http:// www.jstage.jst.go.jp / browse / jpsa
doi:10.2141/ jpsa.0110124
Copyright Ⓒ 2013, Japan Poultry Science Association.
≪Research Note≫
Construction and Analysis of Subtractive cDNA Library of Muscular
Tissue in Hybrid of Chicken-Quail Related to Myogenesis
YaoWei Liang1, Wei Zhen1, MiLa G.L. Jiaerheng1, ZongSheng Zhao1 and HongMei Zhang2
1
Animal Genetics and Breeding Department, Animal Science and Technology College,
Shihezi University, Shihezi, Xinjiang, China, 832000
2
Analysis Department, The First Affiliated Hospital of Medical Colledge of Shihezi University,
Shihezi, Xinjiang, China, 832000
Myogenesis is a complex developmental progress in which a variety of transcription factors play essential roles in
regulating myogenesis. However, the genetics programs that control myogenesis molecular mechanism are poorly
understood. Therefore, meat-breeding and egg-breeding chicken were selected as male parent, Korean quails were
used as female parent for hybridizing. To identify differentially expressed genes between the different hybrids,
suppression subtractive hybridization (SSH) have been undertaken for generating cDNA collections of representative
mRNAs specific to muscle tissue with meat-hybrid versus egg-hybrid. Following SSH, 54 clones were sequenced
and analysed through BLASTX. The results showed that 26 Expressed Sequence Tags (ESTs) found no homology
while the other 22 ESTs found the homology in GenBank and 4 ESTs might have correlation with myogenesis.
Key words: gene expression, hybrid of chicken-quail, muscular tissue, myogenesis, suppression subtractive
hybridization
J. Poult. Sci., 50: 326-331, 2013
Introduction
Myogenesis is a complex developmental progress in which
myogenic regulatory factors (MRFs), mysgenic enhancer
factor 2 (MEF2), Pax3, Pax7, myostatin, fibroblast growth
factor (FGF), and troponin T (TnT) play essential roles in
controlling the expression of muscle-specific genes in different muscle cells and in different developmental phases (Jiang
et al., 1999). However, the genetics programs that control
myogenesis molecular mechanism are still poorly understood.
The interspecific hybridization of chicken (♂) and quail
(♀) has been succeeded in Japan (1983), United States
(1985), Malaysia (1989) (Watanabe et al., 1985) , and the
test was successful for the first time by professor Liao in
Shihezi University in 1992 and China (Liao, 1992). Jiao
(2001) studied that the chicken is characterized with large
somatotype and stronger while the quail is early-maturing,
Received: October 13, 2011, Accepted: March 7, 2013
Released Online Advance Publication: April 25, 2013
Correspondence: Dr. Z.S. Zhao, Animal Genetics and Breeding Department, Animal Science and Technology College, Shihezi University,
Shihezi, Xinjiang, China, 832000. (E-mail: [email protected])
Dr. H.M. Zhang, Analysis Department, The First Affiliated Hospital of
Medical Colledge of shihezi University, Shihezi, Xinjiang, China, 832000.
(E-mail: [email protected])
rapid growth and superior meat, however, chicken-quail
hybrid combines the advantages of chicken and quail
mentioned above, and the skin of hybrid is translucent and
more thin, the feather is more beautiful. Chen et al. (2005)
measured the body size and muscle performance of 9 weeks
chicken, quail and chicken-quail hybrid, the result showed
that there was a significant difference in the parameters of
muscle traits included breast muscle diameter, leg muscle
diameter and water holding capacity between chicken, quail
and chicken-quail hybrid. Through long-term breeding,
broiler and layer have significantly difference in weight and
growth rate. Thus the meat-hybrid and egg-hybrid also have
significantly difference.
Suppression subtractive hybridization (SSH) is a highly
effective method for amplifying differentially expressed
genes. It overcomes the technical limitations of traditional
subtractive hybridization method and dramatically increases
the probability of obtaining low-abundance differentially
expressed cDNA (Diatchenko et al., 1996). In this study,
meat-hybrid and egg-hybrid, breast muscle tissue which
collected through embryonic days 13 (E13, n＝3 per day)
were used as experimental material to construct their
subtractive hybridization libraries for obtaining the information of differentially expressed genes at meat-hybrid and egghybrid. And this will be a foundation for further cloning and
functional analysis of the genes related to myogenesis, even
Liang et al.: Construct Subtractive cDNA Library of Muscle
provide a new aspect of the molecular mechanism of myogenesis.
