从豆科植物的根瘤中直接提取根瘤菌DNA的方法  

ISOLATION OF DNA FROM THE ROOT NODULE OF LEGUME PLANT

作　　者：陈强[1] 张小平[1] 李登煜[1] 陈文新[2] K.Lindstrm Z.Terefework 

机构地区：[1]四川农业大学农学院微生物学系,雅安625000 [2]中国农业大学生物学院微生物学系,北京100094 [3]Department of Applied Chemistryand Microbiology

出　　处：《微生物学通报》2002年第6期63-67,共5页Microbiology China

基　　金：国家自然科学基金资助项目 (No 39970 0 2 9)~~

摘　　要：豆科植物的新鲜根瘤经表面灭菌、破碎后直接加入 2 0 0 μL 4mol/L异硫氰酸胍 (GUTC)裂解液混匀 ,离心去掉根瘤残留物 ,再加入适量RNA酶 ,37℃温育 30min后 ,加入 2 0 μL硅藻土吸附液 ,室温处理 1 5min离心去掉上清 ,沉淀经GUTC裂解液二次处理 ,再分别用洗涤液、 70 %酒精洗涤。最后 ,硅藻土沉淀经真空干燥 ,加入 2 0 μL超纯水洗涤硅藻土沉淀 ,即可获得类菌体根瘤菌DNA。所获得的DNA可以用于AFLP、 1 6SrRNAPCR反应。The fresh root nodule from the legume plant was surface sterilized, and crashed in an Eppendorf tube, then mixed with 200μL of GUTC buffer thoroughly,and spinned briefly,the upper phase was transferred to a new Eppendorf tube, and RNase was added,and the mixture was incubated at 37℃ for 30min, and 20μL of diatomaceous earth solution was added and mixed properly. The mixture was incubated at room temperature for 15min, then centrifuged, and discarded the upper phase. The sediments were treated with GUTC buffer described as above again, and followed by 3 time rinsing of new wash buffer. The sediments were washed by 70% ethanol, and dried in vacuum. 20μL of ultrapure water was used to dissolve the DNA absorbed by diatomaceous earth sediments. After centrifuge, the upper solution was transferred to another tube, and used as template DNA to do the AFLP and 16S rRNA PCR. The results showed that high quality of DNA can be obtained by this method.

关 键 词：豆科植物 直接提取 根瘤菌DNA AFLP指纹图谱 

分 类 号：Q933[生物学—微生物学] S154.38[农业科学—土壤学]