TA3-13是克隆于小麦冷胁迫蛋白基因的截短片段。原核表达的TA3-13蛋白能够诱导烟草产生显著的抗烟草花叶病毒(TMV)的作用。文章将TA3-13基因片段克隆到植物表达载体pBI121上, 构建成转基因重组体pB-3-13, 通过冻融法转化农杆菌EHA105, 构建成转基因侵染菌株。采用叶盘法将pB-3-13转化三生烟草, 经卡那霉素抗性筛选, 获得48株T0代再生植株。通过PCR检测, 鉴定出33株转基因单株, 收获了20株种子作为T1代株系。PCR-Southern杂交结果显示, PCR阳性条带与TA3-13探针有特异性杂交, 说明外源基因被转化到烟草的基因组中。选取两个T1代株系的烟草植株用于各项测定。GUS组织化学活性鉴定和RT-PCR检测结果显示, 外源基因可以成功地表达。接种TMV病毒后, 转基因烟草抗TMV的能力较转空载体烟草提高3~5倍。转基因烟草具有抗TMV侵入和抗病毒病害发展的作用, 同时转基因烟草可以抗细菌软腐病菌的扩展。
TA3-13 is a truncated gene coding for the fragment of wheat (Triticum aestivum L.) cold shock protein WCP1. It has been shown previously that the procaryotically expressed TA3-13 can induce resistance to Tobacco mosaic virus (TMV) when sprayed onto plant leaves. In this study, we constructed an expression vector pB-3-13 by cloning TA3-13 into the bionary vector pBI121 and transformed it into Agrobacterium tumefaciense strain EHA105 via freeze-thaw method. Tobacco (Nicotiana tobacum cv. Xanthi nc.) transformation was performed using the leaf disc infection method. After screening on MS medium containing kanamycin and PCR analysis, 33 T0 plantlets were identified as transgenic. Seeds from twenty T0 plants were collected and planted as T1 lines. Two T1 lines were selected for further characterization. PCR and GUS staining analysis showed that TA3-13 was integrated into the T1 tobacco genome and expressed. When inoculated on leaves, the transgenic tobacco showed significant resistance against TMV and rot pathogen Pectobactrium carotovorum subsp. carotovorum. These results suggest that the expression of TA3-13 in tobacco can induce defense responses to patho-gen infection.
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