抗菌肽Shiva A基因在转基因柑橘无性繁殖后代中的遗传与表达分析

摘要

In order to further clarify the genetic stability and the target traits of the foreign gene in the progeny of transgenic citrus,the asexual reproduction plants of transgenic Newhall navel orange(Citrus sinensis Osbeck)containing bivalent antibacterial peptide gene(Shiva A-cecropin B) were studied.In this study,the genetic stability of Shiva A gene in To,T1,T2 and T3 progenies of transgenic citrus varieties were analyzed by PCR,Southern hybridization,Real-time quantitative PCR and greenhouse disease index statistic to Xathomonas axonopodis pv.Citri(Hasse) Dye.The results showed that the antimicrobial peptides Shiva A gene was existed and expressed in To,T1,T2 and T3 plants.This meant target gene could be stably inherited from one generation to another through asexual propagating.There was difference of Shiva A gene copy number between transgenic To and its asexual propagation.Southern bloting analysis showed that To generation had two hybridizations,but its progenies had only one hybridization consistent with To generation.It could be speculated that the T0 generation plant was a transgenic multicellular mixed cytochimera.In addition,Realtime quantitative PCR results showed that,even if Shiva A gene in the To generation genome was double copy numbers,but its expression level was lower than a single copy of the T1,T2 and T3 generation.Therefore,in this study,exogenous gene expression and transgene copy number was a negative correlation.The results of this study provide some basic date and the material for extending the stable phenotype of transgenic citrus strains in carrying out the safety evaluation of transgenic plants.%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(ShivaA-cecropinB)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ShivaA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,ShivaA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,ShivaA基因在To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.