it give information about the silk worm production and its biochemical and technical studies....

2. Biochemical and
Biotechnological advances in
Sericulture
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3. BIOTECHNOLOGICAL
ADVANCEMENTS IN
SERICULTURE

4. Introduction
 The recent advances in Recombinant DNA techniques have revolutionized biology and
given a fresh impetus to biotechnology techniques of genetic manipulation have found a
place in all spheres of modern biology, both basic and applied.
 Silk considered as the queen of fibres,is a proteineaous in nature.
 The bulk of commercial silk is produced from the mulberry silk worm Bombyx mori of
which there are many strains from bivoltines to multivoltines.
 The other silk worm commercially exploited for silk production are eri (Philosamia spp.)
and tasar.

5. Better races of Silkworms
 Traditionally, strain improvements has used breeding methods for the production of high
yielding starins with such desireable starins, as disease resistance and heat tolerance.
 Where intensive breeding studies have been carried out for long periods of time.
 The traditional approaches appears to be reaching the natural biological limits of
silkworm with regard to growth rate, silk yield and fecundity.

6. Better races of Silkworms

7. Transferring sturdiness genes
 The Indian a native races of silkworms 9 (e.g: the Mysore )strain, though poor
yielder of silk, are much more tolerant to high temperature as well fluctuations in
temperature compared to the high yielding bivoltine strain.
 They are also capable of tolerating of poor quality of mulberry leaves used as
feed and appeared to the more resistance to the locally prevalent diseases.

8. Transferring sturdiness genes
 If the genes are responsible for any of these traits in the native races can be identified and
isolated,they may be transferred to the high yielders transferring fibroin genes.
 The fibroin heavy chain(Fibroin H) gene from B.mori is very large, including intron of 1.2 Killo base
pairs towards the upstream end of the gene and codes for a messenger RNA of 15 Killo bases.
 If the fibroin genes from high quality strain proves to be superior In terms of .quality of silk fibers,
transferring such gene to the more study native races may be a convenient alternative to identifying
and isolating the less tangible gene for studiness.

9. Gene transfer methods:
 It is clearly essential that methods of transfer of foreign genes into
germ line of silkworm be established.
 At the moment there are two ways of doing this.However these are
yet to be perfected in silk worm.

10. Microinjection
 Cloned genes (say fibroin) are directly introduced into eggs or early embroys by microinjection
techniques.
 If such embryos except and integrate the microinjecting DNA into their genome they should
develop into transgenic animals,harboring additional are alternative copies of the gene.
 Electroporation and other methods of direct introduction of DNA into dechorionated eggs may
also be useful for arriving at the same result.

11. Microinjection
 The discovery of transposons like sequences in silkworm DNA(analogues to the P
element of Drosophilla) raises the possibility of exploitation of these sequences to
target and integrate foreing DNA.

12. Virus mediated gene transfer
 The nuclear polyhedrosis virus (NPV) which is pathogenic to silk
worm can be exploited as a vector for introduction of foreign gene.
 Experiments that used in insect derived culture have already made
use of this method.

13. Scanning electron micrographs showing
Bombyx mori NPV

14. Biotechnology in Moriculture
 It is clear that the availability of sturdy silkworms is most important as far as the quality and
yield of silk are concerned.
 As essential prerequisite for sturdiness of silkworms in high- quality mulberry leaves.
 Considering that nearly 90 per cent of silk produced in India is mulberry silk, attempts to
improving the mulberry plant (Morus alba) itself by application of biotechnology are
certainly desirable

15. Biotechnology in Moriculture
 At present, the genetics of pure mulberry species is poorly defined. The methods of plant tissue
culture and clonal propogation of high quality mulberry plants can be undertaken.
 Host plant improvements is even more important in case nonmulberry silkworms.
 Development of alternative food sources and synthetic or semisynthetic diets for mulberry and
nonmulberry silkworms is another area that needs attention.

16. Synthesis of silk proteins in the silk gland
 The silkworm possesses an ability to synthesize large amounts of silk
proteins in its silk gland.
 The mechanism of silk protein synthesis has been extensively studied at
the molecular level . Silk consists of two proteins called fibroin and sericin.
 Fibroin is the main component of silk fibers, while sericin is a kind of glue
protein that coats the surfaces of the fibers.

17.  Fibroin accounts for about 75% of all silk protein and is produced in the posterior silk
gland (PSG).
 The remaining 25% is sericin, which is synthesized in the middle silk plasmid.
 In the helper plasmid, the piggyBac transposase gene is under the control of the
silkworm cytoplasmic actin gene (A3) promoter and possesses only one ITR.

18.  Since transposition of the transgene requires two ITRs, the
transposase gene in the helper plasmid does not insert into the
chromosome of the silkworm.
 A solution containing a mixture of vector and helper plasmid DNAs
is injected into the silkworm eggs at 2-8 h after oviposition and the
hatched larvae are reared until they reach adulthood.
 The adults are then mated with each other or the host strain to
obtain the G1 generation.

