| Radish(Raphanus sativus L.), a root vegetable belonging to the Brassicaceae family with a high value and economic value, is one of the most important root vegetable crops originated from China (Wang&He2005). Cadmium (Cd) is one of the highly toxic heavy metals in environmental pollution. Cd in soil not only seriously affects the quality and product of plants, but also is harmful to human health through the food chain. When accumulated in plant to a certain extent, Cd would produce a series of poison symptom. Roots are the first site that contact with the soil environment, under heavy metal stress, the structure of plants would be affected at cell, tissue and organ levels and caused a series of physiological disorders in photosynthesis, respiration, and nitrogen metabolism. With the long-term adaptation to the environment and evolution, plants have a series of genetic mechanisms of Cd tolerance to reduce or avoid the toxic effects of Cd. In this study, optical microscope and transmission electron microscopy were performed to study the cellular mechanisms of cadmium uptake and accumulation in radish; the key genes involving in Cd accumulation and transport were isolated using T-A clone; and the mRNA differential display reverse transcription technology was employed to isolate the related genes under Cd stress. The results would provide powerful and valuable tools for better understanding the molecular mechanisms of Cd uptake and transport. The main results were summarized as follow:In this study, optical microscopy and transmission electron microscopy were performed to study the cellular mechanisms of cadmium uptake and accumulation in radish;. The results showed that the organization structure was impacted and destructed greately and seriously with the Cd concentration increasing dramatically. Cd affected the changes of ultrastructure of radish roots, at low concentration, the cell wall thickened, the concentration increased, the cell wall broken and the plasmolysis phenmena happened; the nuclear membrance was slightly shrinked at low concentration before nucleolus disintegrated at high concentration; the number of mitochondrial cristae decreased at low concentration, after the concentration increased, mitochondria disintegrated, and the cristae fractured. Observation indicated that at the low concentration in roots, the Cd was mainly distributed in the epidermis, while the concentration grew, it would gradually extend to the internal until proliferated the xylem. At celluar level, Cd was firstly distributed in the cell wall, and then distributed in the vacuole.Differential-display reverse transcription-PCR (DDRT-PCR) Differential-display reverse transcription-PCR was employedapplied to isolate the genes whose transcription was altered with Cd stress. A total of36TDFs were successfully identified, including29TDFs that were high homology homologous with known functional genes. The functions of these identified TDFs were classificated as follow:cell and tissue metabolism, reponse to stress, photosynthesis, transfer function, transcription and translation expression factor and signal transduction. The results indicated that Cd stress is a complex physiological and genetic process that including a variety of biological metabolism are implicated in Cd stress reponses.According to the conserved sequence of T.caerulescens (TcZNT2), a ZIP family member, Zn transporter RsZNT2, was isolated using T-A clone. The DNA full-length of RsZNT2was1636bp, including a complete open reading frame (ORF) of1251bp, encoding416amio acids with a molecular weight of44.13KDa and containing six transmembrance domains. RT-PCR analysis showed that after added Zn, the expression of Cd decreased, which may contribute to the similar structure of Zn and Cd, Cd and Zn share a same absorption transport mechanism, the increase of concentration of Zn reduced the uptake and transport of Cd, at certain extent, Zn and Cd may enter into the plants by a same way. A gene of CDF family was obtained from radish, designed as RsMTP2. DNA full-length was1183bp with1161bp ORF, encoding386amino acids. Analysis exhibits that, the protein contains six transmembrance regions, as a cytoplasmic membrance protein, it associated with Cd uptake and transport. RT-PCR showed that the expression of Cd increased with the increasing concentration, but at400mg-L’1, expression reduced, which suggested that the increasing concentration of Cd induced the expression of RsMTP2to reduce the toxity of Cd. However, the expression level would be reduced after the concentration increased to a certain extent, which indicates that Cd involved in RsMTP2regulation at transctriptional level. |
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