Establishment Of AFLP Technical System On Anther Of Mitochondria DNA In Lycium Barbarum L

There is a widespread phenomenon of male sterility in higher plants, with the development of molecular biology, the research about mechanism of cytoplasmic male sterility has made generous progress. The mitochondria and cytoplasm male sterility has a very close relationship. Studies Sterile and Viable system gene expression differences to reveal the molecular mechanism of male sterility has the vital significance. AFLP molecular markers is a main method of gene expression analysis, has been widely used in plant male sterility function gene cloning and gene expression characteristics analysis, so using AFLP technology is a necessary way to screening different gene in Male sterile line medlar anther mitochondria.This research by Sterile and Viable system anther as experiment material, isolate and extract anther mitochondria and mitochondrial DNA, In order to mitochondrial DNA as a template, set up a suitable for anther of mitochondria DNA-AFLP technology system, in Lycium barbarum L. The results are as follows:1.Using the modified differential centrifugation established on anther of mitochondria in Lycium barbarum L, separation and purification method, through cytological and molecular biological methods for testing, Show that this study separation concentration of mitochondria, and integrity are high purity, full compliance with follow-up mitochondria related molecular biology research.2. Established a set of suitable for anther of mitochondria DNA-AFLP technology system; Using one-step and fractional step method all can obtain the ideal product of Enzyme digestion and connection, but the premise is the quality of template DNA concentration and conform to the requirements, the general template DNA OD260/280ratio must be in1.8to1.9, OD260/230ratio must be greater than2.0at the same time. Connect the enzyme reaction of sample template DNA concentration must not be less than100ng/ul, otherwise it is difficult to appear banding, before the experiment started, must test the template DNA concentration, it is the key to the follow-up experiments go smoothly.3.Established a suitable for anther of mitochondria DNA polyacrylamide gel electrophoresis and silver stain technique.In the process of polyacrylamide gel electrophoresis, temperature in the process of the electrophoresis have more influence on experimental results, electrophoresis temperature of45℃or so more appropriate. Formal electrophoresis temperature keep40-45℃, can obtain the desired effect, or a stripe weak, don’t even appear banding. Operation, the silver stain enhancement temperature strictly control on9-10℃, there will be an ideal band.