綠豆防禦素VrD1之抗蟲活性及其與鉀離子通道作用之分子機制探討

Abstract

各種動植物體中普遍存在防禦素（Defensins）或相關的分子，藉以防禦異物入侵或毒殺獵物。以植物來說，thionins為最有名的例子，可以對抗細菌或真菌的入侵。2002年國人首先在綠豆發現具有抗昆蟲特性(insecticidal activity)之植物防禦素，名為VrCRP (Cysteine-rich Protein of Vigna radiata) ，後稱VrD1 (Vigna radiata defensin 1)。此防禦素屬於cysteine-rich proteins，具有由4對雙硫鍵（disulfide bonds）連結一段α-helix (即cysteine-stabilizing helix) 和三段β-strands的基本結構。 VrD1對綠豆豆象(bruchids; Callosobruchus chinensis or C. maculatus) 具有毒殺的效果，唯VrD1造成綠豆豆象死亡之活性來源及作用機制則尚不十分明瞭。過去我們在完成VrD1及其多種突變種的3-D結構模型(homology structure models)，並分析了其與scorpion toxins的異同後，已經合理推斷出VrD1造成綠豆豆象死亡之活性來源在於分子一側之數個鹼性殘基，並可能使用類似蠍毒中的short toxins之鍵結模式與細胞膜上之鉀或氯離子通道作用;而另一側之數個鹼性殘基則負責抗菌。 所以在本論文中，我們設計了一系列的電生理實驗，希望透過全細胞電位鉗制法 (whole cell voltage clamp method)，試圖找出VrD1作用在秋行軍蟲細胞株 (Sf-21) 的標的分子(target molecules)。實驗所得的結果顯示，VrD1對秋行軍蟲的細胞膜電流產生大規模的抑制情形；再由平衡電位與結構分析逐步歸納出可能的目標，並佐以不同的拮抗物測試，目前已排除是透過氯離子通道產生作用，而更進一步指向以鉀離子通道作為此標的分子。這些發現在動植物間的演化上應具重大意義，甚至在生物性農藥之相關合理設計方面也應有關鍵的幫助。

A variety of evolutionarily related defensin molecules is found in plants and animals. However, little is known about whether defensins are also involved in plant resistance to insect. A cDNA encoding a small cysteine-rich protein designated VrD1 (VrCRP) was isolated in the laboratory of Prof. Ching-San Chen, Academia Sinica, from the bruchid-resistant mungbean -- a well-known important food source in Asia and worldwide. VrD1 is apparently the first reported plant defensin exhibiting in vitro and in vivo insecticidal activity against C. chinensis. The molecular and structural basis of such unique insecticidal activity of VrD1 is not clear. VrD1 has a four-disulfide linkage which connects the cysteine-stabilizing helix and the β–sheet, and can be categorized as a member of the γ-thionin family. From our structural analysis, VrD1 may use the three to four basic residues on one side of the molecule to perform the interaction with its membrane target, which is comprehended as potassium or chloride channel(s). And as further described in this thesis, our electrophysiological results with whole-cell recordings have demonstrated a very large inhibitory effect of VrD1 on the membrane potentials of SF-21 cells. Upon utilization of the channel agonists/antagonists, it is suggested that the insecticidal activity of VrD1 be very possibly related to the modulation or directly targeting at the potassium channels on Sf-21 plasma membranes. The exact channel type(s) is still under intensive investigations and will be the focused concerns in the near future. Not only to provide the necessary information required for the studies of molecular mechanisms, our data should be also very helpful in the development of bioactive insecticides and in the related rational drug designs.