Selection And Preliminary Analysis Of Zn/Cd-resistance Genes Based On RNA-Seq In Hyper-accumulator Arabidopsis Halleri

Hyperaccumulators are characterized by the strong capability of taking up a large amount of heavy metals and translocating them to aboveground part.Due to their outstanding tolerance and accumulation of heavy metals,hyperaccumulators are considered as ideal materials for exploring genetic basis of heavy metal responses and acclimation in plants.Identification of the genes that play vital roles during this process will contribute to the understanding of heavy metal response and acclimation,provide candidate gene resources for genetic breeding of plants with strong acclimation to heavy metal environment,and thereby promote phytoremediation of heavy metal pollution in soil.Arabidopsis halleri,a close species of the model dicot plant Arabidopsis thaliana,is a typical hyperaccumulator with strong tolerance and accumulation of Zn and Cd.In the present study,comparative transcripteome analysis of Zn/Cd responses between A.halleri and its close species Arabidopsis thaliana(A.thaliana)was performed through next-generation sequencing to identify the key genes involved in Zn/Cd response and tolerance.The expression and function of some candidate genes were also analyzed preliminarily.A.halleri and A.thaliana were hydroponically cultured in Zn and Cd excessive solutions for 2 and 8 hours,and the roots were collected for RNA extraction and transcriptome sequencing analysis.The reads were mapped to the reference genomes of both Arabidopsis species for transcript abundance quantification and comparison.The intra-species analysis revealed that more differentially expression genes were detected in both species in response to 2 and 8-hour Zn excessive treatment than Cd.The number of Zn/Cd responsive genes in A.halleri was significantly less than that in A.thaliana.There are 51 overlapping differentially expressed genes in response to Zn and Cd in A.halleri,which are obviously less than the 172 ones in A.thaliana.Therefore,A.halleri displays significant differences from A.thaliana on the expression patterns in response to Zn and Cd stimuli.This may be an important reason for the contrasting heavy metal tolerance and enrichment between A.halleri(hyperaccmulator)and A.thaliana(non-hyperaccumulator).GO enrichment indicated that the terms involved in catalytic activity,binding correlation,transport activity and heavy metal detoxification.As revealed by KEGG pathway analysis,some metabolic pathways associated with heavy metal detoxification,for example,glutathione metabolism,were highlighted.After analyzing the most enriched GSTs in the glutathione metabolic pathway,it was found that the analysis of differentially expressed genes in the species was not comprehensive and requires cross-species analysis.In order to identify the key genes responsible for the strong capability of Zn/Cd tolerance and accumulation in A.halleri,the inter-species pair-wise comparison of gene expression in root tissues were performed between both Arabidopsis species.Firstly,a homologous gene set containing 15221 pairs was constructed.Cross-species analysis of differential gene expression indicated that 1123 genes in A.thaliana and 1138 in A.halleri displayed significantly differential expression.Combined with the differentially expressed genes in cross-species,there were 1102 genes that were not only significantly differentially expressed in the A.thaliana but also in the A.halleri and Significantly differences in cross-species,combined with functional annotation after looking for 97 pairs of resistance to Zn / Cd stress related to key genes.Noticeably,at least 12 genes that are closely associated with metal homeostasis displayed significantly preferential transcript abundance in A.halleri,compared to A.thaliana.These genes were previously reported to regulate cellular trafficking and chelation of ions.Based on the function and expression of these genes,NAS and ZIP gene families were selected for further analysis.NAS genes encode nicotinamide synthase catalyzing the in vivo synthesis of nicotinamide,a compound capable of conjugating with metal cations.It was shown that five NAS members were harbored in the genome of A.halleri,while four in that of A.thaliana.The NAS proteins exhibit highly conserved motives at C-terminus.Validated by qRT-PCR,AhNAS2 and AhNAS3 were significantly induced under Zn/Cd stresses.These results revealed that high correlation of NAS genes with Zn/Cd chelation.Eleven ZIP family genes were found in the A.halleri,and by analyzing their primary,secondary,and tertiary structures,they found that they had a conservative structure of ZIP superfamily,except AhZIP2 and AhZIP3.Combined with the data obtained from transcription and qRT-PCR,it was suggested that AhZIP1 might be less expressed in roots just like AtZIP1 and its transport properties to Zn were stronger than that of Cd.AhZIP7 and its homogeneous genes in A.thaliana AtZIP9 are down-regulated under Zn stress,and it is required to confirm its transport activity of Cd.AhZIP8 and AtZIP10 display the similar response under Zn/Cd,therefore,its is speculated that AhZIP10 may be the transporter of Zn/Cd.In this study,our resutls provided basic information and novel insights for further analysis of molecular mechanism underlying heavy metal tolerance and accumulation of A.halleri,a Zn/Cd hyperaccumulator.