野生稻基因组抗性基因富集文库的构建

The Construction of Wild Rice Genomic Enrichment Library of Resistant Genes

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。

The strategy of cloning new resistance genes of wild rice on the basis of construction of the genomic enrichment library was proposed by way of integrating the techniques of candidate resistance gene cloning, magnetic bead enrichment library and transformation-competent genomic library. Firstly, the transformation-competent genomic library of wild rice was constructed, then the wild rice genomie enrichment library of resistance genes was constructed by the magnetic bead enrichment method with the biotin probe mediated by the recombinant protein Re- cA, which was amplified from wild rice by degenerate primers designed according to resistance gene conservation. The enrichment library was then screened by dot hybridization and positive clones were assayed by end sequencing. Finally, the whole-clone sequencing of the selected positive clones was done and their functions would be verified by transformation. In this research, the transformation - competent genomic libraries with a volume of 1.58×10^-5 clones and 2.68 ×10^-5 clones were constructed for Dongxiang wild rice （ Oryza rufipogen） and Oryza minuta wild rice, respectively. The enrichment library of resistance genes for Dongxiang wild rice was further constructed with a volume of 1 152 clones, from which 12 positive clones were isolated. The whole-clone sequencing of 5 representative positive clones revealed that 4 clones contained the conserved domain of the existing resistance genes, indicating that the strategy is feasible to clone new resistance genes from wild rice by construction of the genomic enrichment library of resistance genes.