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根据两个植物抗病基因N和RPS2中核酸结合位点 (NBS)和富亮氨酸重复区 (LRR)中的保守序列设计了一对特异引物 ,用PCR从具有水稻 (OryzasativaL .)改良所需要的许多优良性状的水稻近缘野生种菰 (Zizanialatifolia(Griseb .)Turcz.exStapf)的基因组DNA中扩增同源片段。PCR产物经克隆后 ,分别以菰和水稻的基因组DNA为探针 ,通过点杂交对所得克隆进行了分析。点杂交结果表明 ,在所分析的 6 0个克隆中有 2个克隆是菰专化的序列 ,即它们与水稻无杂交信号。基因组DNA的Southern杂交进一步证实了这 2个克隆的专化性。为了验证一些可能的“水稻_菰”渐渗杂交系是否确实含有源自供体菰的DNA ,以这 2个克隆为探针 ,与经EcoRⅠ酶切的 5个可能的渐渗杂交系进行了Southern杂交。结果表明 ,这 2个克隆均能检测出其中的一个系含有其同源序列。这一结果为曾经报道的经一种非常规有性杂交方法将菰DNA导入水稻提供了确凿的证据。
A pair of specific primers was designed based on the conserved sequences in NBS and RLE2 of two plant disease resistance genes N and RPS2. A pair of primers was designed and synthesized by PCR from rice plants with Oryza sativa L. Homologous fragments were amplified from the genomic DNA of many desirable traits of wild relatives of Zimbabwe (Zizania latifolia (Griseb.) Turcz. ExStapf). After cloning the PCR products, the clones were analyzed by dot blot using genomic DNA of rice and rice as probe. Dot blotting results showed that two out of the 60 clones analyzed were specific sequences, ie, they did not hybridize to rice. Southern hybridization of genomic DNA further confirmed the specificity of these two clones. In order to verify that some possible “rice-菰” introgression lines do indeed contain DNA from donor 菰, two clones were used as probes to probe into five possible introgression lines digested with EcoRI Southern hybridization. The results showed that both of these two clones could detect that one of them contained its homologous sequence. This result provided conclusive evidence for introducing DNA into rice through an unconventional hybridization method that was reported previously.