Transcriptional Analysis Of Hyperaccumulating Sedum Alfredii Hance And Functional Study Of ZIP Family Genes

The non-essential heavy metal cadmium (Cd) is very toxic. It readily enters the plant tissue, and thus enters human body through food chain. The major concern of Cd pollution is its potential threats to human health with the risks of causing many cancers including the lung, bladder, renal, prostate, and breast cancer. Sedum alfredii Hance is a Cd hyperaccumulator native in China. Investigations into its physiological and biochemical mechanisms of Cd hyperaccumulation would offer valuable insight into heavy metal homeostasis. Besides, genes identified responsible for Cd hyperaccumulation would aid the creation of genetic modified plant materials for phytoremediation. In the past, main efforts were devoted to the physiological mechanisms of hyperaccumulation of S. alfredii. Lacking of genome sequences has greatly hindered the progress toward molecular mechanisms of hyperaccumulation of S. alfredii. In the present research, we employed the RNA-seq (Roche454and Illumina/Solexa) platforms to explore the transcriptome of S. alfredii Hance and to investigate its transcriptional changes in response to high Cd accumulation in shoots. The gene expression in shoots of two contrasting ecotypes of S. alfredii was also examined by quantitative-Real-Time-Polymerase Chain Reaction (q-RT-PCR).Zrt-Irt like Protein (ZIP) family genes were reported mediating the transmembrane uptake of divalent heavy metals. Based on the RNA-seq results, three ZIP family genes in S. alfredii were cloned. The expression pattern of these ZIP genes was analysed by q-RT-PCR.Yeast complementary test was carried out to examine the potential function of above genes in Fe/Zn uptake. The results were as follows:1. The transcriptome of S. alfredii was investigated with Roche454and Illumina/Solexa platforms. Pooling of sequencing runs on Roche454resulted in135,894qualified reads with an average length of286bases; More than47.5million qualified reads were generated on the Illumina/Solexa platforms, which was equal to9.4gigabases (Gb). The454reads were firstly used to generate the transcriptome assembly, and then Illumina reads were mapped to it and assembled. The final assembly with all the contigs obtained generated the transcriptome of S. alfredii shoots with57,162contigs longer than200bp. Based on GO term, these contigs involved metabolic process, macromolecule metabolic process, and binding, catalytic activities. Pathway enrichment of the transcriptome showerd that these contigs involved37different pathways. Among them,6,695contigs were related to signal transduction mechanisms, accounting for11.7%of the total. Besides,671contigs encoding putative transcription factors wer identied, including zinc finger proteins (16.3%), MYB (16.2%), bHLH (12.5%) and bZIP (8.3%);2. Compared to the protein databases from six fully sequenced species, including Arabidopsis thaliana, Vitis vinifera, Medicago truncatula, Zea mays, Oryza sativa, Chlamydomonas reinhardtii, S. alfredii Hance showed a similarity less than40%. Among the57,162contigs in S. alfredii transcriptome,6,176perfect SSRs were found, which would be useful co-dominant, locus specific markers fro DNA fingerprinting, genome mapplying, phylogenetic analysis, etc. Sedum alfredii belongs to Crassulaceae family. Twenty contigs encoding PEPC homologs were found in the transcriptome of Sedum alfredii. Analysis revealed that S. alfredii had both C3and CAM types of PEPCs.3. Long exposure of S. alfredii to Cd was employed in the present work to investigate the molecular mechanisms of Cd hyper-tolerance. Compared with control, totally110contigs were up-regulated and123contigs were down-regulated by Cd treatment. To evaluate the validity of Solexa data,15inducible genes were selected, and there expression level was examined by q-RT-PCR. The expression pattern was generally agreed with Solexa sequencing. Among all the significantly regulated contigs, nearly one half of them encoded unknown proteins. Based on GO term, Cd induced contigs were mostly related to extracullar, amino acid and derivative metabolism, catalytic activity and macromolecule metabolism. Based on KEGG orthologies, these genes were related to biosynthesis of other secondary metabolites, energy metabolism, metabolic diseases and membrane transport. In general, the up-regulated contigs could be grouped into five categories, namely, cell wall deposition and modification, metal transport, metal ligand synthesis and metal-ligand transport, reactive oxygen species detoxification, and transcriptional gene regulation. 4. By q-RT-PCR, gene expression in the shoots of two contrasting ecotypes of S. alfredii was compared. Several genes were selected based on the current RNA-seq results and also previously reported metal tolerance determinants, including cell wall modification, deposition, antioxidants, metal transport and ligand biosynthesis. All primers were designed based on the transcriptome of S. alfredii, PCR results showed that these primers were applicable for both ecotypes of S. alfredii. Based on the induction fold of peroxiredoxin gene,5μM Cd treatment on non-hyperaccumulating ecotype (NHE) of S.. alfredii produced compatible Cd stress as100μM Cd on hyperaccumulating ecotype (HE). Among all the examined contigs, lacccase, cinnamate5-hydroxylase and Nramp2were more highly induced in HE than that in NHE; cellulose synthase, SAM synthase were up-regulated in HE while down-regulated in NHE. In contrast, Nramp3, HMA4and YSL3were more highly induced in NHE than that in HE. Under normal growth condition, the expression level, of genes related to heavy metal transport, cell wall synthase and modification were mostly constitutively higher in HE than those in NHE.5. Based on the transcriptome sequences of hyperaccumulating ecotype of S. alfredii, three contigs encoding ZIP family genes were selected with relatively high expression level. The full length of these contigs were cloned. Blast analysis showed that, Sa_Contig615was most similar to AtZIP4and thus named as SaZIP4. Sa_Contig3033and Sa_Contig22157were accordingly named as SaZIP1and SaZIPll, respectively. Phobius prediction of transmembrane topology revealed8transcmembrane domains in these genes. Among these genes, SaZIP4and SaZIPll fully restored Zinc (Zn) uptake ability and partial iron (Fe) uptake ability in according Zn or Fe uptake deficient strains. The expression of SaZIP4in shoot and SaZIPl in both shoot and root was depreesed with application of Zn. While, application of Cd induced the expression of SaZIPl and SaZIPll in both shoots and roots. These results indicated positive roles of these genes in the transportation of Zn/Cd within S. alfredii.