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通过集成候选抗性基因克隆技术和磁珠富集文库技术以及可转化基因组文库技术,提出了基于野生稻基因组富集文库克隆野生稻新抗性基因的策略。包括构建野生稻可转化基因组文库,利用RecA重组蛋白介导生物素标记的探针对文库进行磁珠富集,构建野生稻基因富集文库,然后对富集文库的克隆进行鉴定分析,包括点杂交和克隆末端测序分析,最后对富集文库的阳性克隆进行全序列测定和功能验证。按照上述方法,构建了东乡普通野生稻和小粒野生稻可转化基因组文库,库容量分别达到1.58×105和2.68×105个克隆,进而构建了东乡野生稻基因组候选抗性基因富集文库,单克隆总数1 152个,对富集文库的菌落原位杂交筛选获得12个阳性克隆,对其中5个阳性克隆全序列测定结果表明,4个阳性克隆含有已克隆抗性基因的保守序列,证实构建候选抗性基因富集文库克隆野生稻新抗性基因的策略是可行的。
By integrating candidate resistance gene cloning technology and magnetic bead enrichment library technology and transformable genomic library technology, a strategy of cloning wild rice new resistance gene based on genomic enrichment of wild rice was proposed. Including the construction of wild rice transformable genomic library, the use of RecA recombinant protein-mediated biotin-labeled probe magnetic enrichment of the library to construct a library of wild rice gene enrichment, and then the enrichment library clones were identified and analyzed, including the point Hybridization and cloning end sequencing analysis, and finally the positive clones enriched libraries were sequenced and functional verification. According to the above method, the genomic library of Dongxiang Oryza sativa and Oryza rufipogon (Oryza sativa L.) was constructed and the library capacity reached 1.58 × 105 and 2.68 × 105 clones, respectively, and then the Dongxiang Oryza sativa genome resistance gene enrichment library was constructed. A total of 1 152, the enrichment library colony in situ hybridization screening of 12 positive clones, of which 5 positive clones full sequence determination results show that 4 positive clones contain the conserved sequence of the cloned resistance gene confirmed the construction candidate Strategies for Enrichment of Novel Genes Involved in Library Cloning of Wild Rice with a Resistance Gene is Possible.