Mizuko Osanai-Futahashi¹, Ken-ichiro Tatematsu¹, Kimiko Yamamoto², Junko Narukawa², Keiro Uchino¹, Takumi Kayukawa³, Tetsuro Shinoda³, Toshiki Tamura¹, Hideki Sezutsu¹

¹Transgenic Silkworm Research Unit, ²Insect Genome Research Unit, ³Insect Growth Regulation Research Unit

［Abstract］

We found a novel transporter family gene that is involved in ommochrome pigment biosynthesis using the red egg mutant silkworm. The Bm-re family gene will be useful to develop a marker that is visible to the naked eye for easy screening of transgenic silkworms. We also developed a method to lighten the coloration of the black pigment in insects using a silkworm gene that inhibits the formation of dark melanin pigments.

［Keywords］

transgenic marker, visible marker, pigments, body color, egg color, insect transgenesis, gene function analysis, recombinant protein production

［Background］

Recently, the studies using transgenic silkworms have been greatly advanced for analysis of gene functions and production of useful materials. Transgenesis of most insects currently relies on using fluorescence markers, which are detectable only under a fluorescence microscope and require laborious procedures. It is therefore necessary to establish easy detectable markers which are visible to the naked eye.

［Results and Discussion］

1. A silkworm mutant "red egg (re)" has crimson-red eggs and dark-red compound eyes of adult, whereas wild-type silkworm has dark lilac eggs and black compound eyes (Fig. 1). Analysis of the re mutant revealed a novel transporter family gene Bm-re, which is involved in ommochrome pigment biosynthesis pathway.
2. The function of Bm-re gene was conserved in moths and beetles. But interestingly, the gene was lost in fruit flies, which may explain why fruit flies have red eyes.
3. Black pigments in insects are thought to be dopamine melanin. Arylalkylamine-N-acetyl transferase of silkworm (Bm-aaNAT) inhibits the synthesis of dopamine melanin. We found that the overexpression of Bm-aaNAT in transgenic insect lightened the coloration in silkworm, fruit fly and ladybird beetle (Fig. 2).
4. Bm-aaNAT gene can be used as a dominant marker for genetic transformation because its expression is visible to the naked eyes. It would therefore facilitate the efficient screening of transgenic individuals at larval as well as adult stages in diverse insect taxa.

1. Using Bm-re gene, development of an egg color marker for the screening of transgenic silkworm is expected. In comparison to existing markers, it facilitates screening even at early stages of development.
2. The Bm-aaNAT gene can be used as a transgenic marker for commercial strains, in which the eyespecific fluorescent markers are difficult to detect because of the dark eggs and eyes. It will further accelerate basic and application researches using transgenic silkworms.

Fig.1. Eggs and compound eyes of the silkworm.(A, C) Wild-type (+re/+re). (B, D) red egg (re) mutant. The egg serosa color of wild-type (+re/+re) has a dark lilac color (A), whereas that of re mutants has a crimson red color (B). The adult compound eye color of wild-type is black (C), whereas that of the re mutant is dark red (D). The re mutant lacks the function of Bm-re gene, which is a novel transporter family involved in ommochrome pigment biosynthesis pathway. The function of this gene was conserved in moths and beetles, although the gene was lost in fruit flies.

Fig.2. Overexpression of Bm-aaNAT alters cuticle coloration in silkworm larvae and other insect orders. (A) Larvae of newly hatched silkworm, B. mori. (B) Adults of fruit fly, Drosophila melanogaster. (C) Larvae of ladybird beetle, Harmonia axryidis. Arylalkylamine-N-acetyl transferase of silkworm (Bm-aaNAT) inhibits the formation of dark melanin pigments. Ubiquitous overexpression of Bm-aaNAT in transgenic insect lightens coloration in silkworm, fruit fly and ladybird beetle (arrowheads in A-C), highlighting the potential usefulness as a dominant marker for transgenesis in diverse insect taxa.

 

[Reference]

1. Osanai-Futahashi M, Tatematsu K, Yamamoto K, Narukawa J, Uchino K, Kayukawa T, Shinoda T, Banno Y, Tamura T, Sezutsu H (2012) Identification of the Bombyx red egg gene reveals the involvement of a novel transporter family gene in the late steps of the insect ommochrome biosynthesis pathway Journal of Biological Chemistry 287(21):17706-17714
2. Osanai-Futahashi M, Ohde T, Hirata J, Uchino K, Futahashi R, Tamura T, Niimi T, Sezutsu H (2012) A visible dominant marker for insect transgenesis Nature Communications 3:1295
3. Patent application# JP-2013-005740 (Japan)