紫花苜蓿线粒体和叶绿体DNA高效提取体系的建立  

The Efficient Extraction System of Alfalfa Mitochondrial and Chloroplast DNA

作　　者：姜少俊 周海辰 金樑[2,3] 宋瑜[1,4] 杨阳[1] 张晓强[1] 王晓娟[2,3] 

机构地区：[1]兰州大学草地农业生态系统国家重点实验室草地农业科技学院,甘肃兰州730020 [2]上海科技馆 [3]上海自然博物馆自然史研究中心,上海200127 [4]中国环境管理干部学院,河北秦皇岛066004

出　　处：《云南农业大学学报（自然科学版）》2015年第5期747-754,共8页Journal of Yunnan Agricultural University:Natural Science

基　　金：国家自然科学基金项目(31270558)

摘　　要：细胞质雄性不育系是作物杂种优势利用的有效途径,分离其不育系线粒体和叶绿体基因组是深入探究细胞质雄性不育分子机制的重要环节,本研究以紫花苜蓿细胞质雄性不育系CMS1和野生对照植株WT为研究材料,选取新鲜叶片经冷冻匀浆、差速离心分离叶绿体和线粒体、DNase I降解核DNA、蛋白酶K裂解线粒体和叶绿体、酚—氯仿—异戊醇去除蛋白质和用乙醇沉淀叶绿体DNA(cpDNA)和线粒体DNA(mtDNA),结果如下:(1)叶绿体提取液中叶绿体数量的平均值为1 500个/m L,而詹纳斯绿B染色观察发现提取液中线粒体结构完整、含量较多;(2)叶片取材量为4 g时,CMS1(WT)的cpDNA平均产率为0.41(0.47)μg/g,而mtDNA的平均产率为1.15(0.83)μg/g;改进叶片取材量为10 g时,两种苜蓿材料的cpDNA和mtDNA的平均产率升高至2.17μg/g和2.22μg/g,并且纯度好(cpDNA和mtDNA的OD260/OD280均保持在2.0左右);延长蛋白酶K消化处理时间(2 h,4 h和6 h)可以提高紫花苜蓿cpDNA和mtDNA的产率,其中消化4 h的mtDNA质量浓度最高(59.9 ng/μL),而cpDNA浓度则在孵育6 h后达到最高值24.3 ng/μL;(3)紫花苜蓿CMS1和WT的mtDNA和cpDNA琼脂糖凝胶电泳检测发现,细胞质基因组条带清晰,无拖尾现象,进一步表明采用以上方法提取的紫花苜蓿mtDNA和cpDNA符合分子遗传学研究的标准和要求。The cytoplasmic male sterility（ CMS） plays an important role in the utilization of heterosis of crops. To separate mitochondria and chloroplast genomes of CMS lines is a necessary step in investigating the funtional mechanisms of cytoplasmic male sterility. In this study,alfalfa（ Medicago sativaL.） cytoplasmic male sterile line CMS1 and its wild type WT were selected as research materials to extract mitochondria DNA（ mtDNA） and chloroplast DNA（ cpDNA）. The fresh leaves were gathered and then frozen homogenized,centrifuged to chloroplasts and mitochondria by differential centrifugation. Then nucleus DNA was degraded by DNase I,and chloroplasts and mitochondria were cracked by proteinase K. After proteins were removed with phenol-chloroform-isoamyl alcohol,the mtDNA and cpDNA were isolated by ethanol precipitation. The main results are as below.（ 1） The average number of chloroplasts in extracting solution was more than 1 500 per milliliter,and mitochondria observed with Jannus Green B dyeing showed a large number with intact structure.（ 2） When 4 g alfalfa leaves were extracted,the average cpDNA productions of CMS1（ WT） were 0. 41（ 0. 47） μg / g,and the average mtDNA yield of CMS1（ WT） were 1. 15（ 0. 83） μg / g. As the extracted leaves of alfalfa increased to10 g,both of average cpDNA and mtD NA yield increased to 2. 17 μg / g and 2. 22 μg / g,respectively,and the quality of them was also good（ the values of OD260/ OD280 of cpDNA and mtD NA samples were around 2. 0）. Extension of the proteinase K digestion time（ 2 h,4 h,6h） could improve the productive rate of cpDNA and mtD NA： proteinase K incubation for 4 h gave the highest concentration of mtD-NA（ 59. 9 ng / μL）,and the highest yield of cpDNA up to 24. 3 ng / μL under 6 h incubation.（ 3）The agarose gel electrophoresis of alfalfa cytoplasmic genomes showed that,all the bands of these cytoplasmic genomes showed no railing phenomenon,which identified high-purity DNA without pollution of RNA furthermor

关 键 词：紫花苜蓿 线粒体DNA 叶绿体DNA 差速离心法 

分 类 号：S541.103.2[农业科学—作物学]