National Institute of Agrobiological Sciences

Other

Takahiro Kikawada

Takashi Okuda

Masahiko Watanabe

Kazuei Mita

Keiko Kadono

Ayako Saito

Yasuhiko Kobayashi

Tetsuya Sakashita

Seichi Wada

Yasushi Kanamori

Tomoo Funayama

Yuichi Nakahara

Kazuyo Watanabe, Legal Representative

An objective of the present invention is to provide polynucleotides encoding insect desiccation resistance proteins, and uses thereof cDNA libraries were produced fromlarvae in a desiccated state, aEST database was constructed, and genes encoding LEA proteins were isolated. This resulted in the successful isolation of three types of novel gene encoding LEA-like proteins (PvLEA1, PvLEA2, and PvLEA3.) When secondary structure predictions and motif searches were performed on the proteins deduced from each of the genes, all three proteins had &#x3b1;-helix-rich structures and LEA_4 motifs, which are characteristic of LEA proteins. Moreover, the recombinant proteins synthesized from PvLEA1, 2 and 3 genes were heat soluble even when boiling, so that PvLEA1, 2 and 3 proteins have highly hydrophilic property as well as plant LEA proteins. Therefore, the three isolated genes were found to be novel-derived LEA genes. Furthermore, introduction of these PvLEA 1, 2 and 3 genes into animal cells successfully conferred desiccation resistance to the cells. The present invention provides the first example of LEA genes isolated from insects.

Insect desiccation resistance genes and uses thereof

An objective of the present invention is to provide polynucleotides encoding insect desiccation resistance proteins, and uses thereof. cDNA libraries were produced fromlarvae in a desiccated state, aEST database was constructed, and genes encoding LEA proteins were isolated. This resulted in the successful isolation of three types of novel gene encoding LEA-like proteins. When secondary structure predictions and motif searches were performed on the proteins deduced from each of the genes, all three proteins had &#x3b1;-helix-rich structures and LEA_4 motifs, which are characteristic of LEA proteins. Moreover, the recombinant proteins synthesized from these genes were heat soluble even when boiling, so that these proteins have hydrophilic properties as high as plant LEA proteins. Therefore, the three isolated genes were found to be novel-derived LEA genes. Furthermore, introduction of these genes into animal cells successfully conferred desiccation resistance to the cells.

There are provided trehalose transporter gene and a method of introducing trehalose into cells by using the gene. Candidates for the trehalose transporter genes were searched inEST, resulting in being obtained cDNA designated as Tret1. Tret1 encodes a 504 amino acid protein with 12 trans-membrane structures. Tret1 expression was induced by desiccation stress and predominant in the fat body. Functional expression of TRET1 inoocytes showed that transport activity was specific for trehalose and independent of extracellular pH and electrochemical membrane potential. The direction of transport of TRET1 was reversible depending on the concentration gradient of trehalose. Apparent Km and Vmax of TRET1 for trehalose were extraordinarily high values. These results indicate that TRET1 is a facilitated, high-capacity trehalose-specific transporter. Tret1 is widespread in insects. Furthermore, TRET1 conferred trehalose permeability upon cells including those of vertebrates as well as insects.

Method of increasing cell permeability to trehalose by recombinantly producing a trehalose transporter

An objective of the present invention is to provide polynucleotides encoding insect desiccation resistance proteins, and uses thereof. cDNA libraries were produced fromlarvae in a desiccated state, aEST database was constructed, and genes encoding LEA proteins were isolated. This resulted in the successful isolation of three types of novel gene encoding LEA-like proteins. When secondary structure predictions and motif searches were performed on the proteins deduced from each of the genes, all three proteins had &#x3b1;-helix-rich structures and LEA_4 motifs, which are characteristic of LEA proteins. Moreover, the recombinant proteins synthesized from these genes were heat soluble even when boiling, so that these proteins have hydrophilic property as high as plant LEA proteins. Therefore, the three isolated genes were found to be novel-derived LEA genes. Furthermore, introduction of these genes into animal cells successfully conferred desiccation resistance to the cells.

This invention provides a method for effectively producing dehydrated larvae for educational materials without disrupting the environment. The cryptobiotic larvae for educational materials can be obtained by dehydrating larvae while gradually reducing humidity in 3 separate stages.

Dehydration procedures for induction of cryptobiosis in insect larvae

A tissue of a multicellular organism is gradually dried during cultivation. After the tissue has been completely dehydrated, water is added to the tissue for its recovery. The tissue of the multicellular organism is submerged in an insect body fluid medium treated with heat, and dried for 48 hours or more.

Growing community of inventors

Tsukuba, Japan

Takahiro Kikawada