
Watanabe, 1983; Sarker, 1993). Our results
showed that supplementation of soy protein to
fifth instar male larvae of B. mori resulted in
higher silk gland weight, synthesis and accumulation of fibroin H and L chains and their respective
rate of synthesis. Previous studies also demonstrated an increase in the silk gland weight in

response to increased concentration of P-soyatose

(Nirmala, 1998). The increase in silk gland weight
during fifth instar development may be a result of
the efficient utilization of increased amounts of
proteins/amino acids and other nutrients in mulberry leaves. This natural food contributes to the
growth of the silkworm, thereby increasing the
weight of larvae and silk glands, leading to the
improvement of the cocoon characteristics in B.
mori (Sarker et al., 1995; Nirmala, 1998). It has
been shown that supplementation with minerals
increase the biochemical contents in fat body and
hemolymph. It also enhances the silk gland parameters (Nirwani and Kaliwal, 1996; Hugar et al.,
1998; Goudar and Kaliwal, 2001; Etebari and Fazilati, 2003; Bhattacharya and Kaliwal, 2005; Kochi
and Kaliwal, 2006; Isaiarasu et al., 2007). Leonardi
et al., (2001) reported that silkworm fed on artificial diet supplemented with leucine methyl ester
(Leu -OMe) gained maximum body weight, at 1218 h compared with the control. Etebari and Bizhannia (2006) showed that there was a decrease in
nitrogen, phosphorus, potassium and protein content of leaves and a significant decrease in total
cocoon production, cocoon weight, pupal weight
and shell weight due to feeding of mulberry leaves

Figure 3. Incorporation rate of 3H leucine into the posterior silk gland proteins after 6h of post-injection, into the control and soyprotein supplemented male larvae of B. mori, in different days during fifth instar development.

P-soyatose mediated gene expression in Bombyx mori

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soyatose per female per day, during the fifth instar

development of B. mori (Nirmala, 2003). Since a
high correlation had been observed between the
silk gland weight and silk production (Fayard,
1978), the accumulation of fibroin H and L chain in
the silk glands and their respective rates of synthesis in the larvae supplemented with soy protein was
observed. The accumulation of fibroin polypeptides
and their respective rate of synthesis were higher in
the supplemented groups than in the control. This is
supported by the findings of Fayard (1978) who
reported a correlation between the silk gland
weight and silk production. Proteins in the hemolymph of silkworm are known to be influenced by
the levels of dietary protein (Watanabe and Horie,
1980). A supportive result was obtained by Nagata
and Kobayashi (1990), who confirmed the findings
and further reported that hemolymph storage proteins were specifically sensitive to the dietary protein content. Krishnan et al., (1995) reported that
the level of storage proteins in the hemolymph was
increased by the increasing concentrations of hydrolyzed soy protein supplementation. Vanisree et
al., (1996) also reported an increase in the levels of
storage proteins in the hemolymph of five races of
B. mori fed with mulberry leaves fortified with
soyprotein. The present study further provides

infested with thrips which impair the nutritional

value of leaves, suggesting it as a good dietary
supplement. Electrophoresis has identified the
fibroin polypeptides with reference to molecular
weight marker proteins, run parallel with the silk
gland proteins. A final confirmation of the polypeptides of fibroin was performed by western blot
analysis. It showed an intact L chain at a molecular weight of the 25 kDa and the bands corresponding to H chain as a streak of multiple bands
from a molecular weight of 350 kDa to 150 kDa.
It is reasonable to suggest that H chain of fibroin
has highly degradable structure.
Patterns of protein synthesis in the silk glands
Although the influence of dietary protein on the
silk production is well established, the higher
accumulation of the silk protein, fibroin, in the
silk gland protein profile in response to dietary
protein is scanty. The fibroin biosynthesis rate
was used as a marker of silk gene expression by
dietary P-soyatose supplementation. Earlier studies indicated that the optimum amount to register an increase in the larval, pupal and shell
weight was obtained by 4 mg of soyprotein per
male per day and 20 mg supplementation of P-

18000

CPM / Pair of Posterior...

silk glands

16000
14000
12000
10000
8000
6000
4000
2000
6

0
5

E
C

H Chain
E
L Chain

Development period (day)

Figure 4. Incorporation rate of radioactivity into the H and L chains of fibroin in the posterior silk glands of control (C) and soyprotein supplemented (E) male larvae of B. mori in different developmental days.

