【摘要】 EcoTILLING(ecotype targeting induced local lesions in genomes)是一种高通量检测和分析自然群体中单核苷酸多态性(single nucleotide polymorphism,SNP)变异的研究方法。由于该技术需要高级、精密的仪器设备和昂贵的荧光标记引物来检测CEL(celery)Ⅰ的酶切产物,极大地限制了该技术的应用。本研究对传统EcoTILLING技术做了适当改进,简化了CELⅠ酶的抽提步骤,采用自然冷却方法制备异源双链产物,普通银染检测CELⅠ酶切产物,酶活检测结果显示CELⅠ粗提液对错配产物切割效果良好。利用EcoTILLING简化技术对18份水稻(Oryza sativa L.)品系Wx基因第1内含子5’端的G/T基因型进行了成功鉴定。此外,对18份水稻材料中一417bp长的Wx基因片段实现了CELⅠ酶切条带分型,经测序验证,酶切带型差异能够准确反映材料间SNP的变异。本研究结果表明,应用EcoTILLING简化技术可有效进行水稻基因型鉴定及SNP检测,是开展水稻功能基因组研究的有力工具。更多还原

【Abstract】 Ecotype targeting induced local lesions in genomes (EcoTILLING) is a high-throughput method of detection and analysis of single nucleotide polymorphism (SNP) variations in natural populations. However, it requires a substantial investment in sophisticated equipment and costly fluorescence labeled primers to detect CELⅠ- cut products that limit its use in laboratories with common financial resources. The technology of traditional EcoTILLING had been appropriately improved in this study. Here, we simplified extraction procedure of the CELⅠ enzyme, prepared heteroduplex DNA products for CELⅠdigestion by use of natural cooling method, and detected the CEL I-cleaved products through general silver staining. The results of activities of crude CELⅠenzyme extracts showed that the activity of CELⅠwas high enough for detection of mismatch bases. Using the simplified EcoTILLING technique, the G/T polymorphism at the first intron 5’ splice site of the Wx gene among 18 rice(Oryza sativa L.) accessions were successfully genotyped. In addition, CELⅠdigested bands of a 417 bp fragment within the Wx gene of the 18 rice accessions were distinguished into several types. On comparisons with sequencing results, CELⅠcut bands could accurately reflect the SNP variations between the materials. The results suggested that the simplified EcoTILLING is an effective method for SNP discovery and genotyping in rice that will provide powerful tool for rice functional genomics research.更多还原