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 amplications 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. References Aghdasi B, Ye K, Resnick A, Huang A and Snyder SH. FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle. Proceedings of The National Academy of Sciences of The United States of America, 98: 2425-2430. 2001. Chen H, Zhao ZS, Li DQ, Jiao LP, Yu CM and Li Y. Studies on the body size and slaughter performance in chicken, quail and chicken-quail hybrid. Heilongjiang Animal Science and Veterinary Medicine, 4: 36-37. 2005. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaus N, Sverdlov ED and Siebert PD. Suppression subtractive hybridization: a method for generating differentially regulated or tissuespecific cDNA probes and libraries. Proceedings of The National Academy of Sciences of The United States of America, 93: 6025-6030. 1996. Duguin JL and Dinauer MC. Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapieinfection. Nucleic Acids Research, 18: 2789-2792. 1990. Hara E, Kato T, Nakada S, Sekiya L and Oda K. Subtractive cDNA cloning using oligo(dT) 30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells. Nucleic Acids Research, 19: 7097-7104. 1991. Hirao M, yonemura I and Miyazaki J-I. Genomic structure of the chicken slow skeletal muscle troponin T gene. Original Research Article, 338: 243-256. 2004. Ivaska J, Pallari HM and Nevo J. Novel functions of vimentin in cell adhesion, migration, and signaling. Experimental Cell Research, 10: 2050-2062. 2007. Jiang YN, Li N and Wu CX. Studies on the molecular biology of myogenesis. Journal of Agricultural Biotechnology, 7: 387391. 1999. Jiao LP. Analysis of the effection in hybrids of Chicken-Quail. The University of ShiHeZi, 4: 69-73. 2001. Kohno K, Izumih and Uchiumi T. The pleiotropic functions of the Y-box-binding protein, YB-1. Bioessays, 25: 691-698. 2003. Li WH, Deng XM, Li N and Wu CX. Isolation of differentially expressed genes in double-muscling large white pig by SSH and Q-PCR strategy. Progress in Biochemistry and Biophysics, 32: 353-358. 2005. Liang P and Pardee AB. Differential display of eukarytic messenger RNA by means of the polymerase chain reaction. Science, 257: 967-971. 1992. Liang et al.: Construct Subtractive cDNA Library of Muscle Liao HR. People’s Daily. 1992. Lisityn N and Wigler M. Cloning the differences between two complex genomes. Science, 259: 946-951. 1993. Perry SV and Troponin T. Genetics,properties and function. Journal of Muscle Reserch and Cell Motility, 19: 575-602. 1998. Prestle J, Janssen PM, Janssen AP, Zeitz O, Lehnart SE, Bruce L, Smith GL and Hasenfuss G. Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca(2+) leak from the sarcoplasmic reticulum and increases contractility. Circulation Research, 88: 188-194. 331 2001. Vater R, Cullen MJ and Harris JB. The expression of vimentin in satellite cells of regeneration skeletal muscle in vivo. Histochemical Journal, 26: 916-928. 1994. Watanabe T and Mizutain M. Domonstration of the maternal inheritance of avian mitochondrial DNA in chicken-quail hybrids. Journal of Experimental Biology, 236: 245-247. 1985. Zot AS and Potter JD. Structure aspects of troponin-tropomyosin regulation of skeletal muscle contraction. Annual Review of Biophysics and Biophysical Chemistry, 16: 535-559. 1987.
© Copyright 2024