Materials and Methods
Animals and Tissue Collection
We selected five meat-breeding cocks (Ankao) and five
egg-breeding cocks (Lohmann Brown) as male parent, and
selected two hundred Korean quails as female parent. We
collected semen from cocks, then input the semen to reproductive tract of quail using artificial insemination technique. Eggs produced by the fertilized quail are meat-hybrid
fertilized eggs and egg-hybrid fertilized eggs. Fertilized
eggs were obtained from birds of Korean quails by crossfertilizing. Eggs were incubated in an incubator at 37±0.5
℃ and 60% humidity. All the breast muscle tissues were
collected through embryonic days 13 (E13, n＝3 per day).
And all the tissues were sampled and transferred into cryotubes and frozen in liquid nitrogen. In addition, institutional
approval was obtained.
RNA Extraction and mRNA Isolation
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). PolyA+ RNA was obtained using
Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocols. The quality of the total
RNA and PolyA+ RNA was tested by 1. 2% agarose gel
electrophoresis, and stored −80℃ until use.
Suppression Subtractive Hybridization
SSH was performed using the PCR-SelectTM cDNA subtraction Kit (Clontech, Palo Alto, CA) following the manufacturer’s instruction. Briefly, the cDNA synthesized from
the breast muscle tissues of the egg-hybrids were used as the
“driver” and that from the meat-hybrids were used as the
“tester” for the forward subtraction. The double strand
cDNA fraction were digested with Rsa Ⅰ to obtain shorter,
blunt-ended molecules and specific adaptors were ligated to
the tester. Tester and driver were hybridized to exclude
common sequences and the resultant cDNA was subjected to
two rounds of PCR for the selective amplification of differentially expressed sequences, using adaptor specific primers.
The cDNA subtraction efficiency was estimated by comparing β-actin cDNA abundance before and after subtraction
through the use of increasing number of PCR cycles.
Subtractive Library Construction
The SSH libraries enriched for differentially expressed
cDNA were constructed by ligating the subtracted cDNAs
into the PMDTM 18-T vector (Takara, Shiga, Japan), transferring them into DH5α Escherichia coli cells that were
plated onto LB agar containing 100 μg/ml ampicillin, 1 mM
isopropyl-β-D-thiogalactopyranoside (IPTG), and 80 μg/ml
5-bromo-4-chloro-3-indolyl bd-galactopyranoside (X-gal),
then incubated at 37℃ overnight to obtain a subtracted expressed sequence tag (EST) band. Individual recombinant
colonies were picked and grown in LB medium containing
amplicillin (50 μg/ml) on 96-well microtiter plates.
Identification of Positive Clones by PCR
Primer paris specific to the sequenced fragments were
provided by PCR-Select cDNA Subtraction Kit (Clontech,
327
Palo Alto, CA) for identifying the clones. The bacteria were
shaken in liquid LB medium overnight and used as the
template. The PCR products were subjected to electrophoresis on a 1.5% agarose gel in a TAE buffer and then visualized
using ethidium bromide staining.
Sequencing and Sequences Analysis
The positive clones were sequenced by the Tsingke Biotch
Co. Ltd (Beijing, China). And the cDNA sequences were
categorized to proper (GO) terms from the Gene Ontology
Consortium annotation categories for molecular functions
and biological processes. A comparative analysis by
BLASTX and functional annotation was performed also.
Result
Extraction of Total RNAs
The test of total RNA integrity with agarose gel electrophoresis showed two bright bands, corresponding to
ribosomal 28S and 18S RNA with a ratio of intensities of 2:1
(Fig. 1). The eletrophoresis gel imaging and documentation
system reveal that the values of A260/A280 were 1.8-2.0.
This indicated that the purity of RNA was satisfactorily high.
Evaluation of Subtraction Efficiency
Difference in amplification patterns between the subtracted and unsubtracted cDNA samples indicative of successful subtraction were visually observed. The subtraction
efficiency was subsequently evaluated by measuring of βactin abundance in the subtracted and unsubtracted populations. For this purpose, portions of the PCR products obtained after 18, 23, 28, and 33 cycles were examined on a
2.0% agarose/EtBr gel (Fig. 2). The amount of β-actin decreased significantly after subtraction, indicating that the
subtraction had worked well.
Construction of Subtractive cDNA Library and Identification of Insert Size
After hybridization and second round PCR products, the
subtractive libraries were ligated to PMD 18-T vector and
transformed into E. coli, the positive clones were screened by
the white/blue colony. One hundred and fourteen clones
were randomly selected. The PCR amplications of cDNA
The result of total RNA electrophoresis. Lane 1
and 2: total RNA of muscle tissue of meet-hybrid. Lane 3
and 4: total RNA of muscle tissue of egg-hybrid.
Fig. 1.
Journal of Poultry Science, 50 (4)
328
Evaluation of subtraction efficiency of subtracted DNA.
