乾豇豆屬植物防禦素基因之功能研究

Abstract

本實驗室利用壓制性扣除雜交法 (suppression subtractive hybridization) 自綠豆 (Vigna radiata) 抗豆象品系VC6089A選殖出和抗豆象性狀有關的植物防禦素 (plant defensin) cDNA，命名為VrCRP (Vigna radiata Cysteine Rich Protein) cDNA，經過大腸桿菌 (Escherichia coli) 系統及嗜甲醇酵母菌 (Pichia pastoris) 系統表現的VrCRP，對豆象 (Callosobruchus chinensis) 及立枯絲核菌 (Rhizoctonia solani) 具有抗性。經純化後得知天然VrCRP的成熟胜

Abstract A novel plant defensin gene named VrCRP (Vigna radiata Cysteine Rich Protein) was previously isolated from a bruchid resistant nearly isogenic line of mungbean Vigna radiate VC6089A by suppression subtractive hybridization, and was expressed in Escherichia coli and Pichia pastoris expression systems. The expressed VrCRP exhibited bruchid (Callosobruchus chinensis) and fungal resistant activities. VrCRP cDNA encodes a 73-amino-acid protein containing a signal peptide of 27 amino acids from Met1 to Ala27 and the mature VrCRP contains 46 amino acids starting from Arg28. This study focuses on the structure and function of VrCRP and VrCRP-like genes. The genomic DNA of VrCRP was isolated from mungbean VC6089A by genomic PCR and its nucleotide sequence determined. The VrCRP genomic DNA contains a 103 bp intron and 3 mismatch base pairs when compared with VrCRP cDNA, hence the genomic DNA was renamed as VrD1 (Vigna radiata Defensin 1). To analyze the transcriptional regulation of VrD1, a 1.7 kb promoter of VrD1 was cloned by genomic walking from VC6089A genomic libraries using gene-specific primers designed from VrD1 genomic DNA. Interestingly, the same VrD1 genomic DNA have also been obtained from five different Vigna radiata varieties by PCR mediated cloning strategy. Southern blot analysis showed that all the five Vigna varities have single VrD1 copy in genome. To prepare VrD1 cDNA, reverse primers were designed to excise the intron sequence from VrD1 genomic DNA by Inverse PCR amplification. The transcriptionally fused 35S promoter/VrD1 cDNA and RP5 promoter/VrD1 cDNA chimeric genes were introduced into tobacco (Nicotiana tabacum) and rice (Oryza sativa) respectively via Agrobacterium tumefaciens mediated transformation. The recombinant mature VrD1 was purified from transgenic tobacco fresh leaves and transgenic rice mature seeds, indicating that VrD1 cDNA was expressed in tobacco and rice plants. The transgenic tobacco will be tested for tobacco cutworm (Spodoptera litura) resistant activity. We also isolated 0.4 kb VrD1-like genomic DNAs using the same primers with genomic PCR from azuki bean (Vigna angularis), black gram (Vigna mungo), rice bean (Vigna umbellata), cultivated Vigna (Vigna glabrescens) and cowpea (Vigna unguiculata), and named VaD1 (Vigna angularis Defensin 1) genomic DNA, VmD1 (Vigna mungo Defensin 1) genomic DNA, VumD1 (Vigna umbellata Defensin 1) genomic DNA, VgD1 (Vigna glabrescens Defensin 1) genomic DNA and VunD1 (Vigna unguiculata Defensin 1) genomic DNA, respectively. All these 6 VrD1-like genomic DNAs contain an intron of about 0.1 kb, 222 bp open reading frame (after intron deletion) encoding a protein of 73 amino acids. All of the 6 VrD1-like proteins are predicted to have a 46-amino-acid mature peptide and a 27-amino-acid signal peptide. These 6 VrD1-like mature peptides are basic and have 8 cysteines located in the same positions as the mungbean VrD1. It is suggested that these six plant defensins have similar 3D structures based on their high homologous amino acid sequences. To understand the structure and function of VrD1-like defensins, we purified mature VaD1 from azuki bean Kao Hsiung No. 6 seeds by cation-exchange chromatography and Superdex Peptide HR 10/30 gel filtration in FPLC system. The fractions containing VaD1 in gel filtration was recognized by anti-VrCRPTSP antiserum. The complete amino acid sequence of the purified VaD1 determined by N-terminal sequencing and tandem mass analysis matched completely the deduced amino acid sequence of VaD1 cDNA starting from Lys28. VaD1 concentration required for 50% growth inhibition (IC50) was determined against 7 plant pathogenic fungi, 3 plant pathogenic bacteria and 15 food pathogenic bacteria. Among the various pathogens tested, the IC50 values for Fusarium oxysporum, Fusarium oxysporum f. sp. pisi, Staphylococcus epidermidis ATCC 14990 CCRC 10785, Salmonella typhimurium ATCC 14028 CCRC 10747 and Xanthomonas campestris pv. vesicatoria were in the range from 30 μg/mL to 143.4 μg/mL. The other pathongens tested were not inhibited by VaD1. The activities of anti-bruchid and protein synthesis inhibition of the purified VaD1 were also analyzed. It appeared that VaD1 was weaker than VrD1 in terms of anti-bruchid and protein synthesis inhibitory activities.