茶树简化EcoTILLING技术的建立

EcoTILLING(Ecotype Targeting Induced Local Lesions IN Genomes)可快速检测自然群体中的SNP(Single Nucleotide Polymorphism)等DNA变异。第一次建立了简化的茶树EcoTILLING技术体系:首先筛选扩增目的基因的特异PCR引物;然后通过CELⅠ酶切PCR产物优化实验,确定酶切时间20 min、10×buffer组成为250 mmol/L Tris-HCl(pH7.4)、150 mmol/L KCl、15 mmol/L MgSO4、4μg/mL BSA和0.04%Triton x-100,反应温度为47.5℃,酶量为1.8μL CJE(Celery Juice Extract)/10μL反应体系时,酶切的谱带信号较强、特异性高;初步确定合适的非变性聚丙烯酰胺凝胶电泳检测条件为凝胶浓度5%、上样量4～5μL、有效分离长度要达到15 cm。利用上述简化的EcoTILLING对6份茶树种质870 bp的PPO基因片段实现了分型,经测序验证酶切产生的谱带与6份种质内部或不同种质间的SNP位置吻合。

Simplification of EcoTILLING Technique for Tea Plant

EcoTILLING(Ecotype Targeting Induced Local Lesions In Genomes) had been used as a SNP(Single Nucleotide Polymorphism) discovery tool to examine DNA variation in natural populations. A simple EcoTILLING technique for tea plant was established. Firstly, specific PCR primers for the amplification of the target gene were screened out. Then through comparison, a fine CELⅠ enzyme digestion could be resulted using 20 min with 10× buffer containing 250 mmol/L Tris-HCl (pH7.4), 150 mmol/L KCl, 15 mmol/L MgSO_4, 4 μg/mL BSA and 0.04%Triton x-100, and 1.8 μL CJE(Celery Juice Extract)/10 μL reacting solution at 47.5℃. Moreover, a good picture could be obtained when using 5% polyacrylamide gels (PAGE), adding 4～5 (L digesting products in PAGE electrophoresis. The 870 bp fragment within the PPO (Polyphenol Oxidase) gene of 6 tea germplasms was distinguished from each other using the simplified EcoTILLING. The CELⅠdigested bands were ascertained that according to the position of SNP in or between the 6 accessions through sequencing verification.