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为了利用Cecropin B(CB)抗菌肽基因提高柑橘对溃疡病的抗性,人工合成了两个含有信号肽的Cecropin B抗菌肽基因PR1aCB和AATCB。洋葱表皮瞬时表达分析表明,与非分泌型CB抗菌肽相比,PR1aCB和AATCB抗菌肽在细胞间隙中优势积累。进一步构建了CaMV 35S调控PR1aCB和AATCB基因的植物表达载体,转化塔罗科血橙(Citrus sinensis Osbeck)上胚轴,获得转基因植株。GUS组织化学染色、PCR和Southern blot分析表明外源基因已成功整合入柑橘基因组。Real-time PCR定量分析表明,抗菌肽基因在转基因植株中成功表达。柑橘叶片离体抗性分析表明,转PR1aCB和AATCB基因植株的抗性显著强于非转基因植株,其抗性水平与高抗品种‘南丰蜜橘’(C.succosa Hort.Ex Tan)和‘四季橘’(C.madurensis)相当。
In order to improve the resistance of citrus to ulcer disease by Cecropin B (CB) antimicrobial peptide gene, two Cecropin B antimicrobial peptide genes PR1aCB and AATCB containing signal peptide were synthesized. Transient expression analysis of onion epidermis showed that PR1aCB and AATCB antimicrobial peptides were predominantly accumulated in the intercellular space compared with non-secreted CB antimicrobial peptides. Further, a plant expression vector for regulating PR1aCB and AATCB gene expression by CaMV 35S was constructed and transformed into the hypocotyl of Citrus sinensis Osbeck to obtain transgenic plants. GUS histochemical staining, PCR and Southern blot analysis showed that exogenous genes have been successfully integrated into the citrus genome. Real-time PCR quantitative analysis showed that the antimicrobial peptide gene was successfully expressed in transgenic plants. In vitro resistance analysis of citrus leaves showed that the resistance of transgenic plants to PR1aCB and AATCB was significantly higher than that of non-transgenic plants. The resistance level of the two plants was significantly higher than that of the high resistant varieties ’C. succosa Hort. Ex Tan and’ Quaternary orange ’(C.madurensis) equivalent.