I. 利用嗜甲醇酵母菌表現系統進行最適化綠豆防禦素VrD1突變株之製備II. 尋找綠豆防禦素VrD1在昆蟲細胞膜上之標的蛋白及其抗蟲機制之探討

Abstract

VrD1 (Vigna radiata defensin 1) is a member of the plant defensin family, containing 46 amino acids and four pairs of disulfide bonds. Isolation of a cDNA encoding a small cysteine-rich protein designated VrCRP (then also known as VrD1) from a bruchid-resistant mungbean revealed the first discovered plant defensin exhibiting both in vitro and in vivo insecticidal and antifungal activities. However, the molecular and structural basis of this unique insecticidal activity of VrD1 is still not fully understood. This thesis is divided into two parts： I. Optimized preparation of VrD1 mutants expressed in Pichia pastoris： Based on the structural and sequence alignment, it is suggested that VrD1, in addition to γ-thionins and several amylase inhibitors, is highly homologous to scorpion toxins, especially the short toxins. We have deduced that VrD1 may utilize a newly found cluster of basic residues on one side of VrD1 molecule to achieve its insecticidal function, whereas another cluster of previously identified basic residues located on the other side of the molecule, which is conserved for all γ-thionins, should be used to achieve the antibacterial/antifugual activities for VrD1 and for all other plant defensins. In order to understand the roles of this newly found cluster of conserved basic residues, we have constructed several expression strains for VrD1 mutants and purified these mutant proteins using the Pichia pastoris system. However, the recent re-examination of our VrD1 expression system showed that some of the previous constructions contained unexpected base frame-shift. On the other hand, regarding the parts with correct sequences, the yields of the consequent protein purification were rather low. This may give rise to the difficulty in reaching sufficient sample size in the experiments of whole-cell recordings performed previously. Therefore, in the present study, we focus our work on two aspects. (1) Reconstruction of the appropriate VrD1 mutants with correct sequences. (2) Increasing the expression levels via optimized preparation of VrD1 mutants expressed in pichia. In the past, we have utilized G418-resistance as a selection marker for high gene copy numbers and potentially for a high expression level. However, this strategy could not ensure the satisfactory expression yields. In the present study, we combine the small-scale expression and the western blotting as checkpoints to improve the screening strategy by exclusion of the false-positive G418-resistant transformants. After transformation of the VrD1-pPIC9K plasmids for R26A and K24A mutants into P. pastoris (SMD1168), seven well-grown transformants on 0.5 mg/mL G418/YPD plates were selected for high copy number and high expression level strains. Using this screening strategy, we obtained Y38 and Y43 for the expression of VrD1-R26A. The western blot results demonstrated that the colony Y38 has significantly higher target protein expression than the colony Y43. Results from the large-scale expressions are in line with those from small-scale expressions. Moreover, using the site-directed mutagenesis, we have successfully generated VrD1-K7A from wild-type VrD1. This will be a convenient method in the preparation for other mutant strains with higher insecticidal and antifungal activities in the future. II. Investigation on the membrane target(s) of VrD1 in insect cells and the molecular mechanism of VrD1 insecticidal activity： Base on sequence and structural alignment, we have postulated that VrD1 may utilized a similar interaction mode as short scorpion toxins to act on insect cell membranes with K+-channel or Cl--channels as molecular targets. Preliminary data has excluded Cl--channels for candidates based on electrophysiological experiments. To study the protein-protein interaction for VrD1 and cell membranes, two major aspects need to be considered. 【1】 Generation of large amounts of VrD1 protein, at least a few milligrams for each experiment. Instead of using pichia expression system, we purify native VrD1 from mung bean seeds. About 5~6 milligrams of VrD1 protein can be produced from 500 grams of mung bean seeds. Mass spectrum has confirmed the correctness of VrD1. 【2】 Choice of appropriate cell-lines that allow VrD1 to interact with its membrane target(s). Previous reports showed that VrD1 can lead to a significant inhibitory effect on Sf21 or Sf9 (Spodoptera frugiperda 21 or 9). However, due to the lack of Spodoptera frugiperda genome database, it will be very difficult to identify unknown VrD1-target candidates from membrane extracts. Therefore, Drosophila S2 cell-line was chosen in this study to perform cell survival assay before the investigation with membrane proteomics.