Comprehensive Analysis Of SaABCCs In Hyperacumulator Sedum Alfredii Hance

China is a large agricultural country,and the soil contains most pollutants including heavy metals.The hard-to-degrade heavy metals,easy enrichment and the transferability of the food chain pose a serious threat to the ecological environment and food safety.Therefore,heavy metals become the "invisible killers" of human life,health and safety.Phytoremediation technology has been a research focus for heavy metal contaminated soil management in recent years.Among them,the Sedum alfredii Hance is one of the few cadmium super-accumulator plants.It can accumulate a large amount of cadmium in the aboveground part without any toxic symptoms,and is a valuable material for soil cadmium contaminated phytoremediation.Meanwhile,the extremely strong cadmium tolerance characteristics of the S.alfredii show that it has abundant genes related to stress resistance,and it is a good species to study the mechanism of plant tolerance to heavy metal.ATP binding box(ATP-binding cassette,ABC)is one of the largest and most functional superfamily of proteins;and is named for its need to complete with the energy released by hydrolysis ATP when transporting substrates across membranes.In plants,the ABC family is thought to be involved in hormones,lipids,metal ions,secondary metabolites and in regulating plant-pathogen interactions.However,the mechanism of heavy metal uptake and transport by the gene family is unclear in the S.alfredii.In this study,full-length transcriptome sequencing is carried out by three-generation sequencing technology(Pacbio,Sequel platform).On this basis,ABCC gene family was identified.We analyzed conserved domains,phylogenetic relationships,tissue-specific expression,and response to cadmium stress of this family.Three Cd transport-related genes(Sa12F279,Sa14F190 and Sa45F39)were selected.We further investigated the function of two genes,Sa14F190 and Sa45F39.The main findings are as follows:1.In this study,full-length transcriptome sequencing were performed by three-generation sequencing technology from the total RNA of six samples of S.alfredii.The results showed that the library produced a total of 10.67 Gb of data,and the sequencing results met the requirements.After clustering and correcting the reads obtained by sequencing,the sequences located on the genome accounted for 91.04%;1472 new genes were predicted to be scattered on 288 Scaffolds.135 803 transcripts were finally got by optimizing the gene structure.In addition,these transcripts were functionally annotated in the Nr,Nt,Pfam,Swiss-Prot,GO,KEGG,and KOG/COG databases.Many important annotations are closely related to stress or resistance.2.A total of 10 ABCC genes were identified in Sedum alfredii Hance,which have highly conserved protein domains and motif arrangements.The gene of an ABC family member was obtained by sequence alignment and named Sa12F279.The Sa12F279 gene is clustered with members of subclass C in evolution,and conforms to the conservative arrangement of the ABC family in the domain structure.The co-expression network analysis of Sa12F279 showed that edge genes are mostly involved in metabolic pathways,intracellular processes,and biological regulation.Transcriptome data showed that the Sa12F279 gene showed a down-regulated response to cadmium stress,and there were differences in response patterns to ABA,saline and drought stress,and the change in expression level was relatively gentle.Sa14F190 and Sa45F39 genes were heterologously expressed in yeast,and it was found that both genes increased the accumulation of Cd in transgenic yeast cells.This result provided support for the possibility that this gene may participate in the regulation of Cd transmembrane transport.The promoter of Sa14F190 gene was expressed in Arabidopsis thaliana.GUS staining showed that the gene was closely related to differentiation.In addition,a MYB binding site was found in the upstream promoter of Sa45F39,and a potential MYB regulatory pathway was obtained by analyzing transcriptome data.This MYB gene Sa5F123 was transferred to A.thaliana to verify its function.Sa5F123 gene in transgenic A.thaliana decreased tolerance to cadmium,but the cadmium content increased significantly;thus MYB transcription factor Sa5F123 gene may play a regulatory role on Sa45F39 gene.