隐地蛋白(cryptogein)基因定点突变及其广谱抗病烟草转化植株的获得

用PCR法从隐地疫霉 (Phytophthoracryptogea)基因组DNA中克隆了cryptogein(Cry)基因。将Cry基因的 13位赖氨酸 (K)突变成缬氨酸 (V) ,获突变基因CryK13V ,并将其构建于CaMV35S启动子控制的植物表达载体上。通过农杆菌介导的叶盘转化法转入烟草 ,经卡那霉素抗性筛选获 33株再生植株 ,PCR检测和Southern杂交分析表明CryK13V基因已整合到烟草基因组中。接种试验结果表明 ,转基因烟草植株对黑胫病菌、赤星病菌和野火病菌等的抗性均有提高。Northern杂交分析表明 ,微弱的CryK13V基因在转化植株中的表达就足以激活PR1和OPBP1等防卫反应相关基因的表达 ,而且表达丰度与转基因植株的抗病性有着一定的正相关性。研究结果还表明 ,隐地蛋白13位上的赖氨酸在诱导细胞死亡中起着关键的作用。

Site-Directed Mutagenesis of Cryptogein Gene(CryK13V) and Generation of Transgenic Tobacco Plants with Nonspecific Disease Resistance

The cryptogein gene was obtained by PCR amplification from genomic DNA of Phytophthora cryptogea (Fig.1). The lysine in site 13 of cryptogein protein was mutated to valine (K13V) through PCR site-directed mutagenesis. The mutant fragment (CryK13V) was confirmed by enzyme digestion and DNA sequencing. We expressed this CryK13V gene under the control of the 35S promoter of cauliflower mosaic virus (CaMV). Then CryK13V gene was fused to the signal peptide sequence of the extracellular pathogenesis-related PR1b protein of tobacco (Fig.2). The chimeric gene was transformed into tobacco by Agrobacterium-mediated transformation method, and 33 kanamycin-resistant plants were obtained. The presence of the transgene in the regenerated plants was confirmed by PCR (Fig.4) and Southern blot analysis (Fig.5). A very low level of CryK13V expression in transgenic plants could activate the expressions of defense-related gene, PR1 and OPBP1 (Fig.7) and the transgenic plants exhibited enhanced resistance to multiple pathogens such as blank shank fungus Phytophthora parasitica var. nicotiana, brown spot fungus Alternaria alternata and wild fire bacterium Pseudomonas syringae pv. tabaci (Fig.6). Our data suggest that the lysine residue at position 13 of cryptogein plays a key role in the induction of hypersensitive cell death, and analysis of cell death and the other defense responses are of potential in genetic manipulation of broad-spectrum resistance against plant pathogens.