| Broussonetia papyifera is an economic plant with good comprehensive value and related to the remediation of heavy metal pollution.At present,there are few studies on the response of B.papyifera to Cd stress.Through the investigation of the tailings area in Xiangtan,Hunan Province,it was found that the B.papyifera tree is an important vegetation resource.This study evaluated the tree resources in different tailings areas,and obtained excellent plants with good performances in germination,physiology,etc,which was used as materials to conduct research on cadmium-resistance.The results are as follows:The sterile seedlings were obtained by disinfecting and sterilizing the superior tree of B.papyrifera,and the tissue culture regeneration system of B.papyrifera was further constructed.The tissue culture seedlings in aseptic and soil-planting conditions were treated with CdCl2 at different concentrations for different time periods,and the physiological responses of B.papyrifera to Cd stress were analyzed.The results showed that in B.papyrifera a large amount of reactive oxygen species(ROS)was accumulated,membrane lipid peroxidation was increased,cell infiltration was enhanced,proline content was added,the activities of antioxidant enzymes(SOD,POD,GST,etc.)were promoted under cadmium stress;suggesting that the cadmium stress response physiology of B.papyrifera involves in ROS metabolism,antioxidant enzyme system reaction,proline metabolism and other important processes.By comprehensively analysis of the physiological responses under different Cd stress concentrations and time,it can be concluded that 6-month-old B.papyrifera is more suitable for correlative analysis with 500 μM CdCl2 stress for 120 h.Therefore,the transcriptomes of B.papyrifera root and leaf were determined by Illumina HiSeq method under the stress treatment with 0 h as control,and a total of 180,678,660 bp(27.1 G)of pure readings were obtained and assembled into 589,487 high quality unigenes,of which 256,025 and 250,251 notes in GO and PFAM items,respectively.A total of 24,414 differentially expressed genes(DEGs)were annotated by GO and 117,547 KEGG-annotated DEGs were at least grouped in 47 KEGG pathways.Many transcription factors(TFs)(AUX/IAA,ARF,bHLH,bZIP,GRAS,NAC,MYB,WRKY-,AP2/EREBP,etc.)and proteins(SOD,POD,V-ATPase,GST,Lea,PPO,etc.)associated with the Cd response of B.papyrifera were screened.At the same time,it is known that Cd stress regulation involves abundant signaling pathways,such as ABA,MAPK,Ca2+,SOS;and it has important relationships with phenylpropanoid biosynthesis,plant hormone signal transduction,and glutathione metabolism,and so on.This not only provides abundant gene sequence resource for studying of the stress mechanism of B.papyrifera,but also provides important candidate genes for exploring the cadmium response mechanism and molecular biology of B.papyrifera and its introgressed species.At the same time,this will help to elucidate the molecular mechanism of Cd stress response of B.papyrifera,and promote the bioremediation of heavy metal contaminated areas by cultivating new tolerant varieties.In order to further study the molecular mechanisms of Cd stress response of B.papyrifera,MYB transcription factors were further screened from the transcriptome,and 38 MYB TFs with complete ORF were comprehensively screened,which were obtained through multiple expression and BLAST analysis,among which including 18 R2R3-MYB genes.These 18 genes exhibited strong transcriptional expression under CdCl2 treatment at different time points(0,3,6,12,24,and 48 h),and they displayed a time-dependent expression and tissue specificity in the roots,stems,and leaves.The results indicated that R2R3-MYB displayed well potential response in the regulation of cadmium stress,which has good research value and prospect.Cluster and comprehensive analysis of the expression levels of 18 R2R3-MYB genes revealed that BpTT2 and BpMYB6 showed higher transcription levels than others.Therefore,BpTT2 and BpMYB6 were selected as the representative gene for revealing the Cd resistance of R2R3-MYBs.The overexpression vectors of BpTT2 and BpMYB6 genes were constructed and overexpressed in B.papyrifera,respectively.The regulation function of Cd response of BpTT2 and BpMYB6 was analyzed by comparison with non-transformed line(NT)and empty vector transformed line(CK).The results showed that overexpression of BpTT2 and BpMYB6 genes could improve plant resistance to Cd stress by regulating ROS metabolism,antioxidant enzyme activity,polyphenolic substance accumulation,proline metabolism,cell stability,and membranization.The transcription levels of the downstream defense-response-related genes were analyzed in BpTT2 and BpMYB6 overexpression lines,which can be effectively up-regulated by the high expression of BpTT2 and BpMYB6 gene.These downstream defense-response-related genes include BpSODl,BpSOD2,BpPOD1,BpPOD47,BpGSTU8,BpGPX-1,BpAPX-1,BpLEA,BpPP1,BpSDH1,BpALDH,BpCHS1,BpFLS,BpDFR1.The results indicated that the BpTT2 and BpMYB6 genes mainly enhance the plant ROS scavenging,reduce the degree of cell damage and cell materialization by regulating the downstream related genes to increase plant antioxidant enzyme activity,polyphenols accumulation,glutathione and proline metabolism,etc,thus the resistance to cadmium stress was improved.The specific performance of BpTT2 and BpMYB6 in the regulation of B.papyrifera Cd stress is different,for the related specific physiological indicators and the specific transcriptional levels of the downstream defense responses in BpTT2 and BpMYB6 gene overexpressing linse were disparated.Overall,BpTT2 could regulate the downstream genes such BpSOD2,BpPOD47,BpGSTU8,BpGPX-1,BpFLS,BpDFR1,BpLea,BpPPl,BpSDHl with higher transcription levels than BpMYB6.At the same time,BpTT2 regulates the expression of downstream genes such as BpGSTU,BpLea,BpDFRl,BpGPX,BpPP1 and BpSDH1,while BpMYB6 controls the expression of BpPOD1 BpSOD1,BpGSTU8,BpGPX,BpAPX-1 and BpSOD2 more strongly than other genes.This not only reflects the differences in the stress resistance of BpTT2 and BpMYB6 in regulation of Cd stress,but also shows that BpTT2 regulates glutathione metabolism,membrane stability,and metabolism of polyphenols in Cd responses highlightly.Compared to other pathways,while BpMYB6 gene regulates Cd response more closely related to the metabolism of antioxidant system than others.In summary,the present study systematically selected the dominant plant of B.papyrifera from tailings area,and the tissue culture system was established.The physiological assessment and transcriptome analysis of B.papyrifera under Cd stress were conducted based on the plant materials.A large number of Cd response related gene resources and metabolic pathways have been obtained,and screened one of the important transcription factors familes—MYB.Then the Cd response expression patterns of R2R3-MYB genes were analyzed,from which two representative genes-BpTT2 and BpMYB6-were identified to further study the Cd-response.The results showed that overexpression of BpTT2 and BpMYB6 can effectively improve the Cd tolerance of B.papyrifera,and involve in different concrete regulatory pathways.This provides an important candidate gene resource for development molecular mechanism of Cd and heavy metal response,and also lays a good foundation for the selection and breeding of resistant varieties to promote the bioremediation of heavy metal contaminated areas. |
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