Preliminary Study On Response Mechanism Of Brassica Campestris L. Hairy Roots To Cadmium Stress Based On Integrated Analysis Of Transcriptome And Proteome

Objective: The response to cadmium stress in the hairy roots of hyperaccumulators Brassia campestris L.is a complex system controlled by multiple quantitative trait loci(QTLs),and its systemic regulatory mechanism is unclear.In order to reveal the regulatory mechanism more comprehensively,we analyzed changes of Brassia campestris L.hairy roots under cadmium stress through morphology,new transcription annotation,proteomic sequencing and transcriptome ensemble analysis in this study.The thesis hopes to provide a theoretical basis for the genetic modification of practical Cd hyperaccumulator.Methods: In this study,the cadmium stress was simulated by liquid suspension culture of the Brassia campestris L.hairy roots.The Brassia campestris L.hairy roots were treated with 0 and 200 ?mol/L cadmium for 24 h,and the changes of individual hairy roots,cell level and ultrastructure were observed.At the same time,new transcript annotation,Isobaric Tags(IBT)quantitative proteome and integrated analysis were used to study the molecular mechanism of Brassia campestris L.hairy root response to short-term cadmium stress.Results:(1)The cells of Brassia campestris L.hairy roots was damaged and cell morphology changed under cadmium stress.Through direct observation and the scanning electron microscope(SEM),it was found that the hairy roots were browned and the lateral roots were reduced.Semi-thin sections revealed that cadmium stress damaged the apical cell integrity of hairy roots.Through transmission electron microscopy(TEM),it was found that cadmium stress thickened the cell wall of hairy roots,diffused nucleoli and swollen mitochondria.(2)New transcripts were annotated after transcriptome sequencing.The proteomes of Brassica oleracea,Brassica cretica,Eutrema yunnanense,Camelina sativa,Arabidopsis lyrata,Arabidopsis thaliana and Capsella rubella were clustered to establish Brassica database.Then 866 new transcripts were compared with the established Brassica database,and 68% of them were annotated in Brassica Oleracea.GO analysis showed that the new transcripts were mainly enriched in the items related to metal ion absorption,binding and transport.(3)Proteome analysis of Brassia campestris L.hairy roots in response to Cd stress identified 1424 differential proteins(DEPs),of which 973 DEPs were down-regulated and 451 DEPs were up-regulated.GO analysis showed that DEPs were mainly enriched in binding,catalytic and transporter activity items.DEPs are mainly annotated in the COG database on signal transduction metabolism,protein post-translational modification,protein turnover and molecular chaperones.KEGG analysis showed that DEPs were enriched in MAPK signal transduction pathway,phenylalanine metabolism,spliceosome,and plant-pathogen interaction.Protein interaction analysis showed that the response process mainly involves the interaction of related proteins in the pathways of binding,signal transduction,protein translation and metabolism Through subcellular localization analysis,DEPs are mainly distributed in chloroplasts,nuclei,endoplasmic reticulum,plasma membrane,mitochondria and vacuoles.(4)Transcriptome and proteomic integration analysis of Brassia campestris L.hairy roots in response to Cd stress revealed 118 DEGs/DEPs with a correlation coefficient of 0.1874.The transcriptome and proteome expressions have the same correlation coefficient and the opposite expression correlation coefficient are 0.6076 and-0.5210,respectively.DEGs/DEPs with the same expression trend in transcriptome and proteome expressions were mainly involved in amino acid transport and metabolism,signal transduction metabolism,lipid transport and metabolism,enzyme transport and metabolism,cell wall/membrane and resistance metabolism.The DEGs/DEPs with the opposite trend of transcriptome and proteome expressions are mainly involved in carbohydrate transport and metabolism,amino acid transport and metabolism,translation and ribosomal structure,and biogenesis.Pathway analysis found that only glutathione metabolism,plant-pathogen interaction and plant hormone signal transduction have significant significance.In-depth analysis of significant metabolic pathways,candidate genes Br GSTU13,Br GSTU25,Br GSTU12,Br GCLC,Br CDPKX2,Br RBOH10,Br CALM5,Br FLS2,Br BAK1,Br GID1,Br DELLA and Br TCH4 were screened out.Conclusions:The effects of cadmium stress on Brassia campestris L.hairy roots were significant in individual level,cell level,subcell level and molecular level.Transcriptome and proteome integration analysis found that DEGs / DEPs were mainly enriched in spliceosomes,plant-pathogen interaction,cysteine and methionine metabolism,plant-pathogen interaction,MAPK signal transduction and phenylalanine metabolism.Further analysis revealed that glutathione metabolism,plant-pathogen interaction and plant hormone signal transduction pathways have significant significance,and 12 candidate genes including Br GSTU13 were screened out.The results provide an important theoretical basis for the construction of cadmium supertolerance/enrichment transgenic Brassia campestris L..