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information on the dietary influence on protein

synthesis in B. mori. Dietary protein not only
influences the storage protein levels but also
synthesis and accumulation of fibroin H and L
chains in B. mori larvae reared with soy protein.
Our results also suggest that low levels of soyprotein incorporated into the diets of B. mori
larvae would be a good nitrogenous supplementary protein as it enhances and accelerates the synthesis of proteins in the silk glands.
Influence of dietary protein on the transcription
of fibroin
The most impressive feat with PSG cells is the
efficient programming of the transcription of the
fibroin gene, which is only 0.0022% of its total
DNA. Biometrical analysis of the silk production
in different B. mori strains in relation to the development of the proteosynthetic apparatus demonstrated that specific mRNA content determines
the amount of fibroin synthesized (Strydom et al.,
1977). Muthumani (1995) observed that supplementation of soyprotein resulted in higher levels
of small fibroin protein (P25) gene transcripts. In
the current study, the rate of fibroin transcription

increased by about approximately 18% in the supplemented groups, when compared with controls.
These results support the findings that the dietary
proteins stimulate changes at the transcriptional
level to improve the silk synthesis. Thus, it is reasonable to suggest that the rate of transcription of
the fibroin gene may have been increased by soyprotein supplementation. Some studies showed that
the fibroin modulator binding protein (FMBP-1)
regulates the specificity of the fibroin gene expression together with some other enhancer binding
proteins such as Bm fork head (Suzuki et al., 1990;
Takiya et al., 1997, 2005). Ranjan et al., (1993),
Tafuri and Wolffe (1993) and Bouvet and Wolffe
(1994) concluded that Bombyx Y box binding
protein (BYB) is also one of the candidates in regulating the activity of FMBP-1 and expression of the
fibroin gene. A more recent study showed that the
expression of BYB in B. mori from fourth to fifth
instar was regulated in a tissue- and a stage- dependent manner (Nisitha and Takiya, 2005).
We conclude that P-soyatose supplementation
results in an increased transcription of fibroin
mRNA leading to an increased silk production of
B. mori. This is supported by the findings of Horie
and Watanable (1983) who reported that different

Figure 5 Northern hybridization of total RNA from the posterior silk glands with probe for the H chain of fibroin. Upper panel
indicates the levels of fibroin H chain mRNA in the control (C) and soyprotein supplemented (E) larvae of B. mori during fifth instar
development. Lower panel indicates the ethidium bromide staining of RNA on 1% formaldehyde agarose gel. Lanes 2-6 represents
the developmental period of fifth instars in days.

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Figure 6 Northern hybridization of total RNA from the posterior silk glands with probe for the L chain of fibroin. Upper panel indicates the levels of fibroin L chain mRNA in the control (C) and soyprotein supplemented (E) larvae of B. mori during fifth instar
development. Lower panel indicates ethidium bromide staining of RNA on 1% formaldehyde agarose gel.

kind of proteins influenced growth and hemolymph components in this economically important insect. This may be attributed to increased
signal of amino acids in the system following
efficiently, which might be released upon digestion of the mulberry and the supplements (Horie
et al., 1971; Kamioka et al., 1971). Our results
clearly infer that the entire protein synthesis machinery of the silk gland, functions for the silk
production during fifth instar, is in support of the
suggestion of Noguchi (1974) that silk formation
in the spinning period is mostly controlled by the
nutrient reserves. Therefore, in the present study,
these reserves may be supplied by enriched mulberry leaves, and in excess by the supplemented
soyprotein, which acts as a signal for the higher
transcription of the fibroin gene, resulting in an
enhanced synthesis of the silk protein as suggested by Subburathinam et al., (1996) for nutritional fortificated mulberry leaves. Our results
demonstrated that soyprotein extract supplementation is of importance in regulating the fibroin
gene expression at transcriptional level. This
would be a practical contribution to sericulture
for maximizing economic traits of economically
important insect, B. mori.
Acknowledgements
The authors are grateful to the International
Foundation for Science (IFS), Sweden to M.K
(B/2520) and the Council for Scientific and Industrial Research (CSIR) (37[1054]) 00/EMR-II),
India for their financial support.

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