| In this paper,two different genotypes of K326(cadmium leaf accumulation type)and N.rustica(cadmium root accumulation type)were studied.The cadmium stress response genes and metabolic pathways were identified based on transcriptomics analysis,and the possible cadmium transport proteins were screened.The family genes were analyzed by qRT-PCR analysis under different cadmium stress conditions,and the candidate genes related to cadmium accumulation were found.The bioinformatics analysis of the key cadmium genes was also obtained.The main results of the study are as follows.(1)The RNA-seq sequencing data of this experiment is of high quality and fully meets the quality requirements of transcriptome research for sequencing depth and accuracy.Under the cadmium stress condition,there was no contamination in the high-throughput sequencing process of the transcriptome of K326 and N.rustica.The 12 samples obtained after a series of quality control met the requirements of subsequent analysis,and the test reference genome was selected appropriately,and the obtained Clean reads could be obtained.Compare it effectively.The Pearson correlation coefficient R2 between the four sample biological replicates was greater than 0.92,and the correlation between gene expression levels was high.At the same time,three up-regulated genes and three down-regulated genes were detected before and after K326 cadmium treatment for gene expression verification.The results were consistent with the results of transcriptome sequencing analysis in qRT-PCR and transcriptome sequencing.The expression changes in the same trend.(2)The expression of genes in roots of K326 and N.rustica under cadmium stress was compared by cadmium stress response gene expression analysis.The results showed that the differential expression of the two tobacco cultivars was significant,and the differential genes were more.Compared with yellow flower tobacco,K326 is more sensitive,rapid and effective in response to cadmium,and its differentially expressed genes before and after cadmium treatment increased significantly.Compared with the two control groups,there were 3276 unique differential gene expressions in K326 and N.rustica after cadmium treatment.These expression genes are likely to cause different cadmium accumulation in the two tobacco varieties.The GO functional enrichment analysis and KEGG metabolic pathway analysis of differentially expressed genes showed that the cadmium stress produced a positive response to various secondary metabolic pathways such as redox process and sulfur metabolism in tobacco.Under cadmium stress in GO functional enrichment analysis,especially before and after K326 cadmium treatment,the differential genes were mainly enriched in the cell redox homeostasis,oxidoreductase activity and antioxidant activity unit,and the expression of related genes was significantly up-regulated.Before and after cadmium treatment in KEGG metabolic pathway analysis,Pathway,which is significantly enriched by DEGs,is mainly involved in thiamin metabolism and sulfur metabolism,and is closely related to abiotic stresses such as cadmium.Plant Pathway,which is significantly enriched by DEGs,is mainly ribosomes and plant pathogens.The interaction was consistent with the down-regulation of the expression of ribonucleoprotein complex,ribosome structural component,ribosome,translation and other related genes in the results of GO enrichment analysis.(3)Based on the analysis of metal ion stress response genes by transcriptomics,9 possible cadmium transporter family genes were screened.Based on the comparative analysis of the expression levels of differentially expressed genes in transcriptomes and their functional annotations,combined with BLASTP results,9 metal ion transporter genes,including the ZIP transporter family and NRAMP,which were significantly up-regulated or down-regulated under cadmium stress conditions,were successfully screened.Transporter family,YSL transporter family and HMA transporter family.(4)Multiple transmembrane transporters were selected to correlate with cadmium stress response in tobacco.qRT-PCR analysis showed that the expression levels of multiple transporter genes in ZIP family,NRAMP family,YSL family and HMA family were significant under cadmium stress.Changes,and different expression patterns between the two varieties,initially confirmed that the proteins encoded by these genes can participate in the regulation of cadmium absorption and transport in tobacco,of which,the HMA family of genes is most obvious.The expression level of HMA2a in K326 was significantly up-regulated.and it was significantly down-regulated in N.rustica.The expression of HMA2β in K326 was significantly higher than that in N.rustica,and the expression level of HMA 2β increased with the increase of cadmium stress.The expression level of HMA2β was significantly lower than that of the control.HMA2 is responsible for the transport of cadmium from root to leaf.K326 plant has the characteristics of high accumulation of cadmium in leaves.It may be the result of up-regulation of HMA2a gene and HMA2β gene,and the ability of HMA2β to transport cadmium is stronger than that of HMA2α.The high expression of ZIP1 and NRAMP3 may be the reason for the high accumulation of cadmium in the roots of N.rustica.(5)The experiment is particularly concerned with the expression differences of genes involved in the pathway of cadmium transport from root to shoot.Therefore,the bioinformatics analysis of the obtained NtHMA2α and NtHMA2β genes showed that the NtHMA2α and NtHMA2βgenes contained a complete open reading frame of 3876 bp and 4077 bp,respectively,which were localized on the plasma membrane.The results of phylogenetic analysis showed that NtHMA2α was more closely related to forest tobacco,and NtHMA2β was more closely related to fluffy tobacco,which may be derived from each parent.The encoded proteins all contain a conserved domain of the HMA2 protein family,in particular two domains associated with metal cadmium transport. |
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