嶋田 透

Silkworms have played an important agricultural role in supporting Japan’s modernization, and traditionally, Japan has led the world as a repository of silkworm bioresources. The silkworm is a small and highly domesticated insect, which is ideal as a laboratory tool, although it is a bioresource that is relatively infrequently used in experiments at present. In this review, we describe the potential for silkworm resources to contribute to life sciences.

カイコのゲノムは、2004年に日本と中国のグループにより独立にドラフト配列が発表された後、その両国間で共同研究が進められ、2008年に精密なゲノム情報が公表された。カイコには、より高度な機構に依存し、かつカイコだけに特徴的にみられる現象、たとえば卵での越冬休眠やクワへの寄主特異性などがある。これらが、はたしてカイコだけが持つ遺伝子によって支配されているのか、あるいは他の昆虫と共有する遺伝子のマイナーチェンジによって実現しているのか、読者にとって興味のあるところであろう。本稿では、カイコのゲノムの特異性と、生物としてのカイコの特性の一つであるクワへの依存性を関連づける研究について紹介することにする。未開拓、未解明の研究領域であり、以下に述べる内容には筆者の推察が含まれていることを予めお断りしたい。

カイコの遺伝学は、外山亀太郎博士によってメンデルの法則の成立が動物で初めて確かめられてから、すでに100年を超える歴史を作ってきた。この間に数百の突然変異が発見され、それらは連関関係および組換え価に基づいて28の連関群に配置されている。一方で、Watson and CrickによるDNAの二重らせん構造の発見以降の分子生物学の興隆期には、真核生物の遺伝子のクローニングとしては最も早い成功例の一つである、鈴木義昭博士らによるフィブロイン遺伝子の単離をはじめ、富野士良博士らによる体液タンパク質遺伝子に関する研究や山下興亜博士らによる休眠ホルモン遺伝子の単離など、日本人がカイコを用いて世界の分子遺伝学をリードする成果を挙げてきた。

All insects are gonochoristic, and their sex is usually genetically determined. The genetic and molecular mechanism as to how sex is determined has been discussed in detail for Drosophila (Schütt and Nöthiger 2000), and recently, sex-determining genes have been identified in the honeybee Apis mellifera (Hasselmann et al. 2008).

カイコ生殖巣に存在するsmall RNAの特性と機能。多くの生物のゲノム解読が完了した昨今、「ゲノム上に存在するタンパク質非コード領域が持つ機能」は、ますます興味深い問題のひとつである。カイコゲノムのほぼ80％、ヒトゲノムでは実に98％が、反復配列をはじめとするタンパク質非コード領域であると認識されている。長い間、これらの領域は「ジャンクDNA」などと呼ばれ、生物にとって不要なものであると見なされてきた。しかしながら、最近になって、これら非コード領域が活発に転写され、転写された非コードRNAが重要な生物機能を有する、ということが分かってきた。我々が最もよく知っている重要な非コードRNAとしては、tRNAやrRNAが挙げられるが、大きな注目が集まっているのは、長さが21-30塩基程度の小さなRNA、small RNAの機能である。ここでは、動物のsmall RNAの基本的な機能に関して概説するとともに、筆者らが最近得た、カイコにおけるsmall RNA研究についての結果を紹介する。

Bombyx mori, the domesticated silkworm, is a major insect model for research, and the first lepidopteran for which draft genome sequences became available in 2004. Two independent data sets from whole-genome shotgun sequencing were merged and assembled together with newly obtained fosmid- and BAC-end sequences. The remarkably improved new assembly is presented here. The 8.5-fold sequence coverage of an estimated 432 Mb genome was assembled into scaffolds with an N50 size of ∼3.7 Mb; the largest scaffold was 14.5 million base pairs. With help of a high-density SNP linkage map, we anchored 87% of the scaffold sequences to all 28 chromosomes. A particular feature was the high repetitive sequence content estimated to be 43.6% and that consisted mainly of transposable elements. We predicted 14,623 gene models based on a GLEAN-based algorithm, a more accurate prediction than the previous gene models for this species. Over three thousand silkworm genes have no homologs in other insect or vertebrate genomes. Some insights into gene evolution and into characteristic biological processes are presented here and in other papers in this issue. The massive silk production correlates with the existence of specific tRNA clusters, and of several sericin genes assembled in a cluster. The silkworm's adaptation to feeding on mulberry leaves, which contain toxic alkaloids, is likely linked to the presence of new-type sucrase genes, apparently acquired from bacteria. The silkworm genome also revealed the cascade of genes involved in the juvenile hormone biosynthesis pathway, and a large number of cuticular protein genes. © 2008 Elsevier Ltd. All rights reserved.

Baculovirus chitinases (V-CHIAs) play a crucial role in the terminal liquefaction of virus-infected larvae after death. Although v-chiAs from nucleopolyhedroviruses (NPVs) have been well characterized, little is known about v-chiAs from granuloviruses (GVs). We characterized the v-chiA of Cydia pomonella GV (CpGV) by constructing a recombinant Bombyx mori NPV (BmNPV) in which BmNPV v-chiA was replaced by CpGV v-chiA (103CpGV virus). CpGV v-chiA encoded an approximately 70-kDa chitinase with an exo-type substrate preference. CpGV V-CHIA lacked a C-terminal KDEL endoplasmic reticulum retention motif and was suggested to be a secretory protein. Terminal host liquefaction of B. mori larvae and proper folding of BmNPV-encoded cysteine protease (BmNPV V-CATH) were observed following infection with 103CpGV, indicating that CpGV v-chiA is able to compensate for the absence of its BmNPV counterpart. Our data suggest that the molecular interaction between V-CHIA and V-CATH may be conserved across a broad range of lepidopteran GVs and NPVs.

Following baculovirus infection, the level of host messenger RNA (mRNA) declines and the synthesis of host proteins is shutoff in virus-infected cells. To comprehensively understand the regulation of host gene expression by Bombyx mori nucleopolyhedrovirus (BmNPV), we took a complementary DNA (cDNA) subtraction approach. Northern blot analysis was then performed to confirm whether obtained clones are differentially expressed. From these analyses, we obtained 4 down-, 7 up- and 2 non-regulated host gene candidates during the early stage of infection. Sequence analyses showed that these may encode proteins involved in transcription, cell cycle, cell adhesion, protein degradation, apoptosis or energy metabolism. We then measured the ADP/ATP ratio to address the response of host energy metabolism upon baculovirus infection. The ADP/ATP ratio showed cell death-like rapid increase following BmNPV infection with a transient decrease during 12-24 h post-infection (p.i.). Taken together, our results suggest that general events in host cells are controlled at the level of mRNA expression by BmNPV infection.