Lane1-4 PCRs with β-actin gene were perform on the subtracted DNA.
Lane5-8 PCRs with β-actin gene were perform on the unsubtracted
DNA. Lane 1 and 5: 18 cycles. Lane 2 and 6: 23 cycles. Lane 3 and 7:
28 cycles. Lane 4 and 8: 33 cycles. Lane M: marker (5000 bp).
Fig. 2.
Fig. 3. Identification of the inserted cDNA fragments in subtractive
cDNA libraries by PCR. Lane1-13: the randomly picked clones from
the subtractive cNDA libraries. Lane M: marker (2000 bp).
inserts then revealed that 105 clones were insert-containing
clones. And the cloned insert sizes ranged from 250 to 700
base pairs (bp) (Fig. 3) with an average fragment size of 426
bp. The single exogenous fragment of the library were over
90%, suggesting that the cDNA subtraction was successful.
Sequencing and Analysis of Clones from the SSH Library
Even though all the clones of subtractive libraries were
screened by dot blot hybridization, only those clones with
strong hybridization signal were sequenced. These differentially expressed ESTs were then analyzed by homology
analysis in GenBank database. Finally, 26 unigenes were
obtained from the subtractive cDNA library (Table 1).
Among these genes, 4 (15.4%) might have correlation with
myogenesis, while the other 22 (84.6%) were classified as
unknown-function genes.
Discussion
In higher eukaryotes, biological processes such as cellular
growth and organogenesis are mediated by programs of
differential gene expression. To understand the molecular
regulation of these processes, the relevant subsets of differentially expressed genes of interest must be identified,
cloned and studied in detail. SSH has been a powerful
approach to identify and isolate cDNAs of differentially
expressed genes (Duguin et al., 1990; Hara et al., 1991).
SSH is a new phenotype cloning technology after the mRNA
differential display (Liang et al., 1992) and representational
difference analysis (Lisityn et al., 1993). And SSH is used
to selectively amplify target cDNA fragments (differentially
expressed) and simultaneously suppress nontarget DNA
amplification (Diatchenko et al., 1996).
Myogenesis is a complex developmental progress in which
a variety of genes conduct a precise control in transcription
level, a variety of signal pathways were composed by kinds
of transcription factors control myogenesis molecular mechanism. Using the SSH, subtractive cDNA library of muscular tissue in double muscling and non-double muscling
large white pig was constructed by Li et al. (2005). In this
Liang et al.: Construct Subtractive cDNA Library of Muscle
Table 1.
Clone #
001
329
Differentially expressed genes identified in the chicken-quail hybrids by SSH
Sequence similarity via Blast searching
GenBank
Accession No.
Species
E-valuable
Identity
(%)
M22158.1
Gallus gallus
2.00E-156
93
NM_204641.1
Gallus gallus
0
91
L13032.1
Gallus gallus
0
92
M15852.1
Gallus gallus
0
100
003
Chicken skeletal muscle troponin T
variant Tnt-4
Gallus gallus FK506 binding protein
3, 25 kDa (FKBP3)
Gallus gallus YB-1 protein
004
Chicken vimentin gene
005
Gallus gallus BAC clone CH261-36N8
from chromosome z
Gallus gallus BAC clone CH261-162G20
from chromosome w
Gallus gallus BAC clone CH261-90G19
from chromosome w
Coturnix japonica W chromosome
AC192717.3
Gallus gallus
0
92
AC186171.3
Gallus gallus
0
95
AC205936.1
Gallus gallus
1.00E-106
95
AB189144.1
Gallus gallus
1.00E-147
91
Gallus gallus BAC clone CH261-98P16
from chromosome z
Gallus gallus BAC clone CH261-187N23
from chromosome w
Gallus gallus BAC clone CH261-167F2
from chromosome z
Gallus gallus BAC clone TAM31-49F22
from chromosome w
Gallus gallus BAC clone CH261-18A24
from chromosome w
Gallus gallus BAC clone CH261-172N8
from chromosome w
Gallus gallus BAC clone CH261-75N4
from chromosome w
Gallus gallus BAC clone CH261-161K24
from chromosome z
Gallus gallus BAC clone CH261-36N8
from chromosome z
Gallus gallus BAC clone CH261-131E6
from chromosome w
Gallus gallus BAC clone CH261-98P16
from chromosome z
Gallus gallus BAC clone CH261-162G20
from chromosome w
Gallus gallus BAC clone CH261-162G20
from chromosome w
Gallus gallus BAC clone CH261-168J14
from chromosome z
Gallus gallus BAC clone TAM32-10H12
from chromosome z
Gallus gallus BAC clone CH261-29M22
from chromosome unknown
Gallus gallus BAC clone TAM32-52P23
from chromosome z
Gallus gallus BAC clone CH261-163J22
from chromosome z
AC215792.3
Gallus gallus
0
93
AC186812.3
Gallus gallus
1.00E-138
95
AC192779.2
Gallus gallus
0
95
AC175394.3
Gallus gallus
6.00E-152
95
AC186851.2
