甘蔗叶绿体DNA的提取方法及其验证分析

Extracting method for sugarcane chloroplast DNA and its verification

[目的]探索适于普通实验室条件的甘蔗叶绿体DNA（cpDNA）提取方法,为甘蔗叶绿体分子生物学研究提供参考.[方法]参考水稻、小麦等作物cpDNA的DNase Ⅰ差速离心处理法,采用改良的DNase Ⅰ差速离心法提纯甘蔗cpDNA,并对其trnL基因序列进行比对分析,证实所提取cpDNA的可行性.[结果]采用改良的DNase Ⅰ差速离心处理法提取能得到量多、杂质少的cpDNA,用Eppendorf蛋白核酸测定仪检测,平均每克甘蔗样可提取2.71 μg cpDNA,OD260/OD280为1.79～1.90.对提取的cpDNA的trnL基因序列进行比对分析,证实参试的甘蔗栽培种、斑割复合体后代及其回交材料的叶绿体trnL基因与已报道的甘蔗栽培种NCo 310、SP-80-3280和割手密HN0046的trnL基因相似度均在99.0％以上,并在381、386、389、392、393、400、488、490 bp等8个位点上检测出碱基突变、插入和缺失现象.[结论]采用改良的DNase Ⅰ差速离心处理法提取甘蔗叶绿体DNA具有可行性,提取的甘蔗cpDNA完全可以用于后续的分子水平研究.

[Objective]A lab-friendly extracting method for sugarcane cpDNA was explored based on sugarcane traits in order to provide references for molecular biology research of cpDNA. [Method]Referring to DNase I differential centrifugation treatment of rice and wheat cpDNA, sugarcane cpDNA was extracted and purified using the modified procedure, tmL gene se- quencing was compared and analyzed to verify the feasibility of cpDNA extraction. [Result] High quality （OD260/OD280：1.79- 1.90） and high amount of cpDNA （2.71 μg per gram of fresh leaves） was isolated and detected by Eppendorf tester of pro- tein nucleic acid. The tmL genes in extracted cpDNA were comparatively analyzed. The results showed that tmL genes of the tested species （cuhispecies, Saecharum spontaneum hybrid and backcross） were 99% homologous with the reported cultivated species NCo 310, SP-80-3280 and Saccharum spontaneum HN0046. Base mutation, insertion and deficiency occurred in eight loci like 381, 386, 389, 392, 393, 400, 488, 490 bp. [Conclusion]This modified method can serve as an efficient cpDNA extraction procedure for further molecular research.