The Proteomics Analysis Of Response To Cadmium Stress In Radish (Raphanus Sativus L.)

Radish(Raphanus sativus L) is a vital root vagetable which originated from China. One of the most undercover and undercontrolled pollution sources is heavy metal which includes Cd, a bio-toxic one of it. It will threat people's health, while excessive Cd leaks into the environment, participating in the circle of water-soil-biology system, and intaken by animals and humanbeings as a proportion of food chain. With more and more serious environmental pollution, cadmium pollution has seriously affected the security of radish production. Currently, the response mechanism to cadmium-stress in plant has become an important subject.Radish advanced inbread lines'NAU-RG'which preserved in our lab was prepared for marerials. The method of protein extraction was optimized and the system of it was established. After it,2-DE was performed to study the differential expression of proteins extracted from radish leaves and root tissues under different concentration of Cd solution and then the Mass Spectrometry was performed to analyse the functions of the proteins which are obviously different. At last, the sqRT-PCR was used to analyse the Cd-stress respond genes in transcriptional level and clone the key genes during the Cd-stress. The main results are as followed:1. Five methods of plants protein extraction were used in radish leaves for extracting the protein, and were optimized at the same time to establish the technology system of protein preparation in radish leaves and root tissues. It is obvious that the Tris-HCl/TCA-Acetone adapts to protein extraction of leaves, and the modified phenol-extraction was also optimized by orthogonal test method.2.The radish seedlings were cultivated in the nutrient solution,containing 0mg·L-1CdCl2·2.5H2O,50mg·L-1CdCl2·2.5H2O and 100mg·L-1CdCl2·2.5H2O.The 2-DE technique was applied to analyse the case of proteome respond under the condition of Cd stress. There were 10 protein spots differentially expressed, in which one spot was expressed by the induction of Cd stress, two spots were up-regulated, five spots were down-regulated, one spot was dismissed, and the last one was up-regulated when the Cd concentrate was low, and vice versa. We found 31 differently expressed protein spots in root proteins,in which 23 were successfully indentified by MALDI-TOF/TOF. The database search showed that the defense related proteins accounted for 56.5%,cell metabolism related protein 30.4% and Signal transduction related proteins accounted for 8.7%.3.The expressional analysis of the sqRT-PCR reveals that after the Cd stress, three genes (RsPDI,RsPGK,RsSOD) changed on the transcriptional level. The genes encode SOD were cloned and the full length of cDNA and the entire ORF were gained.In our study, the establishment of protein extraction system have important implications for proteomic analysis of radish.The difference expression protein images have been constructed and some proteins identified by MS.Several Cd-stress response genes have been analyzed by sqRT-PCR and TA cloning.These results provided theoretical basis for the revealing of Cd-stress response mechanism.