The Proteomic Analysis of Fleshy Tap Root Development and Germplasm Identification in Radish (Raphanus Sativus L.)
|School||Nanjing Agricultural College|
|Keywords||Radish freshy tap root expansion protein extraction 2-DE Seed protein|
Radish (Raphanus sativus L.), belonged to Cruciferae, Raphanus and originated from china, was an important vegetable worldwide. Tap root is the main product organs of radish. The study of freshy tap root development had a long period of morphological, physiological and biochemical description. In the protein level, the protemics study on mechanism of the freshy tap root development is still blank. Therefore, this study from total protein extracting of fleshy tap root and staining methods, established suitable for Radish proteomics technology system, and then researched the changes of the protein in the fleshy tap root development. The results will provide certain foundmation to reveal the mechanism fleshy tap root thicken at the level of protein, as well as the quality and production of radish and other important horticultural traits for genetic improvement of the provision of technical and theoretical basis. The major findings are as follows:The protein of tap root was prepared using trichloroacetic acid-acetone precipitation, phenol extraction, TCA-acetone-phenol extraction method. Then the comparison was studied on the pure protein yield of unit weight tap root, the result revealed that TCA-acetone extraction method had large impurities in protein sample, not suitable for the extraction of protein in fleshy tap root; phenol extraction and TCA-acetone-phenol extraction methods had better results in those comparation. The two methods are suitable for the radish fleshy root total protein extraction.The method of protein detection in the two-dimensional electrophoresis is particular important. Three stained methods, classical Coomassie brilliant blue staining (CBB-R250), colloidal Coomassie brilliant blue staining(CBB G-250)and silver staining studied were compared with the sensitivity of dyeing, background color, horizontal and vertical stripes, fake points, the experimental operational, reagent purity requirements, time, and downstream analysis of MS compatibility,etc. the results suggested that the silver staining could be used firstly to find out the difference explained spots, and then the CBB-R250 was used to quantitative and mass spectrometry.With White High-shoot ratio’NAU-XXXS’and red low-shoot ratio’NAU-LLYH’as accessions, the total protein were extracted from fleshy tap root of belly-breaking stage, leaf growth stage, thicken stage to carry out two-dimensional electrophoresis, and analysed using PD Quest 8.0 image analysis software for quantitative analysis. The results show that protein expression changes exsit in the two accessions during tap root development.16 differental protein spots was detected in high Shoot ratio’NAU-XXXS’, including 5 protein spots from a broken belly begins as a period of downward expression.5 protein spots showed upward,2 protein spots only in the fleshy roots thicken before hatching the expression of 4 protein spots were cut after the first rise.18 differential spots were detected in low-shoot ratio’NAU-LLYH’, of which 7 protein spots showed upward,3 protein spots downword,5 protein spots showed the first increase then decrease 3 protein spots high expression fleshy tap root of belly-breaking and thicken stage.10 protein spots were identified by MALDI TOF-TOF, transporter and elongation factor 1-beta and so on. These differential expression proteins spots were relevant with the freshy tap root development.The seed proteins of 32 heat-resistant radish accessions were analyzed with Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to estimate their polymorphism. A total of 41 protein bands were detected, of which 25 (61%) were polymorphic. Each accession could produce 28 to 39 protein bands, their profiles were different with each other and all the accessions could be distinguished. All accessions could be clustered into two major groups of white and red roots at the similarity index of 0.76 with UPGMA. The protein band similarity coefficient ranges from 0.64 to 0.98 and the average is 0.79. The results revealed that the cluster analysis of seed protein was highly in accordance with the radish root color, and the low genetic diversity and the narrow genetic basis existed in these heat-resistant radish accessions.