20. Systems for producing recombinant proteins in
the silk gland
 The system for producing recombinant proteins in the transgenic
silkworm uses the
 silk synthesis system in the silk gland
 silk genes that are strongly expressed in the silk gland.
 Until now, production systems using the fibroin L and H chain and FHX
genes have been used for production in the PSG.

21. Recombinant protein production system using the
fibroin L chain gene:
 The first expression system for the production of recombinant proteins uses the fibroin L chain
gene.
 The system has been applied to the production of human collagen and enhanced green
fluorescent protein (EGFP).
 The expression vector contains the promoter and full cDNA sequence of the fibroin L chain gene.

22.  The fibroin L gene promoter of the fusion gene works well without the help of
other factors,
 Fibroin contains three different proteins called fibroin heavy (H) and light (L)
chains and fibrohexamarin (FHX), which are produced in molar ratio of 6:6:1,
respectively .
 The character of silk as a fiber is determined by the large fibroin H chain, whose
molecular weight is 350-400 kDa.

23.  The fibroin L chain and FHX are small proteins with molecular weights of about
25 kDa.
 The three molecules form a large complex called the fibroin elementary unit,
whose molecular weight is 2.3 MDa.
 Joining of the fibroin H and L chains by an S-S linkage is required for the
formation of the complex and the secretion of the fibroin proteins from the silk
gland cell to the lumen of the PSG.

24.  The fibroin H, L, and FHX genes are single-copy genes and the DNA sequences
regulating their PSG-specific expression has been analyzed by in vitro transcription
of the cloned gene .
 The fibroin secreted into the lumen of the PSG moves to the MSG, where it
accumulates prior to spinning.

26. Biochemical Studies on the
Formation of silk protein

27.  Biosynthesis of the silk proteins in silkworms has been studied by a
few research workers but little is known of the formation of the silk
proteins in the course of the silkworm growth.
 About 70 per cent is directly derived from the proteins of the
mulberry leaves which is a direct method of formation.
 But some part of the silk proteins, about 30 per cent is also derived
from the tissue proteins and body fluid proteins (indirect
formation).

28.  Direct formation of the silk proteins takes place during the period between
the 4th day of the 5th instar to maturity.
 Indirect formation takes place during the period from the 8th day of the 5th
instar to just before termination of the spinning, after reaching maturity.

29. After mulberry leaves labeled
with C14 or amino acids
labeled with C14 were given
per os to the silkworm larva
in the different stages, these
silkworms were reared on
non-radioactive mulberry
leaves until they produced
their cocoons.
These cocoon fibres were unwound,
and the distribution of C14 in each
silk filament, the length of which was
about 1,200 m, was investigated.
If the silk proteins are
directly derived from amino
acids of the mulberry leaves,
it would be expected that a
great amount of C14 given to
the silkworm would appear

30. Experiment:
The radioactive mulberry
leaves were prepared by
placing a mulberry tree for
5 hours in an atmosphere
containing C14O2.
The radioactivity of
these leaves was
4,134 count/sec/cm2.
A radioautograph,
shows that glycine,
serine, glutamic acid,
aspartic acid,
leucine,
phenylalanine,
tyrosine, valine,
proline, lysine etc.
contain C14 in their
molecules.
This fact shows that
the radioactivity of
the mulberry leaves
is due to the
existence of the
C14in the molecules
of the constituent
amino acids of the
mulberry leaves.

32.  2) Amino Acids labelled with C14:
Amino acids are labelled with the C14.
 3) Administration of Radioactive Mulberry leaves and Radioactive
Amino Acids to the Silkworm:
Silkworm is administrated with the radioactive mulberry leaves and radioactive
amino acids in different stages from the 5th instar to the end of 5th instar.
These silkworms were reared on normal mulberry leaves until they produce
cocoons.

33. 4) Distribution of C14 on silk filament:
 All the cocoons produce by the silkworms that are given
radioactive mulberry leaves and radioactive amino acids
at different stages were unwound with the help of an
instrument and this filament was cut into pieces and
were degummed with the help of distilled water and
sodium carbonate solution.
 Each length was washed with ethanol and ether, and
dried. Each part obtained from the silk filament was
rearranged on a sheet in the order obtained from one
cocoon.