Gallus gallus
0
95
AC173212.3
Gallus gallus
3.00E-29
95
AC175832.2
Gallus gallus
1.00E-163
94
AC202790.4
Gallus gallus
0
92
AC192717.3
Gallus gallus
0
92
AC182256.2
Gallus gallus
4.00E-163
95
AC215792.3
Gallus gallus
3.00E-160
93
AC186171.3
Gallus gallus
0
93
AC186171.3
Gallus gallus
1.00E-150
92
AC188391.2
Gallus gallus
2.00E-156
93
AC201860.3
Gallus gallus
0
94
AC145927.3
Gallus gallus
7.00E-95
94
AC186550.3
Gallus gallus
0
93
AC187766.3
Gallus gallus
0
92
002
006
007
008
009
010
011
012
013
014
015
016
017
018
019
020
021
022
023
024
025
026
330
Journal of Poultry Science, 50 (4)
investigation, CAMK2, RYRI and IGFBP7 which related to
myogenesis were screened, these genes might be associated
with the double-muscling character (Li et al., 2005). In the
present study, subtractive cDNA library of muscular tissue in
hybrids of chicken-quail were constructed successfully
which strongly suggested that screening the differentially
expressed genes in two kinds of hybrids by using SSH is
feasible.
In the present investigation, 4 function genes-FK506 binding protein 3 (FKBP3), TnT, Vimentin and Y-Box binding
protein-and 22 unknown-function genes were screened.
Troponin T (TnT) was a key protein for Ca2+-sensitive molecular switching of muscle contraction. In vertebrates, three
TnT genes have been identified, which produce isoforms
characteristic of cardiac, fast skeletal, and slow skeletal
muscles (Hirao et al., 2004). And TnT interacts with troponin C (TnC), troponin I (TnI) and actin, which plays a
central role on calcium-mediate signal system of thin filaments of striated muscle in vertebrates (Zot et al., 1987;
Perry et al., 1998).
Besides the activity of Peptidyl-prolyl cis-trans isomerase
(PPIase), FK506 has some other functions, including regulating the activity of Ca2+ channel, regulation of cell cycle
and function on ontogeny (Aghdasi et al., 2001; Prestle et
al., 2001). A further study is needed to confirm that FK506
regulates myogenesis through the functions mentioned.
Y-box binding protein family, existing in both lower and
higher eukaryotes, has a number of biological functions. Ybox binding protein is a kind of transcription factor which
specifically interacts with the Y-box (CTGATTGGCCAA)
of enhancer and promoter of target gene (Kohno et al.,
2003).Therefore Y-box binding protein may impact myogengesis.
Vimentin is the major intermediate filament protein of
mesenchymal cells. Ivaska J et al. (2007) studied that several key functions for vimentin were not obvious at first
sight. Vimentin emerges as an organizer of a number of
critical proteins involved in attachment, migration, and cell
signaling. The highly dynamic and complex phosphorylation of vimentin seems to be a likely regulator mechanism for
these functions. The implicated novel vimentin functions
have broad ramifications into many different aspects of cell
physiology, cellular interactions, and organ homeostasis
(Ivaska et al., 2007). Vater et al. (1994) investigated the
expression of the vimentin in skeletal muscle during a cycle
of degeneration and regeneration. Venom from the Australian tiger snake, Notechis scutatus scutatus, was used to
initiate the breakdown of the soleus muscle of young, mature
rats in vivo. They discovered that vimentin was absent in
control adult muscle fibres, but was identified in activated
satellite cells 12 h after venom assault. The amount of this
protein rose during the early stages of regeneration, reaching
its peak at 2-3 days. At this time, the expression of musclespecific intermediate filament protein, desmin, began. As
the abundance of desmin increased with the maturation of the
regenerating myofibres, the abundance of vimentin declined
until it was no longer detectable in mature regenerated fibres
(Vater et al., 1994). Vimentin plays an important role during
satellite cell activation in the early stages of regeneration.
And vimentin may play an important role during the progress
of myogenesis.
There are still a lot of unknow ESTs in the present study,
no homologous sequence can be found from Genebank nucleotide database, these genes represent the unknown genes,
which may be good candidates for re-examination of expression or function relevant to myogenesis in future research. So, there is a still a long way to go to illuminate the
molecular mechanism of myogenesis.
Acknowledgments
This work was funded by the research grants from Ministry of Science and Technology of China.
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