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本研究以野生型三生烟(Nicotiana tabacum cv.Xanthi nc.)及T_2、T_3代转hpa Xm(即hpa1Xm)基因烟草和转hpa Xm N-端缺失突变体(标记为hpa Xm△LP)烟草为试材,对目标基因的整合表达、叶绿素含量及防御酶活性进行检测。结果表明,卡那霉素抗性筛选、PCR及RT-PCR检测证明,外源目的基因hpa Xm和hpa Xm△LP整合到烟草基因组中,在转录水平上正常表达,且两个世代中稳定遗传。叶绿素含量测定中,转基因烟草含量显著高于野生型植株,同种转基因植株T_2代含量显著高于T_3代,而转hpa Xm基因烟草的总叶绿素含量比转hpa Xm△LP基因烟草的高,无显著性差异。防御酶活性的测定,表明转基因烟草的PAL、POD和PPO活性皆显著高于野生型植株;而转hpa Xm基因烟草的PAL和POD活性显著高于转hpa Xm△LP基因烟草;同品种转基因烟草中,T_2代PAL活性显著高于T_3代。说明hpa1Xm基因在烟草中表达能显著提高其叶绿素含量及抗病防御酶活性水平,且不同世代的转基因烟草之间表现有所差异。初步探索了hpa Xm的功能、遗传稳定性及信号肽类似序列对hpa Xm的表达及功能的影响。
In the present study, Nicotiana tabacum cv. Xanthi nc. And T_2, T_3 transgenic hpa Xm (hpa1Xm) transgenic tobacco and transgenic hpa Xm N-terminal deletion mutant (labeled as hpa Xm △ LP) As the test material, the integrated expression of target genes, chlorophyll content and defense enzyme activity were detected. The results showed that kanamycin resistance screening, PCR and RT-PCR test showed that the exogenous genes hpa Xm and hpa Xm △ LP were integrated into the tobacco genome and expressed normally at the transcriptional level, and stable in both generations . The chlorophyll content of transgenic tobacco was significantly higher than that of wild type plants. The content of T_2 generation of transgenic plants was significantly higher than that of T_3 generation, while the total chlorophyll content of transgenic tobacco with hpa Xm gene was higher than that of transgenic tobacco with hpa Xm △ LP gene. Significant difference. The activities of defense enzymes showed that the activities of PAL, POD and PPO in transgenic tobacco were significantly higher than those in wild type plants. The activities of PAL and POD in transgenic tobacco with hpa Xm were significantly higher than those in transgenic tobacco with transgenic hpa Xm △ LP. , The activity of PAL in T_2 generation was significantly higher than that in T_3 generation. The results showed that the expression of hpa1Xm gene in tobacco significantly increased the content of chlorophyll and the level of defense and defense enzyme activity, and the performance of different generations of transgenic tobacco was different. The function and genetic stability of hpa Xm and the effect of hpa Xm on the function of hpa Xm were explored tentatively.