Study On Cd-induced Stress Responses And Transcriptome In Hairy Roots Of Transgenic Brasssica Campestris L.

PURPOSE:According to surveys of the Ministry of Ecological Environment and the Ministry of Land and Resources,cadmium pollution is currently the most serious and widespread heavy metal pollution in China.Plant is one of the most important ways for heavy metals flowing into the human body,and is also the most crucial carrier of phytoremediation engineering for heavy metal contaminated soil.Brassica campestris L.is one of the typical cadmium hyperaccumulators with the advantages of short growth cycle,large biomass and strong adaptability.As the main organ in touch with heavy metals,root is the first locus to start stress response.With many merits such as rapid growth,large biomass,no seasonal restriction and unaffected by rhizospheric microorganisms,transgenic hairy root is physically very close to the real root.With transgenic Brassica campestris L.hairy roots as experimental materials,this paper studies their physiological response features under different cadmium concentrations and in different time as well as enrichment capacities of cadmium and necessary elements.Using method of bioinformatics,the paper comprehensively analyses the transcriptome sequencing data about how transgenic Brssica campestris L.is in response to cadmium stress,and explores its key genes and metabolic pathways,thus to provide solid theoretical basis for the subsequent transgenic modification and the construction of practical cadmium hyperaccumulation Brassica campestris L.METHODS:The transgenic Brassica campestris L.hairy roots induced by A4 agrobacterium were used as experimental material.And the stable transgenic hairy roots system was established by MS suspension culture method.The physiological response of the transgenic Brassica campestris L.hairy roots were detected under different cadmium concentrations(0,25,50,100,200,400 ?mol/L)and times(1 d and 7 d),which included growth state,biomass,reactive oxygen species(ROS)accumulation,antioxidant enzymes activity variation,cell apoptosis,cadmium and other essential elements contents.Then the optimum cadmium concentration and time of transcriptome sequencing were determined by statistical analysis.The transcriptome differences of transgenic Brassica campestris L.hairy roots before and after cadmium stress were detected by second generation sequencing technology.Through bioinformatics analysis,the significant difference expression genes were selected,and the Gene Ontoloty(GO)function and KEGG Pathway enrichment analysis were carried out.Then parts of differential genes were verified in RNA level by Real Time PCR.And genes of the key metabolic pathways were verified in protein level.RESULTS:In 30 ds' growth cycle,transgenic Brassica campestris L.hairy roots underwent the first 10 ds' logarithm growth,10 ds' stationary phase and 10 ds' aging phase.The results of cadmium stress experiments showed that the growth of hairy roots had no significant difference under low cadmium concentrations(below 100 ?mol/L)stress,while obviously inhibited under high cadmium concentrations(more than 100 ?mol/L)stress.And the fresh weight of transgenic Brassica campestris L.hairy roots reached maximum of 4.34 g after 7 d's 25 ?mol/L cadmium stress.The content of ROS in transgenic Brassica campestris L.hairy roots increased with the increasing concentrations of cadmium.The antioxidant enzymes activities of transgenic Brassica campestris L.hairy roots(superoxide dismutase,SOD;peroxidase,POD;catalase,CAT)decreased first and then increased with the increasing cadmium concentrations under 1 d's stress,while under 7 d's stress the trend was the opposite.PI(propidium iodide)staining and the content of MDA(malonaldehyde)indicated that the cellular damage was more serious along with the increase of the cadmium concentrations.With the increase of cadmium concentrations,cadmium content of transgenic Brassica campestris L.hairy roots increased,and reached maximum of 2.97 mg/g after 7 d's 400?mol/L cadmium stress.The iron content also increased significantly with the maximal value of 14.52 mg/g under 1 d's cadmium stress,while there was no significant difference under 7 d's cadmium stress.The potassium content under 7 d's cadmium stress is 1.6 times of the 1 d's stress(15.73 mg/g).The study showed that the physiological response of transgenic Brassica campestris L.hairy roots was correlated with the concentration and time of cadmium stress.At the same time,cadmium stress caused the metabolic disorders of iron and potassium in the hairy roots,but the hairy roots of Brassica campestris L.had better enrichment effect on cadmium.The statistical analysis results showed the difference of transgenic Brassica campestris L.hairy roots between 1 d's 200 ?mol/L cadmium stress group and the control group was significant,and it was suitable for transcriptome sequencing(p<0.05).The RNA quality control showed that the RNA was of good quality,and the total amount could meet the requirements of two or more times building blocks which satisfied the sequencing requirements.The comparison rate of each sample was 64.97%,and the uniformity ratio between samples showed that the data was comparable.Correlation analysis and cluster analysis showed that the correlation coefficient of each sample in the treatment group and the control group was above 96%.The statistical results of DEseq2 showed that 1,564 genes were up-regulated and 830 were down-regulated.Eight significant enrichment GO terms and six significant enrichment metabolic pathways were found through GO function classification and enrichment analysis,KEGG biological Pathway classification and enrichment analysis.According to GO information,Pathway information and FC value,eight significant difference genes were verified(103845713,103874543,103835040,103872346,103875293,103867458,103855019,103870505)by Real Time PCR.The results showed that the transcriptional levels of these 8 genes were consistent with those of the transcriptome sequencing.The Western blotting experiments showed that cadmium stress resulted in expression in great quantity of glutathione synthetase and glutathione-S-transferase in transgenic Brassica campestris L.hairy roots.CONCLUSION:Transgenic Brassica campestris L.hairy root can replace the roots of complete plant to do the studies of heavy metal cadmium stress.The experimental results of transcriptome sequencing and Western Blotting showed that glutathione synthetase and glutathione-S-transferase might have played important roles in the process of transgenic Brassica campestris L.hairy roots' response to cadmium stress.