cDNA libraries were constructed from different
tissues of adult and juvenile black tiger shrimp Penaeus monodon. The
details of tissues used for library construction were shown in the Table below.
For standard cDNA libraries, total RNA was extracted from tissues of interest
using TRI-REAGENT (Molecular Research Center). Messenger (m) RNA was further
purified using a QuickPrep micro mRNA Purification Kit (Amersham
Pharmacia Biotech). Five micrograms of mRNA were subjected to complementary cDNA
synthesis using an Uni-Zap XR cDNA synthesis kit (CLONTECH). Size-selected cDNAs
(> 500 bp) were ligated to dephosphorelated Eco RI/Xho I-digested Uni-Zap
vector and packaged in vitro. The titer of the primary library was
estimated. The library was then amplified and subsequently converted to the
pBluescript libraries by in vivo excision. EST clones were randomly
picked and subjected to nucleotide sequencing using MegaBace1000 (Amersham)
Normalized cDNA library was constructed from standard cDNA library established
previously. The normalization method is a reassociation-kinetics-based approach
involving hybridization of a 20-fold excess of cDNA inserts excised from a
plasmid DNA preparation of the standard EST library with the library itself in
the form of single-stranded circles (Bonaldo, Lennon and Soares, 1996; Carninci
et al., 2000). The cDNA inserts were labelled with biotin. The remaining
single-stranded plasmids of rarely expressed genes were removed from the hybrid
by using streptavidin beads and were converted to double-strands by using Pfu
polymerase (Promega). The double-stranded cDNA inserts were then cloned into the
bacterial host to propagate a normalized library.
Tester and driver mRNA were synthesized first and second strand cDNA. Then the cDNA were digested
with Rsa I. This digested driver cDNA served as the experimental driver
cDNA. The tester cDNA was ligated with adapters to create tester cDNAs for
subtraction. cDNAs from tester (target) and those from driver were subjected to
subtractive hybridization and selective PCR amplification using a PCR-Select
cDNA Subtraction Kit (Clontech). The subtractive fragments were further
screened using a PCR-Select Differential Screening Kit (Clontech) and cloned
into the pGEM-T vector (Promega)
|
Library
Name |
code |
Tissue |
Titer |
No. of EST |
|
Antennal
gland-normalized |
AG-N-N01 |
Antennal
gland of broodstock wild shrimp |
--- |
966 |
|
Brain and
Thoracic ganglia |
BT-N-S01 |
Brain and
thoracic ganglia of wild mature female shrimp (Ang-Sila, Chonburi) |
3.20E+06 |
499 |
|
Eyestalk
library 1 |
ES-N-S01 |
Eyestalk
neural tissues of wild females with late 3rd- 4th stage of ovarian
development |
2.50E+05 |
138 |
|
Eyestalk
library 2 |
ES-N-S02 |
2.00E+05 |
489 |
|
Eyestalk
library 3 |
ES-N-S03 |
Eyestalk
neural tissues of wild females with 0-1st stage of ovarian development |
5.00E+04 |
965 |
|
Gill
|
GL-N-S01 |
Gill of
juvenile cultured shrimp |
6.00E+05 |
1,011 |
|
Gill-heat
stress |
GL-H-S01 |
Gill of
juvenile cultured shrimp treated with 34.5-36ºC seawater for 4 h |
5.93E+06 |
893 |
|
Gill-salt
stress 1 |
GL-ST-S01 |
Gill of
juvenile cultured shrimp maintained under 3 ppt salinity for 96 h, 1, 2 and
4 weeks |
5.93E+06 |
221 |
|
Gill-salt
stress 2-mtDNA subtracted |
GL-ST-S02 |
--- |
413 |
|
Gill-subtractive
1 |
GL-N-STH01 |
Gill of juvenile shrimp cultured in normal and heat stress condition (treated
with 34.5-36ºC seawater for 4 h) |
--- |
163 |
|
Gill-subtractive
2 |
GL-N-STC02 |
--- |
166 |
|
Gill-Epipodite
|
GlEp-N-S01 |
Gill and
Epipodite of juvenile cultured shrimp |
6.00E+06 |
1,641 |
|
Gill-Epipodite-normalize
|
GlEp-N-N01 |
--- |
2,717 |
|
Heart
|
HT-N-S01 |
Heart of
juvenile cultured shrimp |
1.55E+06 |
280 |
|
Hematopoietic tissue |
HPO-N-S01 |
Hematopoietic tissues of adult SPF shrimp obtained from Broodstock
Domesticated Program |
4.70E+05 |
904 |
|
Hemocyte
|
HC-N-S01 |
Hemocytes
of juvenile
cultured shrimp |
4.00E+06 |
594 |
|
Hemocyte-normalilzed
|
HC-N-N01 |
--- |
11,008 |
|
Hemocyte-heat
stressed |
HC-H-S01 |
Hemocytes
of juvenile
cultured shrimp exposed to heat-stress |
5.00E+05 |
1,054 |
|
Hemocyte-Vibrio
harveyi challenged |
HC-V-S01 |
Hemocytes
of juvenile
cultured shrimp injected with Vibrio
harveyi |
2.50E+05 |
443 |
|
Hemocyte-WSSV
challenged |
HC-W-S01 |
Hemocytes
of juvenile SPF shrimp obtained from Broodstock Domesticated Program
injected with WSSV |
1.00E+05 |
484 |
|
Hepatopancreas |
HPa-N-S01 |
Hepatopancrease of juvenile
cultured shrimp |
2.50E+05 |
598 |
|
Hepatopancreas-normalized 1 |
HPa-N-N01 |
--- |
699 |
|
Hepatopancreas-normalized 2 |
HPa-N-N02 |
--- |
570 |
|
Hepatopancreas-normalized 3 |
HPa-N-N03 |
--- |
1,972 |
|
Hepatopancreas-normalized 4 |
HPa-N-N04 |
--- |
1,580 |
|
Intestine
|
IN-N-S01 |
Intestine
of juvenile cultured shrimp |
5.70E+06 |
1,129 |
|
Lymphoid
organ |
LP-N-S01 |
Lymphoid
organs of juvenile
cultured shrimp |
3.20E+05 |
408 |
|
Lymphoid
organ- Vibrio challenged |
LP-V-S01 |
Lymphoid
organs of juvenile
cultured shrimp injected with Vibrio
harveyi |
3.10E+06 |
625 |
|
Lymphoid
organ- YHV challenged |
LP-Y-S01 |
Lymphoid
organs of juvenile
cultured shrimp injected with YHV |
2.50E+05 |
720 |
|
Lymphoid
organ-normalized |
LP-N-N01 |
Lymphoid
organs of juvenile
cultured shrimp |
--- |
949 |
|
Ovary
|
OV-N-S01 |
Vitellogenic ovaries of wild shrimp with 2nd stage of ovarian development |
4.00E+06 |
2,316 |
|
Ovary-normalized
|
OV-N-N01 |
--- |
1,756 |
|
Ovary-substractive
1 |
OV-N-ST01 |
Ovaries of
female broodstock shrimp having GSI = 5.689% and 1.434% |
--- |
198 |
|
Ovary-substractive
2 |
OV-N-ST02 |
--- |
192 |
|
Testes
|
TT-N-S01 |
Testis of
broodstock cultured shrimp |
5.66E+06 |
889 |
|
Testes-subtractive
1 |
TT-N-ST01 |
Testes of
male broodstock and juveniles shrimp |
--- |
168 |
|
Testes-subtractive
2 |
TT-N-ST02 |
Testes of
male broodstock and juveniles shrimp |
--- |
183 |
