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为进一步明确外源基因在转基因柑橘无性繁殖后代中的遗传稳定性及目标性状表现,本研究以转双价抗菌肽基因(Shiva A-cecropin B)纽荷尔脐橙(Citrus Sinensis Osbeck)无性繁殖后代植株为材料,通过PCR扩增,Southern杂交和实时定量PCR检测,以及温室抗病性评价分析等,对抗菌肽Shiva A基因在转基因柑橘后代株系中的遗传稳定性进行了研究。结果表明,外源抗菌肽Shiva A基因在转基因及其无性后代中能够稳定整合与表达。Southern杂交分析表明,Shiva A基因在无性繁殖后代基因组中为1个拷贝,而在T0代中为2个拷贝,由此推测T0代植株为混合嵌合体。定量PCR分析表明,Shiva A基因在T0代中的表达水平低于其无性繁殖后代。本研究中外源基因的表达与拷贝数的多少成负相关。研究结果为开展转基因柑橘安全性评价提供了有用的实验材料,积累了有效的实验数据。
In order to further clarify the genetic stability and target traits of the exogenous genes in the cloned offspring of transgenic citrus, in this study, the offspring of Shiva A-cecropin B cloned in Citrus Sinensis Osbeck Plants were used as materials. The genetic stability of Shiva A gene in transgenic citrus progenies was studied by PCR amplification, Southern hybridization and real-time quantitative PCR assay, and greenhouse disease resistance analysis. The results showed that the exogenous antimicrobial peptide Shiva A gene can be stably integrated and expressed in the transgenic and its ancillary progeny. Southern hybridization analysis showed that the Shiva A gene was one copy in the genome of the offspring and two copies in the T0 generation, suggesting that the T0 generation plants were mixed chimeras. Quantitative PCR analysis showed that the expression level of Shiva A gene in T0 generation was lower than that of its clonal generation. In this study, the expression of foreign genes is negatively correlated with the number of copies. The results provided a useful experimental material for the safety evaluation of transgenic citrus, and accumulated effective experimental data.