| Polybroinated diphenyl ethers(PBDEs)are the environmental organic pollutants of concern,as not only accumulate in water and soil,but also accumulate through the substance chain.PBDEs have a certain negative effect on the immune system,reproductive system and growth and development of organisms,which has attracted much attention.ABC(ATP-binding Cassette)transporter as an important transporter in organism,participates in the absorption,accumulation and excretion of a variety of toxic substances.It has become a hot spot in the study of the uptake,accumulation and excretion of harmful substances,including heavy metals and pesticides.However,there are few reports on the study of PBDEs and other environmental persistent organic pollutants.In this paper,the W.anomalus was used to study the tolerance of W.anomalus to BDE-3,the least brominated 4-bromodiphenyl ether(BDE-3)in PBDEs.Through the transcriptome analysis of W.anomalus treated with BDE-3,the related gene of ABC transporter was discovered,and whether the important ABC transporter mediated W.anomalus tolerance to BDE-3 was analyzed and identified.The main results are as follows:(1)Growth analysis of W.anomalus under BDE-3 stress:the W.anomalus showed different growth patterns under different concentrations of BDE-3 in inorganic salt and YPD.In the treatment group of inorganic salt medium,there was no lag period and the W.anomalus directly entered the logarithmic period;the growth of W.anomalus was better under the condition of adding different concentrations of BDE-3 in the inorganic salt medium than that of the blank control,among which the logarithmic periods of 100 mg/L,200 mg/L and 300 mg/L were close,and had obvious stable periods.400 mg/L and 500 mg/L The two treatments of BDE-3 of mg/L entered a stable period before and after 48h.Under the two conditions,W.anomalus growth was better than other treatments.Among them,the treatment of BDE-3 of 400mg/L had the best effect,while the treatment of BDE-3 of 500mg/L showed a certain inhibition effect;the W.anomalus absorbance of BDE-3 at the turning point of 48h was significantly higher than that of the blank control(P<0.05),and the treatment of 400mg/L had the best effect.In YPD test group,different concentrations of BDE-3 treatment also had a significant impact on the W.anomalus system,and the growth of the whole under each treatment condition was better than that of the corresponding inorganic salt treatment;the growth curve of blank control W.anomalus showed the inflexion points of the four periods of W.anomalus entering delay,logarithm,stability and decay;the growth curve of W.anomalus under other different concentrations of BDE-3 treatment was similar to that of blank control However,the turning points of logarithmic and stable periods were not obvious,and all treatments entered the decline period at the same time under the condition of YPD nutrition culture.The number of W.anomalus(absorbance)in different concentration of BDE-3 treatment at 48 hours was significantly lower than that in the blank control(P<0.05),which had a certain inhibitory effect.(2)Transcriptome analysis under BDE-3 stress:a total of 136 genes with logFC≥2 or ≤-2 were screened from 3628 differentially expressed genes.The 69 up-regulated annotation genes mainly include three aspects:metabolic factor,sterol synthesis and transporter,including Wapdr15 gene,a member of ABCG transporter family.The ABCG subfamily proteins in other fungi are generally related to drug resistance,and Wapdr15 is significantly up-regulated compared with other genes under the induction of BDE-3.It is speculated that Wapdr15 may participate in the efflux of BDE-3,thus mediating the tolerance mechanism of W.anomalus to BDE-3.(3)Response analysis of Wapdr15 gene to BDE-3:the change of Wapdr15 gene expression under different concentration of BDE-3 was verified by fluorescence quantitative experiment.It was found that Wapdr15 gene expression was up regulated under BDE-3 stress,and Wapdr15 gene expression was up regulated under 300 mg/L and 400 mg/L BDE-3 stress,among which 400 mg/L was up regulated The expression of Wapdr15 gene was up-regulated by BDE-3,which was 3.07 times higher than that of the blank control group.This indicated that Wapdr15 gene responded to BDE-3 induction.(4)Cloning and bioinformatics analysis of Wapdr15 gene:Based on the results of transcriptome sequencing,Wapdr15 gene sequence was obtained and amplified in W.anomalus genome.Wapdr15 gene is 4536bp in length,encodes 1511 amino acids,and has no intron in its gene structure.WAPDR15 protein is a typical transmembrane protein with a complete TMD1-NBD1-TMD2-NBD2 structure.Phylogenetic tree analysis of WAPDR15 and other ABC proteins showed that Wapdr15 was closely related to Arabidopsis.(5)Function analysis of Wapdr15 gene:the vector of Wapdr15 gene was constructed,transformed by agrobacterium mediated tobacco,and the transgenic plants were successfully screened.The results showed that Wapdr15 gene could improve the tolerance of tobacco leaves to BDE-3.In conclusion,W.anomalus has high tolerance to BDE-3 treatment,and can be used as the only carbon source to maintain the growth of W.anomalus cells.The expression of Wapdr15 gene in the W.anomalus was significantly up-regulated by BDE-3,The tolerance of transgenic tobacco with Wapdr15 gene to BDE-3 was significantly enhanced.In this study,the detoxification mechanism of BDE-3 was preliminarily studied,Wapdr15 ABC transporter gene can be used for genetic transformation of poplar and other woody plants including P.tomentosa,which lays a foundation for the innovation of poplar germplasm in PBDEs in repairable environment. |
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