基于聚丙烯酰胺凝胶电泳的棉花EcoTILLING技术体系创建

Establishment of PAGE-based EcoTILLING technique in cotton

以棉花纤维发育相关基因GhSUS1为目标基因,从EcoTILLING技术中常用的关键酶CELⅠ的提取、活性检测、目标基因引物设计及酶切等方面入手,开展了棉花EcoTILLING技术体系的创建研究。结果表明:所提取的CELⅠ活性高,稳定性好。在42℃、酶切体系为8μL PCR扩增产物中加入2μL粗酶液,与缓冲液等量混合,酶切45 min,能有效地对单核苷酸多态性(single nucleotide polymorphism,SNP)所在的异源双链错配位点进行酶切。对棉花GhSUS1基因设计A和D染色体亚组特异性引物进行等位基因的多态性分析,结果表明:EcoTILLING结果与测序结果一致,说明建立的基于PAGE的Eco-TILLING技术体系稳定可靠,可用于棉花复等位基因发掘研究。

EcoTILLING originated from TILLING has been employed to investigate allele polymorphisms in plant natural populations.In order to develop PAGE-based EcoTILLING technique at low cost for single nucleotide polymorphism（SNP）discovery and beneficial allele mining in tetraploid cotton,the key steps were extraction and activity test of the enzyme,CELⅠ,and design of gene-specific primers.The result indicated that highly active and stable CELⅠenzyme obtained was able to effectively digest mismatched base pairs at 42 ℃ for 45 minutes in the mixture of 8 μL of PCR products,2 μL of celery juice extract and 10 μL of buffer where the SNP exists in heteroduplexes.Gene-specific primers were designed followed by SNAPER softwere for sub-genome well-worked.The stability and reliability of EcoTILLING technique established was examined by sequencing for discovery of the allelomorphism of GhSUS1 gene related to fiber development among cotton varieties.The result showed that a stable and reliable PAGE-based EcoTILLING technique was established,allowing us to further mine favorable multi-alleles in cotton.