盐爪爪核酸提取方法研究

Study on Extraction Method of Nucleic Acid in Kalidium foliatum(Pall.) Moq.

为了提取盐生植物盐爪爪高质量的核酸,为其基因工程研究、实现盐渍土的生物改良以及解决土壤盐渍化这一世界性的生态问题奠定基础,笔者采用CTAB法、SDS法和改良尿素法用于盐爪爪DNA提取,在DNA提取方法的基础上,增加了乙醇和氯化锂特异沉淀步骤用于提取RNA,采用RT-PCR技术验证了该法提取RNA的质量。研究结果显示:3种DNA提取方法中,SDS法提取DNA的浓度较大,OD260/OD280和OD260/OD230均在正常范围之内;3种RNA提取方法中,SDS-LiCl沉淀法的OD260/OD280在1.8～2.1之间,OD260/OD230大于2.20,说明杂质去除较完全,浓度也较大;以SDS-LiCl沉淀法提取的碱蓬RNA为模板,进行胆碱单加氧酶基因的RT-PCR扩增,能清楚地看到在700～1500bp处有一清晰的扩增条带,并且其扩增量随着不同盐浓度(0、100、250、350、500mmol/L)变化,条带显示由暗到亮再变暗的趋势。确定了SDS法为盐爪爪核酸的最佳提取方法,所得结论为盐爪爪耐盐分子机理的研究奠定了基础,同时为其他植物的核酸提取提供借鉴。

In order to extracting high-quality nucleic acid in Kalidium foliatum（Pall.）Moq.,and to laying a foundation for its gene engineering research,biology improvement of saline soil and resolving soil salinization-an ecological problem in a world,The CTAB,SDS and improved urine method were adopted to extracting DNA of Kalidium foliatum（Pall.）Moq.,and added a step which employ alcohol and LiCl to precipitate particularly RNA,RT-PCR technology was adopted to examine quality of the RNA from SDS-LiCl method;Research results showed：in three extraction methods of DNA,concentration of the DNA from SDS method was larger,OD260/OD280and OD260/OD230were in normal range;in three extraction methods,the OD260/OD280 of the RNA from SDS-LiCl was between 1.8 and 2.1,the OD260/OD230was larger than 2.20,this meant that the impurity in RNA was entirely gotten rid of and the concentration of the RNA from SDS-LiCl method was larger;a trap between 700 bp and 1 500 bp was very clear in RT-PCR research of Choline monooxygenase（CMO）gene,the amplifying quantity changed with different salt concentration（0,100,250,350,500 mmol/L）,and the trap showed a trend from dim to bright,again to dim.The SDS was determined as the best method of nucleic acid extraction in Kalidium foliatum（Pall.）Moq.,the result will lay the foundation for research of salt-resistant molecule mechanism,and will also provide useful experience for nucleic acid extraction of the other plant.