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TRANSCRIPTOME-WIDE DISCOVERY OF INNATE IMMUNE-RELATED GENES AND MOLECULAR CHARACTERIZATION OF THE INFLAMMATORY/APOPTOTIC PATHWAY INMANILA CLAM (Ruditapesphilippinarum)

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Abstract
바지락은 전세계적으로 매우 중요한 양식 이매패류 종으로 한국을 비롯한 동아시아 지역과, 유럽 및 북미에서 넓게 서식하고 있다. 최근 바지락의 대량 폐사에 대한 연구가 보고되고 있다. 이러한 바지락의 대량 패사는 다양한 환경 요인, 환경오염, 그리고 감염 등이 그 원인으로 보고되었으며, 그중에서 특히 brown ring disease (BRD)와 perkinsus 감염증이 중요 원인으로 알려져 있다. BRD와 perkinsus 감염증과 같은 병원체에 의한 대량패사는 바지락의 생산량에 커다란 영향을 미치고 있어 경제적 손실 또한 커지고 있기 때문에 이러한 문제의 해결을 위하여 바지락의 면역체계에 대한 연구에 대한 관심이증가하고 있다. 병원체에 의한 바지락내의 면역반응을 이해하기 위해서는 분자 수준에서의 접근이 필요한데, 이는 해당 종에 대한 유전정보가 필수적이다. 하지만 NCBIGenBanK database에 등록되어 있는 바지락의 유전정보는 다른 양식 이매패류종인 굴이나 가리비 등에 비하여 매우 적은 7%에 불과한 실정이다.
바지락을 포함한 연체동물은 후천면역이 결핍되어 있다고 알려져 있다.그렇기 때문에 바지락과 같은 연체동물에서의 선천면역은 매우 중요한 역할을 하지만 해양연체동물의 선천면역반응에 대한메커니즘 연구는 매우 미비한 실정이다. 반면 포유류에서는 광범위하게 연구가 이루어지고 있으며, 이러한 자료는 연체동물의 면역 시스템을 연구하기 위한 방향을 제시하고 있다. 바지락과 같은 무척추동물에서는 선천면역이 외부 병원체감염으로부터 방어하기위한주요시스템으로작용한다. 이런이유로, 분자수준에서다양한병원체의감염에대한면역반응에대한이해는매우중요하다. 이연구에서는바지락의면역체계에대한이해를위하여부족한유전정보를확보하고자 454 Genome Sequencer Flex titanium을이용하여바지락의transcriptome 을분석하였고, 이를통하여 117,347개의유전자에대한정보를확보하였고, 이중에서 NCBI에등록된단백질들과의비교를통하여 24,327개의 unigene을확보하였다. 확보한 24,327개의 unigene에대한 data로부터중요한면역관련유전자로알려진MyD88, IκB, TNFα와세포사멸에중요한조절자로작용하는것으로알려진두개의 Bcl-2 family 단백질의전체서열을확보하고분자적특성및면역자극을통한발현변화를관찰하였다.
바지락의 RpMyD88은 1416 bp의 ORF로구성되어있으며, 471개의아미노산을암호화하고있으며, RpMyD88의아미노산서열에서 MyD88단백질의전형적인특징인 death doman과 TIR도메인을확인할수있었다. RpMyD88의아미노산서열은가리비 MyD88과 27%로가장높은 identity를나타내었다. RpMyD88은바지락의모든조직에서발현이관찰되었으며, 특히주요면역유전자인 hemocyte와 gill 그리고 mantle에서높은발현량을나타내었다. 반면 siphon, foot, adductor muscle에서는매우적은양의발현을나타내었다. RpMyD88은Vibrio tapetis와 LPS를주사한바지락의 hemocyte와 gill모두에서유의적인발현증가를나타내었으며, 이러한서열특성및유전자발현에관련된결과들은바지락에서도포유류와같은 MyD88 dependent signaling pathway가존재하며 RpMyD88역시박테리아에대한선천면역반응에서중요한역할을수행하고있을것으로예상할수있었다.
NF-kB signaling pathway는선천면역시스템에서가장중요한구성요소중의하나로, NF-kB의활성은 inhibitor of NF-κB (IκB)와의물리적상호작용에의하여조절되게된다. IκB가 NF-κB에결합하게되면 NF-κB의 nuclear localization signal을막음으로써 NF-κB를불활화상태로 Cytoplasm에가둬두게된다. 3장에서는 NF-κB의활성을조절하는 IκB의분자적특성과발현정보를나타내었다. 바지락 IκB (Rp-IκB)의 cDNA는 1032 bp 의 ORF로구성되어있고, 343개의아미노산을암호화하였다. Rp-IκB 단백질은IκB단백질의전형적인특징인 IκB degradation motif, PEST sequence 그리고 6개의 ankyrin repeats로구성되어있었다. 또한계통분류학적분석을통하여무척추동물의 IκB 단백질들과그룹을이루는것을확인하였다. Rp-IκB는모든조직에서지속적으로발현을나타내었으며, 주요면역세포인 hemocyte와 gill에서V. tapetis와 LPS 자극에의하여유의적인발현증가를나타내었다. 이러한결과는포유류에서 NF-κB의 key 조절자인 IκB와같이 Rp-IκB가박테리아의침임에대하여중요한역할을수행할것으로예상할수있었고, 이는 Rp-IκB 는포유류의 IκB orthologs들과비슷한기능을가지고있음을뒷받침하고있다.
