红花cDNA-AFLP限制性内切酶的选择及银染体系的建立

Choice of restriction endonuclease and establishment of silver-staining method in cDNA-AFLP System for Carthamus tinctorius

目的:建立适于红花cDNA-AFLP分析酶切组合及银染技术体系,为进一步进行红花重要性状相关基因研究奠定基础。方法:对PstI/MseI和MseI/EcoRI两种限制性内切酶组合酶切效率进行比较,并探讨测序板处理中亲和硅烷和剥离硅烷的用量、测序胶中TEMED的用量、预电泳、温度因素、显色液的配置等银染过程中较关键的几个因素。结果:选用PstI/MseI限制性内切酶组合酶切效率较高,测序板应彻底清洁,TEMED量应适中,应进行30 min预电泳,整个铺板电泳过程保持温度恒定,选用4℃冷藏的碳酸钠做显色液效果好,综合以上几方面能显著提高胶图的质量。结论:本研究建立了适于红花的cDNA-AFLP银染体系,PstI/MseI较MseI/EcoRI酶切组合具有更高的多态性率,能更全面地揭示红花研究材料间更为丰富的遗传变异性;银染过程中成功克服了以往试验中常见的撕胶、背景太深、条纹不清晰等问题。

Objective： To establishing an appropriate combination of restriction endonuclease and silver-staining system for cDNA- AFLP analysis in Carthamus tinctorius L,. Methods：The efficiency of combination of PstI/MseI and MseI/EcoRI is compared. Several key factors in silver-staining including the affiliation and dissection dosage in sequencing plate processing, TEMED dosage in gel, preeleetrophoresis, temperature factor, staining liquid configuration etc are researched and discussed. Results ： Enzyme efficiency of PstI/ MseI is higher than MseI/EcoRI. Sequencing plate should be cleaned thoroughly. TEMED volume should be moderate. 30mins pre-electrophoresis should be carried out. Tenlperature should be constant through the whole electrophoresis process. Conclusion：Thls research has found a high effective restriction endonuclease combination can further discover the genetic background of safflower and has established an appropriate silver-staining system which has overcome several problems in staining process.