35. 5) Radioactivity of the amino acids isolated from radioactive silk:
 The silk produced by the silkworm given the radioactive mulberry leaves the distribution of C14
on the silk filaments produced by the silkworms having consumed the radioactive mulberry leaves
in different stages from the beginning of the 5th instar to the end was investigated.
 It was seen that the first 100 m of the silk filament unwound from one cocoon is made from the
mulberry leaves that the silkworm ate on the 4th day of the 5th instar: the section from 200 m to
400 m from those consumed on the 6th day of the 5th instar, and the section from 700 m to 1,000
m from those consumed on the 8th day of the 5th instar, namely just before reaching maturity.
 This experimental result seems to suggest that a great amount of the silk produced by one
silkworm is directly derived from the mulberry leaves, and that this direct formation of the silk
takes place during the period from the 4th day of the 5th instar.

36. Recent trends and Uses of
silk

37. Silk (a natural protein fiber)
 Silk is a natural protein fiber, some forms of which can be woven into textiles.
 The best-known type of silk is obtained from the cocoons of the larvae of the mulberry
silkworm Bombyx mori reared in captivity (sericulture).
 Silks are produced by several other insects, but generally only the silk of moth caterpillars
has been used for textile manufacturing.

38. adult insects such as web spinners produce silk,
Silk production production also occurs in Hymenoptera (bees,
wasps, and ants
arthropod produce silk, most notably various arachnids such as
spiders

39. Chemical Properties of Silk:
 Silk emitted by the silkworm consists of two main protein
 sericin and fibroin,
 fibroin being the structural center of the silk,
 serecin being the sticky material surrounding it.

40. Silk is an amazing ingredient:
 Silk Amino Acids are natural water-soluble source of acid derived from liquid
silk; these acids help moisture penetrate the skin and aid in skin healing.
 Silk Amino Acids add a 'silky' lather to soaps and also have moisture binding
properties.
 This luxurious recipe blends the silky and softening properties of liquid Silk
amino acids in our mild soap base to create an exquisite, silky, smooth, rich lather.

41. The use of Silk in cosmetics:
 Research has found that silk has moisture retention qualities and
UV ray blocking properties.
 So it is used in cosmetics.

42. Silk Cream (100% Fragrance Free):
 Smiss Silk Cream is a natural beauty cream for the face.
 It contains pure natural silk.
 Smiss Silk Cream protects the skin from pollutants, windburn,
sunburn and is especially suitable for sensitive skin

43. Smiss Natural Silk Lotion
 Smiss Natural Silk Lotion contains rich silk protein and
silk peptide, which keep your skin soft and moist.
 Regular use will help keep the skin soft, silky-smooth and
always youthful looking.
 Smiss Natural Silk Lotion is for all types of skin.

44. Silk Baby Cream:
 One million mothers proclaim that Smiss baby cream is a
safe and effective products.
 Smiss baby cream is made from high quality nutritional
elements, natural silk peptide and raw greasy lanolin
 which work together to protects the baby's tender skin.

45. SILK toothpaste:
 It is a pure natural health protection toothpaste.
 It contains the surface growth factors of the activated natural material EGF
and silk.
 In this way, it helps cells grow, absorb nutrition, sterilize, diminish
inflammation, prevent bleeding, ease pain, and heal wounds.

46. Conclusion and Future Prospects
 Transgenic silkworms can be used as bioreactors to produce recombinant proteins. They can be easily
generated using the DNA transposon piggyBac as a vector by injecting helper and vector plasmid DNA into
eggs just after oviposition.
 The recombinant protein production systems were constructed using silk genes expressed in the silk gland.
The expression system in the PSG is suitable for the production of genetically modified silk, and the silk made
by transgenic silkworms can be used to produce fabrics and biomaterials for medical purposes.
 The system in the MSG is suitable for the production of recombinant proteins that can be used for
pharmaceutical purposes. The latter system can yield up to 4 mg of recombinant protein per silkworm. This
yield is quite high compared to other systems and producing the transgenic silkworms is much cheaper and
faster than producing transgenic livestock.

47.  Posttranslational modification and folding of the proteins is more similar to that in mammals
compared to that in microorganisms or plants.
 The protein produced in the MSG forms the S-S linkage between the recombinant proteins and
is secreted into the cocoon. The N-linked glycans of proteins produced in cultured insect cells
and baculoviruses are generally of the paucimanose type, with the α-1,3 and α-1,6 fucose sugars
being attached to the core N-acetylglucosamine. In contrast, the protein produced in the MSG
does not have the α-1,3 fucose sugars and the end of the sugar chain contains N-
acetylglucosamine, indicating that glycosylation in the MSG is more similar to that in
mammalian cells

48.  This is an important reason for producing recombinant proteins in the silkworm, although
improvements to the system are still required.
 To increase the protein yields, analysis of the factors controlling protein expression and
translational efficiency is necessary, as are studies to modify glycosylation.
 The development of a method for targeting and replacing endogenous silk genes is also
important for the production of recombinant silks and increasing protein yields.
 In fact, targeted gene disruption can be achieved by injecting zinc finger nuclease mRNA.
Moreover, the development of a system for rearing transgenic silkworms in the field is important
for commercialization of the this industry.