수염풍뎅이(Polyphylla laticollis manchurica)는 과거에는 흔히 발견되었으나, 1970년대 이후 한반도 내 개체수 가 급격히 감소하여 2005년 환경부에 의해 멸종위기 야생생물 Ⅰ급으로 지정되었다. 또한 해당종의 분자생물학적 연구는 멸종위기종이라는 특성으로 인해 제한적으로 진행되었다. 그로 인해 NCBI 등 공공 데이터베이스에서 제공되는 서열정보들 또한 부족한 실정이다. 이 연구는 이러한 한계를 극복하고 수염풍뎅이의 유전적 특성을 규명하기 위해 생물정보학적 기술을 활용하여 전사체 분석을 진행하였다. Illumina HiSeq 2500 플랫폼을 사용하여 53,433,048개의 RNA reads를 얻었으며, Trinity와 TGICL을 이용한 De novo 어셈블리 분석을 통해 18,172개의 unigenes를 생성하였다. 생성된 unigenes는 GO, KOG, KEGG, PANM DB를 활용하여 annotation을 진행하였다. 그 결과, GO 분석에서는 ‘binding and catalytic activities’와 관련된 항목이 높은 발현을 보였으며, KOG 분석의 경우 ‘Cellular Processes and Signals’ 범주가 높은 비율을 나타내었다. KEGG 분석을 통해 2,118개의 unigenes가 metabolic 카테고리에 annotation된 것을 확인하였다. SSR 모티프 분석에서는 AT/AT (42.90%) 모티프, AAT/ATT (13.13%) 모티프 순으로 많이 나타나는 것을 확인하였다. 이 연구를 통해 분석한 결과 들을 이용하여 유전자원 및 종 정보를 실시간 제공 및 정보 공유가 가능하도록 Database 및 web-interface를 구축하 였으며, 이러한 자료들은 국내 멸종위기종인 수염풍뎅이의 고유한 유전적 특성을 발굴 및 확보할 수 있는 기반자 료로써 활용될 수 있을 것으로 사료된다.

CD63, a member of tetraspanin membrane protein family, plays pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic ‘Cys-Cys-Gly’ motif and ‘Cys188’ residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcript was upregulated the maximum 4.5 fold in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also made significant increase in the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146 Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5’-flanking region. BLAST and phylogenetic analysis reveals that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic helices, including a short helix 3’. The ‘helix-short helix-helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly up-regulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.