포유류의 TNFα의 homology를바지락으로부터확인하였고, RpTNFα로명명하였다. RpTNFα는다른선행연구에서보고된 TNFα의전형적인특징인 Transmembrane과 TNF-signature를가지고있었다. qRT-PCR을통하여조직특이적발현을분석한결과모든조직에서발현이관찰되었으며, V. tapetis, LPS 그리고 poly I:C를주사하였을때이에반응하여 hemocyte와 gill에서유의적인발현증가를나타내었다.
Bcl-2는Mitochondrial membrane permiabilzation (MMP)의억제를통하여세포사멸을억제하는 Anti-apoptotic Bcl-2 sub-family다.Bax는pro-apoptotic Bcl-2 subfamily다. GS-flx transcriptome 서열에기초하여, Bcl-2 와 Bax like 단백질의 orthologue로예상되는 cDNA전체서열을바지락에서확인하였고, 이서열들은 Bcl-2 homology domain과 transmembrane (only RpBcl-2)을포함하고있었다. Real time PCR을통하여분석한조직특이적발현에서는모든조직에서발현이관찰되었으며, gill에서가장높은발현을나타내었다. V. tapetis를주사한후 gill과 hemocyte에서유의적인발현변화를나타내었다. 이러한결과는 RpBcl2와 RpBax가바지락의면역반에서포유류의 apoptotic regulator들과비슷한기능수행과함께면역에매우깊이관련있는것을나타내고있다.
Manila clam, Ruditapes philippinarum, is an important aquaculture species worldwide. Recent mass mortalities of manila clam landings, believed to be due to microbial infection, have significantly impacted production and sparked interest in immune-related research of this species. Genetic information is important to better understand the molecular basis for the host immune response against pathogen. However, genomic data of clam form just 7% in bivalve genomic database. The manila clam is an economically important species, but to date only a few fully-annotated genomic data are available in genomic databases.
The manila clam is known to lack an adaptive immune system, but itsmechanisms of innate immunity have yet to be fully elucidated.In contrast, the innate immune system in mammals has been extensively studied and this data may be used to provide significant insights into the immune system used by molluscs.The innate immune system is the main host defense in manila clam like invertebrates. Hence, it is important to understand the immune responses and their mechanisms against different microbial challenge at molecular level. In this study, in order to fill the gap in transcriptome sequence data abailable for the manila clam, transcriptome analysis was performed by Next-Generation Sequenicng (NGS) technology, furthermore molecular analysis of immune genes in manila clam, Ruditapes phillipinarum was investigated using transcription profiling.
Myeloid differentiation factor 88 (MyD88) is a universal adaptor protein which is required for signal transduction of TLR/IL-1R family. In this study, a novel molluscan MyD88 family member protein (named as RpMyD88) was identified from manila clam, Ruditapesphilippinarum. It was identified using BLAST algorithm from GS-FLXTMsequencing data. The cDNA of RpMyD88 consists of 1416 bp open reading frame (ORF) encoding 471 amino acid residues. The RpMyD88 contains death domain and Toll/interleukin-1 receptor (TIR) domain which are typical features of MyD88 family proteins. The predicted amino acid sequence of RpMyD88 shares 27% identity with scallop MyD88. The expression level of RpMyD88 mRNA was investigated in healthy and challenged clams by quantitative real-time RT-PCR. The RpMyD88 gene expression is ubiquitous in all selected tissues. The RpMyD88 mRNA was strongly expressed in hemocyte, gill and mantle. In contrast, it was weakly expressed in siphon, foot and adductor muscle. RpMyD88 was up-regulated in gill and hemocyte after immune challenge with both Vibrio tapetis and LPS challenge. All results considered, sequence characterization, comparison and gene expression data suggesting that MyD88 dependent signaling pathway is presence in manila clam and RpMyD88 plays an important role in innate immune response against bacteria.
