Induction Of Hairy Roots Of Brassica Campestris L. By Agrobacterium Carrying GshB Gene

Purpose:The heavy metal cadmium (Cd) was one of heavy metal elements with the biological toxicity.Along with the "three wastes" emissions to the soil, the mobility of Cd was very strong in the soil-plant system.The repair of the contaminated soil by Cd becomes research hotspot all over the world.The phytoremediation had potential application on repairing the contaminated soil by Cd, and the Cd hyperaccumulation plant was the key of the technology.Therefore, to strengthen the research on the Cd hyperaccumulation plants was the basis of promoting the application of phytoremediation in the Cd contaminated soil.Brassica campestris L. was selected for the research due to its fast growth and easy access, and gshB gene encoding Glutathione Synthetase(GS) was selected as exogenous gene.The aim of the research was to establish transgenic hairy root system mediated by Agrobacterium technology and provided a simple and reliable experimental system for the study of the interaction between hairy roots and Cd.Methods:Heterologous gene transformation mediated by Agrobacterium was common method of plant transgenic technology.This paper used wild type Agrobacterium and Agrobacterium containing gshB to transform Brassica campestris L. to establish wild type and gshB transgenic hairy root.In order to induce Brassica campestris L. hairy root and obtain high transformation rate,effects of four important induction factors,including Agrobacterium rhizogenes strains (ATCC11325 and ATCC15834),explants (cotyledon and stem segment),infection time (10 min and 30 min),auxin (2,4-D and NAA) were investigated by using the Ls(2’) orthogonal experiments.The recombination pRI101-gshB plasmid was transformed into Agrobacterium ATCC11325 by using heat shock.PCR and RT-PCR technique were used to confirm that the key gene gshB and rolC had been intergrated into DNA of Brassica campestris L. hairy root.Cd stress mechanism of the Brassica campestris L. hairy root was studied finally. The hairy root with gshB gene and wild hairy roots were cultured on solid MS medium containing 50 μmol/L Cd and without Cd in the darkness. Biomass of hairy root, hairy root of peroxidase (POD) and superoxide dismutase (SOD) activity, malondialdehyde (MDA) content would be measured after 7 days. PI staining detected the permeability of plant cell membrane under Cd stress.Results:According to the variance analysis,the interaction of the Agrobacterium strains and infection time as well as the explants type showed significant effects on the induction of hairy root.The orthogonal experiment results displayed that the induction rate of hairy root reached 73% when the leaf was precultured on the MS medium for 48 h and infected for 10 min by the Agrobacterium rhizogenes ATCC11325 along with 0.3 mg/L of 2,4-D.PCR amplification results revealed that rolC genes of Ri plasmid of Agrobactterium rhizogenes had been integrated into the plant genome of Brassica campestris L.hairy root, which indicated that Brassica campestris L.hairy root had been induced successfully.Based on optimizational conditions of hairy root induction by orthogonal test, Brassica campestris L. hairy root carrying gshB gene was induced successfully using Agrobacterium ATCC11325 containing the recombinant pRl101-gshB plasmid infecting the cotyledons of Brassica campestris L.PCR results showed that the DNA genome of the hairy root contained about 960 bp fragments,which proved that the gshB fragment had been integrated into the genome of Brassica campestris Z.The cDNA sequence of the genome of Brassica campestris L. hairy root was obtained by RT-PCR.PCR results showed that the cDNA sequence contained 960 bp fragments,which indicated that the foreign gene had transcribed in the Brassica campestris L.hairy root.Experiments under the Cd stress showed that growth of transgenic hairy root and wild type hairy root were inhibited under 50 μmol/L Cd. The activity of SOD and POD enzyme increased in wild-type hairy root and transgenic hairy root as well as the content of MDA under 50μmol/L Cd. Besides, the degree of PI staining deepened under 50 μmol/L Cd. The increased proportion of SOD and POD activity in the transgenic hairy root was higher than that of wild-type hairy root, but the content of MDA and PI staining degree was lower than wild-type hairy root. This may be overexpression of gshB gene in the hairy root to improve the ability of chelating Cd, which reduced the toxicity of Cd in the hairy root.The Brassica campestris L. hairy root system carrying gshB gene was established successfully, which provided a better system for exploring the mechanism of Cd enrichment by the Brassica campestris L. hairy root.