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Expression of silk gene in response to P-soyatose - Journal of Cell ...

A Ahmed Journal Mansour of Cell and Molecular Biology 6(2): 163-174, 2007

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

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 3 H 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 beşinci evre erkek larvasında P-soyatoz (hidrolize edilmiş soya

proteini) takviyesine cevap olarak ipek geni ekspresyonu

Özet

Research Article 163

Bombyx mori L. ipek bezleri, kozaya dönüşmeden önceki dönemde fazla miktarda ipek proteinlerin üretiminin

modellenmesinde kullanılmıştır. Sentezlenen ipek proteinlerinin miktarı, ipek larvaların beslenmesi

geliştirilerek arttırılabilir. Bu çalışma B. mori erkek larvasının beşinci instar döneminde P-soyatoz (hidrolize

edilmiş soya proteini) takviyesi ile ipek gen ekspresyonundaki değişimleri sunmaktadır. Soya protein takviyeli

gruptaki ipek bezi proteinlerinin Western blot analizi, H ve L zincirleri için yoğun boyanma göstermiştir.

P-soyatozla beslenmiş erkek larvada protein sentezi, kontrol grubuna göre artmıştır. Bu durum, Psoyatozla

besili larvadaki arka ipek bezlerindeki 3 H leucine işaretlemesi ile kendini göstermektedir. Northern

blot analizi de ayrıca soya protein takviyesinin ipek fibroin sentezini, dolayısıyla fibroin mRNA seviyesini

arttırdığını kanıtlamıştır. Bu sonuç, B.mori’nin ekonomik açıdan faydalı olacak özelliklerinin güçlendirilmesini

yansıtmaktadır.

Anahtar Sözcükler: Bombyx mori , ipek fibroin, P-soyatoz, besin takviyesi, Northern blotlama

_______________________________________________________________________________________


A 164

Raman et al.

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

27±2˚C and a relative humidity of 75±5% (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.


Extraction of silk proteins from the silk gland and

cocoon

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% β-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.

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.

P-soyatose mediated gene expression in Bombyx mori 165

A

Labeling of proteins in vivo in the posterior silk

gland

Intra-coelomic injection of 10µCi 3 H 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 3 H leucine incorporation into the

posterior silk gland proteins

To determine the amount of radioactivity incorporated

into the silk glands, an aliquot (5µl) 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 3 H 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


A 166

Raman et al.

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

µg 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 10 6 cpm/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 3 H 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).


larvae. A similar trend in the incorporation of

radioactivity ( 3 H) 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 3 H Leucine incorporation into fibroin

polypeptides

The incorporation of 3 H 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

P-soyatose mediated gene expression in Bombyx mori 167

A

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 6 th

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 4 th day.


A 168

Raman et al.

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 12-

18 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 3 H 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.


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-

CPM / Pair of Posterior……...

silk glands

18000

16000

14000

12000

10000

8000

6000

4000

2000

0

E

H Chain

C

E

L Chain

P-soyatose mediated gene expression in Bombyx mori 169

A

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

C

3

4

5

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.

6


A 170

Raman et al.

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.


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.

P-soyatose mediated gene expression in Bombyx mori 171

A

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.

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