The nuclear factor-kappaB(NF-κB) signaling pathway is one of the most importantcomponentsof the innate immune system and its activity is regulated by physical interaction with theinhibitor of NF-κB (IκB). Upon binding, IκB protein masks the nuclear localization signal (NLS) of NF-κB, thereby sequesteringinactive NF-kB inthe cytoplasm. Thus, elucidating the expression profile of such NF-kBinhibitors in the agriculturally-important manila clam will advance our understanding of the species' immune response to pathogenic threatsfor preventing and controlling molluscan diseases. Our investigations led to the identification a novel IκB (Rp-IκB) from the manila clam, Ruditapes philippinarum. The Rp-IκB cDNA is comprised of a 1032 bp ORF, which encodes 343 amino acids residues with a predicted molecular mass of 38 kDa. Rp-IκB protein exhibited typical features of IκB protein family members, including the IκB degradation motif, PEST sequence and six ankyrin repeats. Phylogenetic analysis showed that manila clam and other known molluscan IκB proteins grouped together in the invertebrate cluster. Tissue specific expression analysis revealed that Rp-IκB was ubiquitiously expressed in all tested tissues. Significant up-regulation of Rp-IκBexpression was observed in gill and hemocytesfollowing bacterial immune challenge with Vibrio tapetis and purified lipopolysaccharide endotoxin.These results indicated that as a key regulator of NF-κB in mammals, Rp-IκB might play an important role in manila clam defense against bacterial infection. Thus, Rp-IκB appears to function similarly to mammalian IκB family orthologs.
The RpTNF amino acid sequences showed their characteristic TNF family signature and N-terminal transmembrane domains. Phylogenic analysis results showed that AbTNF-α was closely related with their invertebrate counterparts. qRT-PCR results showed that RpTNF-α transcripts were constitutively expressed in Clam hemocytes, gills, mantle, adductor muscle, Siphon and foot in a tissue-specific manner. Transcription level of RpTNF-α was significantly (p<0.05) up-regulated in gills and hemocytes by Vibrio tapetis and LPS challenge.
Bcl-2 is conserved anti-apoptotic protein that belongs to the anti-apoptotic Bcl-2 sub-family, which inhibits cell death by preventing mitochondrial membrane permeabilization (MMP). Bax is belongs to the pro-apoptotic Bcl-2 sub-family protein. Given the anti-apoptotic functions of these proteins in vertebrate and the involvement of apoptotic regulation in immune responses, we studied the sequences of this gene and its transcript expression in manila clam during innate immune responses to V. tapetis stimuli.Based on GS-FLX transcriptome sequence data, we identified full-length cDNA sequences of putative orthologues of manila clam Bcl-2 and Bax-like protein. The analysis of manila clam cDNA sequences, and comparisons of the clam deduced amino acid sequences to putative orthologues in other species, revealed the presence of highly conserved Bcl-2 homology (BH) and trnasmembrane(TM) domain (only RpBcl-2) in manila clam sequences. Quantitative RT-PCR was used to study tissue specific expression in six tissues of un-challenged clam. We observed that constitutively expression of these genes in all examined tissues and both genes highly expressed in gill. In challenge of V. tapetis , RpBcl-2 and RpBax mRNA expression were significantly up-regulated in gill and hemocytes.
Author(s)
이영득
Issued Date
2012
Awarded Date
2012. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000006084
Alternative Author(s)
Lee, Youngdeuk
Affiliation
제주대학교
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
CHAPTER I: Characterization of manila clam transcriptome using 454-pyrosequencing approaches 1
1.1 INTRODUCTION 2
1.2 MATERIALS AND METHODS 5
1.3 RESULTS AND DISCUSSION 5
CHAPTER II: Characterization of novel molluscan MyD88 family protein from manila clam 11
2.1 INTRODUCTION 12
2.2 MATERIALS AND METHODS 14
2.3 RESULTS 18
2.4 DISCUSSION 20
CHAPTER III: Immune response-related gene expression of a novel molluscan IκB protein member from manila clam 31
3.1 INTRODUCTION 32
3.2 MATERIALS AND METHODS 34
3.3 RESULTS 38
3.4 DISCUSSION 40
CHAPTER IV: Molecular characterization and expression analysis of molluscan TNFα homologue from manila clam 51
4.1 INTRODUCTION 52
4.2 MATERIALS AND METHODS 53
4.3 RESULTS 57
4.4 DISCUSSION 60
CHAPTER V: Characterization of cDNAs of key genes involved in apoptosis in manila clam 70
5.1 INTRODUCTION 71
5.2 MATERIALS AND METHODS 72
5.3 RESULTS 75
5.4 DISCUSSION 79
Reference 92
감사의글 102
Degree
Doctor
Publisher
제주대학교 대학원
Citation
이영득. (2012). TRANSCRIPTOME-WIDE DISCOVERY OF INNATE IMMUNE-RELATED GENES AND MOLECULAR CHARACTERIZATION OF THE INFLAMMATORY/APOPTOTIC PATHWAY INMANILA CLAM (Ruditapesphilippinarum)
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General Graduate School > Marine Life Sciences
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