| PURPOSE:There are kinds of hyperaccumulators in nature that can enrich the heavy metal Cadmium (Cd) effectively. They can survive in the environment with a certain concentration of Cd pollution, uptaking and immobilizing Cd through metabolic mechanisms. At present, there are some disadvantages in known Cd hyperaccumulators, such as low enrichment coefficient, limited enrichment amount and so on, which affect the practical application. With the development of gene engineering technology, modifying the Cd hyperaccumulators by means of genetic engineering technologies is an important way to realize the practical application of phytoremediation of environment polluted by heavy metal Cd.We choose the Cd hyperaccumulator Solanum nigrum L. which widely distributed and easily available as the plant material in our genetically modification study. Agrobacterium-mediated technology was used to establish IRT1 gene modified Solanum nigrum L. hairy roots system, and the effects of Cd enrichment and the possible mechanism of IRT1 gene modified Solanum nigrum L. hairy roots are discussed. We hope to provide a reliable theoretical basis for plant regeneration and application of IRT1 gene modified Solanum nigrum L.Methods:Three Cd hyperaccumulators were chosen for hairy roots induction, which are Solanum nigrum L., Brassica campestris L. and Brassica juncea. Solanum nigrum L. is determined to serve as the target plant through the preliminary study of Cd enrichment ability and mechanism of the hairy roots. The conditions of Solanum nigrum L. hairy root induction were optimized further. The effects of important induction factors were investigated, including the Agrobacterium rhizogenes strains, naphthalene acetic acid (NAA) concentrations when the explants were pre-cultured on the medium, different sources of the plant material, as well as the explants types.On the basis of successful establishment of Solanum nigrum L. hairy roots, the foreign IRT1 gene associated with Cd accumulation was transferred into ATCC15834 Agrobacterium rhizogenes strains by pRI 101 plasmid. Leaf explants of Solanum nigrum L. were infected by the IRT1 gene modified ATCC15834 bacterium suspension and IRT1 gene modified hairy roots was induced and obtained. PCR was used to check the existence of rol B gene and foreign IRT1 gene in hairy roots tissue. Western blot technique was used to examine the protein expression of IRT1 gene after the stable IRT1 transgenic hairy roots line of Solanum nigrum L. was established.Finally, IRT1 transgenic hairy roots were used for Cd enrichment experiments.The IRT1 gene modified hairy roots and wild hairy roots were cultured on liquid MS medium containing different concentrations of Cd in darkness. The growth state and biomass of hairy roots, Cd content absorbed by hairy roots, and also the situation of ROS accumulation, cell apoptosis, anti oxidative enzyme system activity as well as the expression of IRT1 protein were measured after 14 days Cd treatment. The effects of IRT1 gene on improving the ability of Cd tolerance of Solanum nigrum L. hairy roots were discussed.Results:According to the comparison of rooting frequency of hairy roots, the growth status of hairy roots line, and also the ability of Cd enrichment of hairy roots system, Solanum nigrum L. hairy roots performed best in biomass and Cd enrichment ability. The fresh biomass of Solanum nigrum L. hairy roots can reach 2.897 g and dry weght 0.437 g, and the Cd content of hairy roots absorbed is 745.023μg/g after 14 days cultivation in the MS liquid medium of Cd concentration of 100μmol/L. So Solanum nigrum L. was chosen for further genetically modification.The optimition of Solanum nigrum L. hairy roots induction conditions showed the highest transformation frequency (more than 90%) was obtained when leaves of three-weeks-old in vitro seedlings were infected with A.rhizogenes strain ATCC15834(leaves were pre-cultured on MS medium supplemented with 0.6 mg/L NAA before infected).Gene sequencing revealed that recombinant plasmid pRI101-IRT1 was built successfully. The IRT1 gene modified hairy roots were acquired by leaves infected by IRT1 gene modified ATCC15834 strains. The results of PCR showed 423 bp and 1044 bp stripes, proving that the rol B gene and foreign IRT1 gene had been integrated into the hairy roots tissue of Solanum nigrum L.Cd stress experiments indicated there are no significant differences in all aspects of evaluation index with IRT1 gene modified hairy roots and wild hairy roots of Solanum nigrum L. under the control condition without cadmium treatment; with the increase of Cd concentration in the solution, IRT1 gene modified hairy roots and wild hairy roots of Solanum nigrum L. can both tolerate the Cd in the solution effectively, but IRT1 gene modified hairy roots showed better tolerance ability than wild hairy roots. Under different concentrations of Cd, compared to the wild hiary roots, the growth state of IRT1 gene modified hairy roots is better. Compared to the hiary roots under the condition without cadmium treatment, the fresh weight of wild hiary roots was reduced by 26.2% and dry weight 18%, and the fresh weight of IRT1 gene modified hairy roots was reduced by only 6.0% and dry weight 2.5% in the mean while after 14 days cultivation in the MS liquid medium in the Cd concentration of 100 μmol/L. The results showed that the biomass of IRT1 gene modified hiary roots affected less by Cd toxicity than wild hiary roots. The enrichment content of Cd reached 745.023 μg/g in wild hiary roots and 886.759 μg/g in IRT1 gene modified hairy roots at 100 μmol/L Cd, which indicated that the IRT1 gene modified hairy roots performed better in Cd enrichment. What’s more, compared to wild hairy roots, the activities of antioxidant enzymes (CAT, SOD, POD) of IRT1 gene modified hairy roots are more stronger, the expression of IRT1 protein is higher, ROS accumulation and apoptosis were lower. Above results revealed that IRT1 gene modified hairy roots received less damage than wild hairy roots. All the results showed that IRT1 gene can improve the tolerance of Solanum nigrum L. hairy roots on Cd.The establishment of the growth independent transgenic hairy root system can serve as the reliable experimental system for researchers to study the mechanism of heavy metal adsorption, the results would provided the applicable and theoretical basis for obtaining IRT1-transgenic regeneration plant of Solanum nigrum L. in the future. |
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