본 연구는 우리나라 해안에서 널리 서식 중인 해양 자원 중 하나인 전복(Haliotis discus hannai) 의 차세대염기서열분석 데이터 기반으로 선별한 신규 펩타이드의 항암 활성을 평가한 연구이 다. 펩타이드의 항암 활성은 교모세포종 세포주인 SNU-489에서 농도 의존적으로 처리 시간에 비례하여 증가하였으며, 200 μM로 48시간 처리하였을 때 암 세포 사멸율이 67%로 가장 높게 나타났다. 반면 정상 세포인 HaCaT에서 가장 높은 세포 사멸율은 18%로 농도 의존적이었으나 처리 시간과는 무관하였다. 또한 신규 펩타이드의 항암 메커니즘 과정을 밝히기 위해 세포자 멸괴사(Necroptosis) 관련 유전자의 발현 변화를 qRT-PCR 방법을 통해 검증하였다. RIPK3는 신 규 펩타이드 처리군에서 200 μM 처리 시 9배 이상 발현 증가, MLKL는 100 μM 처리군에서 대조군 대비 2배 이상 유의미하게 발현이 증가되었다. 이러한 결과로 미루어 볼 때, 전복 유래 신규 펩타이드는 암 세포 특이적으로 세포 독성을 가지며, 세포자멸괴사 메커니즘을 통해 암 세포 사멸을 일으키는 것으로 추측되므로 신규 펩타이드가 추후 교모세포종 치료제의 후보 물질로 활용될 수 있을 것으로 사료된다.

In recent years, high-throughput next-generation sequencing (NGS) techniques have provided fascinating opportunities to understand the biology of non-model organisms, especially insect species. The decrease in sequencing costs and extensive sequencing services from NGS providers has brought many entomologists to be involved in genome sequencing. However, poor planning can lead to extremely fragmented genome assemblies which prevents high quality gene annotation and other desired analyses. Insect genomes can be problematic to assemble, due to combinations of high polymorphism, inability to breed for genome homozygosity, and small physical sizes limiting the quantity of DNA able to be isolated from a single individual. Given to the rapid development of host resistance to multiple classes of insecticides, it is indispensable to study the comprehensive genomic information of insects. Recent advances in sequencing technology and assembly strategies can able to fetch breakthroughs in deciphering the genetic information of insects. Here, we present the cost effective high throughput genome sequencing and assembly strategies for insect species in respects to taxonomy, evolutionary history, immune response, drug development, insect host-virus interactions and pest management etc.

The small brown planthopper (SBPH), Laodelphax striatellus Fallén (Hemiptera: Delphacidae) is one of the major insect pest against rice, Oryza sativa L. in Korea. High density of SBPH could cause severe damage on rice plant by directly sucking and indirectly transmitting viral pathogens, Rice stripe virus and Rice streaked dwarf virus. As a preliminary study for de novo whole-genome sequencing of SBPH, we investigated 6 transcriptomes isolated from different developmental stages, sex, and tissue (egg, 1st ~ 3rd nymphs, 4th ~ 5th nymphs, female and male adults, salivary gland). Clean-sequence data of 19.3 Gb were obtained from total 47.8 Gb raw data after adaptor and quality trimming (Q30) and overlapped reads joining. As a suitable assembler, Bridger was selected based on the results of reference mapping (93.45%) and CEGMA completeness (95.97%). Finally, we obtained 158,207 reads (size range: 201 ~ 22,162 bp; Mean size: 1,048.04 bp; N50: 2,417 bp) after clustering the assembly results by CD-HIT-EST (similarity threshold: 99%). Based on these results, we are conducting further studies such as transcript expression pattern among different developmental stages and gene annotation.

Leptotrombidium pallidum is the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. To understand the molecular mechanism of L. pallidum, we sequenced the whole genome using Illumina sequencing technology. Totally four genomic libraries with different insert sizes ranging from 280 bp to 8 kb were used to generate 45.1 Gb of genome in the combination of paired-end and mate-pairs sequencing reads. Quality filtering and correction of paired-end reads for very small and/or bad-quality sequences yielded 26.9 Gb of high-quality sequences, which are used to estimate the genome size as 175 Mbusing kmer methods and assembled into a 193.7 Mb genomic sequence scaffolds with N50 length of 92,945 bp. Furthermore, 94% of CEGMA completeness score were obtained from genome scaffold assembly. To facilitate gene annotation, we used a combination of de novo and homology based tools to predict gene models in the chigger mite genome. A combination of evidence-based and de novo approaches predicted 15,842 high-confidence protein-coding genes with an average transcript length of 1,511 bp and 2.4 exons per gene which corresponds to about 12.4% total gene length. Bacterial endosymbiosis are very common in mite species and can range from mutualistic to pathogenic associations. Henceforth, the endosymbionts in L. pallidum were predicted using the NCBI microbial draft genomes and mitochondrial genome. Besides, this L. pallidum draft genome can be used as a significant reference for comparative genomic studies across mite species.

Leptotrombidium pallidum and Leptotrombidium scutellare are the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. Before these organisms can be subjected to whole-genome sequencing, the genome sizes of L. pallidum and L. scutellare were estimated by a method based on quantitative real-time PCR. In addition, k-mer analysis of the genome sequences obtained from Illumina sequencing was conducted to verify the mutual compatibility and reliability of results. The genome sizes estimated by qPCR were 191.3±7 Mb for L. pallidum and 262.1±13 Mb for L. scutellare. The estimated genome sizes based on k-mer analysis were 175.5 Mb for L. pallidum and 286.6 Mb for L. scutellare. The estimates from two independent methods were mutually complementary and in a similar range to those of other Acariform mites. The relatively small genome size would facilitate genome analysis, which could contribute to understanding Arachnida genome evolution and mite vector competence and provide key information for scrub typhus prevention.