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作 者 ：欧阳波;龙芳;张扬勇;叶志彪

期 刊 ：武汉植物学研究 2006年 24卷 1期 页码：12-16

关键词：NPTⅡ；卡那霉素；多重PCR；转基因番茄；遗传分析；

Keywords:NPTⅡ, Kanamycin, Multiplex-PCR, Transgenictomato, Geneticanalysis,

摘 要 ：利用卡那霉素喷施方法对带有NPTⅡ标记基因和双价外源基因(烟草渗调蛋白基因AP24和菜豆几丁质酶基因Chi)的转基因番茄的3个世代在温室或田间环境进行规模化筛选,成功获得8个单拷贝转基因纯合植株。含有单个拷贝NPTⅡ标记基因的转基因株系后代,对卡那霉素的抗感分离符合3:1的孟德尔分离比例,T₂代中一些株系表现为对卡那霉素全抗,表明这些株系的外源基因已经纯合,这一结果在T₃代中进一步得到证实。但对于含有两个拷贝外源基因的转基因株系,外源基因的遗传则比较复杂。同时,结合Km喷施和多重PCR技术对外源基因的异常遗传进行了初步分析。用PCR分析进一步证实了该方法的准确性,该方法是对转基因番茄进行大规模、快速遗传分析的理想方法。

Abstract:Transgenic tomato plants with tobacco osmotin gene AP24 and bean basic chitinase gene Chi together with the NPTⅡ marker gene were screened by kanamycin spraying test for several generations.The transgenic offspring of single copy lines showed the expected 3 :1 segregation ratio,and absence segregation patterns for some lines were detected in T₂ seedlings,which indicated they were homozygous lines.Some of the homozygous lines were further checked on the T₃ generation.More than 8 different homozygous transgenic lines were obtained in this experiment.However,as to double copy transgenic lines,the inheritance of trangenes are complex.Multiplex-PCR analysis was introduced into investigation of abnormal transgene segregation.The accuracy of this method was confirmed by PCR analysis,it is an optimal method in rapid and large-scale screening of transgenic tomato plants with NPTⅡ marker in open field,and this method may be extend to other crops easily.

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