【摘要】 【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2.71μg 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。更多还原

【Abstract】 【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.trnL gene sequencing 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 protein nucleic acid.The trnL genes in extracted cpDNA were comparatively analyzed.The results showed that trnL genes of the tested species(cultispecies,Saccharum 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.更多还原