氏 名 | エリザベス アトングル Elizabeth Atungulu |
本籍(国籍) | ケニア |
学位の種類 | 博士 (農学) | 学位記番号 | 連研 第335号 |
学位授与年月日 | 平成17年9月30日 | 学位授与の要件 | 学位規則第4条第1項該当 課程博士 |
研究科及び専攻 | 連合農学研究科 生物環境科学専攻 | ||
学位論文題目 | Identification and Functional Analysis of Arrest-associated Proteins in the Leaf Beetle Gastrophysa atrocyanea ( コガタルリハムシにおける休止関連タンパク質の同定と機能解析 ) |
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論文の内容の要旨 | |||
Diapause can occur during any developmental stage in insects and can be facultative (induced by various environmental stimuli) or obligatory. Molecular studies on insect diapause are still limited. By comprehensively comparing proteins associated with insect diapause and other forms of dormancy, Denlinger(2000,2002) proposed that a subset of heat shock proteins(HSP) is a common feature in dormancy. As for adult diapause, diapause-associated transcripts have recently been charactorized from the Colorado potato beetle Lepinotarsa decemlineata (Yocum,2003). Information collected from this species and other studies will provide molecular understanding of facultive adult diapause. The leaf beetles, Gastrophysa atrocyanea hibernates in the adult stages and mates in spring.
Newly emerged adults burrow into the soil(approximately 10 cm) and begin diapause after one week
feeding on leaf dock weeds.
The beetles remain under obligatory diapause from June until the spring in the following year.
To present, some biochemical analyses on this adult diapause report a diapause-specific
peptido composed of 41 amino acids including 6 cysteins isolated from diapausing adults(Tanaka et al.,
1998,2003).
A23kDa protein belonging to small heat shock family isolated as a constitutive protein expressed
even in the absence of stress throughout the periods of both diapause and pre-diapause(Sakata et al.,
2003).
In the non-diapausing phase, an active phase associated-protein has also been identified(Fujita et al.,
2005).
Here, an arrest associated protein(21kDa) was electrophoretically identified by tricine SDS-PAGE and
some properties of the protein were examined.
Further experiments evaluated and compared the functions of both 21kDa and 23kDa proteins.
To understand the molecular mechanism of the obligatory adult diapause of the leaf beetle,
the results obtained propose that the two small heat shock proteins display a possible double
chaperone function against heat-based environmental adversity. The results are summarized as follows: Gene products of small heat shock/α-crystallin proteins are constitutively expressed at normal temperature in several organisms such as Artemia (Liang and MacRae, 1999), Drosophila (Michaud et al., 1997),Caenorhabditis (Linder et al.,1996; Leroux et al., 1997),Xenopus (Heikkia et al., 1997), vertebrates (Loones et al., 1997; Mehlen et al.,1997) and plants (Carranco et al., 1997). Prior to periods of growth arrest in insects and shrimps, sHsp transcripts are highly expressed and cellular components are protected from stress-induced denaturation (Liang and MacRae, 1999; Denlinger, 2002). In humans, the sHsp27 protein plays a major role in the increased thermal resistance, as shown in rodent cells transfected with the human Hsp gene (Landry et al., 1989). The combination of human sHsps 27 and 20 results in a slow, temperature-dependent formation of hetero- oligomeric complexes in vitro (Bukach et al., 2003). However, the role of non-induced sHsps on intrinsic thermotolerance in organism without transfected Escherichia coli-sHsp has yet to be reported, as has a possible double chaperone function. Despite the deficiency in genetic manipulability of an entomoresource, the leaf beetle Gastrophysa atrocyanea as compared with Drosophila or Caenorhabditis, we found that gene silencing induced by double-stranded RNAs is a useful tool for the analysis of a diapause-specific peptide in diapausing adults (Tanaka and Suzuki, 2005). Consequently, a strong correlation observed between the in vitro chaperone function and in vivo thermotolerance analysis results support a possible double chaperone function critical for the survival of the leaf beetles against higher temperatures. On the basis of the presently available data, constitutive sHsp 21 and 23 may be involved in overcoming unsatisfactory season during the extended adult diapause. (日本語訳) そこで、本研究では現在報告されているペプチドとは異なる、休眠期に特異的に出現するタンパク質21kDaの 単離・同定を行い、さらにこの21kDaと23kDaの熱ショックタンパク質の機能について解析した。 また、2種のシャペロンタンパク質の発現抑制にも成功し、この遺伝子ノックアウトの休眠成虫では、 熱に弱いことを明らかにした。 このことから、昆虫が夏期に土中に潜り高温を回避するという戦略を獲得した新しいモデルとして提案した。 1.休眠特異的タンパク質21kDaをSDS-PAGEで分離し、PVDF膜に転写した後、目的のタンパク質のバンドを切り出し、
プロテアーゼ消化を行った。さらに、HPLCで分離精製し、得られたペプチド断片の配列を同定した。 以上のように、本研究ではコガタルリハムシの休眠期成虫からスモールシャペロンタンパク質21kDaを同定し、 完全長のcDNAの塩基配列を明らかにした。 さらに21kDa と 23kDaの相互関係においてはin vivoで高温に対してダブルシャペロン活性を示すことが 示唆された。 以上の結果から、これらのシャペロンタンパク質は、コガタルリハムシの休眠成虫において過酷な環境を 克服するため重要な役割を果たすと考える。 |