| Summary: | 博士 === 國立臺灣大學 === 農業化學研究所 === 90 === VrArc genomic clone was isolated from a bruchid resistant wild mung bean, Vigna radiata TC1966, encoding a 265-amino-acid protein with a signal peptide of 21 amino acids. VrArc is completely identical to arcelin-1 which has been shown to be an insect resistant protein. A cDNA encoding a small cysteine-rich protein designated as VrCRP was isolated from a bruchid resistant nearly isogenic line of mung bean Vigna radiata VC6089A. VrArc protein was obtained by overexpression of signal peptide truncated VrArc (VrArcsp) in a pQE expression system. VrCRP (GenBank accession no. AF326687) encodes a protein of 73 amino acids, containing a predicted 22 amino acid signal peptide and 8 cysteines. VrCRP protein was obtained by overexpression of signal peptide truncated VrCRP (VrCRPsp) in an IMPACT (intein-mediated purification with an affinity chitin-binding tag) expression system. The purified VrCRPsp was identified by its molecular mass (5,944 Da) and N-terminal amino acid sequencing. Similar to a number of cysteine-rich proteins. There is a cystine stabilized -helix motif (CSH motif) in sequence of VrCRP protein that shows amino acid sequence homology to plant defensins. Artificial mung bean seeds containing 0.2% VrCRPsp killed larvae of the bruchid Callosobruchus chinensis at first instar stage. A VrCRPsp level as low as 0.06% was sufficient to delay larval development. VrCRPsp is toxic to E. coli and completely arrested growth of insect cells (Spodoptera frugiperda, Sf21) at a low concentration of 3.42 M. VrCRPsp is also a potent inhibitor of protein synthesis and arrests growth of a fungus Rhizoctonia solani. In situ hybridization revealed that VrCRP mRNA is most abundant in endosperm cells, followed by parenchyma cells. Immunolocalization indicated that VrCRP protein was predominantly present in parenchyma cells of the seed coat.
VrCRP was also purified directly from mung bean VC6089A by a procedure involving a two-column system comprising an anion-exchange (DEAE) and a cation-exchange (CM) columns, and Superdex Peptide HR10/30 gel filtration in FPLC system. The fractions containing VrCRP in the chromatography were recognized by the polyclonal anti-recombinant VrCRPsp antiserum. The first 41 amino acids of the purified VrCRP determined by N-terminal amino acid sequencing were completely consistent with the deduced amino acid sequence of VrCRP cDNA starting from Arg28. The results indicated that there is a signal peptide from Met1 to Ala27, and the mature VrCRP contains 46 amino acids. The calculated molecular mass and pI value of the purified VrCRP were 4,824 Da and 8.62, respectively. The anti-bruchid activity of mung bean VrCRP is similar to the recombinant VrCRPsp.
Based on the similarity of amino acid sequence with other plant defensins, biological activities and the protein localization, it is suggested that VrCRP may act as an evolutionary barrier in mung bean seeds that is important in coping with invasion of pathogens and herbivores.
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