Identification Of Micrornas And Nramp Gene Family Associated With Chromium Uptake And Accumulation In Radish (Raphanus Sativus L.)

Heavy metal pollution has endangered the global ecological environment and human health seriously.Chromium(Cr)is one of the most toxic heavy metals,which not only inhibits the plant growth and development,but also poses a significant threat to human health via the food chain.Radish(Raphanus sativus L.)is an important Brassicaceae root vegetable crop with high nutritional and medicinal value in the world.Since the plant roots were the first vulnerable parts directly exposed to metal-contaminated soils,it's of vital significance to explore the molecular regulatory networks of HM tolerance and homeostasis in radish.Previous studies have revealed that miRNA-guided gene regulation could play critical roles in plant response to HM stresses.Recently,Cr-responsive microRNAs in vegetable crops as well as the isolation and functional verification analysis of heavy metal-responsive Nramp gene family have not been reported.And the molecular characterization and regulatory mechanisms of Cr uptake and accumulation in radish remain in its infancy.This study would be conducted to isolate Cr-responsive miRNAs and their targets at the genome-wide level as well as a number of heavy metal-responsive genes.These results would provide valuable information for analyzing the characteristics of molecular genetics of heavy metal uptake and accumulation in radish.The main achievements obtained were as follows.1.To systematically isolate and dissect Cr-responsive miRNAs and their targets at the global level in radish roots,two sRNA libraries derived from Cr-free(CK)and Cr-treated(Cr200)roots were constructed.With Solexa sequencing,81 known and 72 novel miRNAs were identified,from which 54 known and 16 novel miRNAs were significantly differentially expressed under Cr stress.RT-qPCR analysis showed that a set of Cr-responsive miRNAs were differentially expressed in different Cr treatment times(0,6,12,24,48 and 96 h)and some miRNAs might play crucial roles in plant response to HM stress by negatively regulating their corresponding targets in radish.Most target genes for Cr-responsive miRNAs encode different TF families which might regulate corresponding HM-related transcriptional processes in plants.Based on the comprehensive identification of Cr-responsive miRNAs and analysis of their corresponding target genes,a schematic model of tolerance mechanism and regulatory networks associated with Cr stress response in radish was proposed.After entry into the plant cell by the actions of several Iron transporter-like proteins,excessive ROS was generated.Meanwhile,Cr6+ could activate the detoxification mechanisms and signaling molecules as well as chaperones,which could eventually enhance the level of adaptation and/or tolerance to Cr stress.After sensing the Cr6+ ions,the plant cells could activated some HM stress responsive hormones and signaling molecules to decrease Cr6+-induced excess ROS level and oxidative damage.Moreover,some metal transporters such as SPL,ABCs,YSL and HMA were activated to transport Cr6+ out of the cell and/or translocate them into the vacuole.In summary,the co-expression of Cr stress responsive genes could enhance the level of defense or tolerance to Cr stress and alleviate the phytotoxicity of Cr6+ in radish.2.Using a radish advanced inbred line 'NAU-YH',four members of Nramp family were successfully isolated.The full cDNA sequences of RsNrampl,RsNramp2,RsNramp4 and RsNramp6 were 1,670,1,529,1,550 and 1,450 bp,and the lengths of ORFs were 1,602,1,489,1,533 and 1,410 bp with encoding 533,537,511 and 469 amino acids,respectively.The deduced amino sequences of Nramp in radish shared high homology with the Nramp proteins of Brassica napus,B.oleracea and B.rapa.RT-PCR and RT-qPCR analysis revealed that the expression patterns of Nramp gene family in radish showed differential expression patterns under different Cr treatment times,different heavy metals and different concentrations of Cr.The expression patterns of RsNrampl and RsNramp6 were significantly up-regulated with Cr treatment time increased and maximized at 24 h.RsNrampl and RsNramp6 had lower expression profiles under Cd and Pb than Cr treatment.The relative expression level of RsNramp4 was higher under Cd than Cr and Pb treatment.The expression pattern of RsNramp4 was up-regulated with Cr concentration increased and maximized at 400 mg·L"1.In addition,four transgenic over-expression vectors were constructed and transformed into tobacco leaves using Agrobacterium-mediated leaf disc transformation method,and 33 transgenic lines were obtained.These results would provide valuable information for understanding and validating the roles of Nramp gene family in HM-responsive regulatory networks in radish.