| Berberine is a common quaternary ammonium isoquinoline alkaloid.Using Arabidopsis thaliana L.as experimental material,taking the thalianol and marneral cluster as main object,to explore the target of berberine's earliest response at the molecular level by RNA-Seq and iTRAQ,in order to elucidation of herbicidal mechanism of berberine.To better understand the inhibitory effect of berberine on plant roots and determine the lowest concentration and earliest time point of berberine,we based on IC500 of berberine on fresh weight and root length of A.thaliana(6 mg·L-11 and 0.25 mg·L-1,respectively)to treat DR5-GFP A.thaliana mutant with 0.25 mg·L-11 berberine for 2 h,2.5 h and 3 h,6 mg·L-1berberine for 0.5 h,1 h,respectively.After observing by confocal laser,the result showed the distribution of auxin began to change at 0.25 mg·L-1,3 h and 6 mg·L-1,1 h.In order to further reveal the action mechanism of berberine at molecular level and find out the target of berberine's earliest response,RNA-Seq and protein iTRAQ techniques were used to explore and analyze the molecular mechanism of berberine.In the result of RNA-Seq analysis,422 up-regulated genes were obtained,of which 403 genes were down-regulated.The down-regulation gene was the focus of this study.In combination with gene function annotation,GO function analysis and KEGG Pathway functional analysis of differential expression genes,there were 20 significant differences metabolic pathways?P<0.05?.Among the most significant metabolic pathways,the sesquiterpenoid and triterpenoid biosynthesis and triterpenoid biosynsisis pathway is the focus of this study.There are 59genes in this pathway,accounting for 0.31%of the annotated genes in all pathways,of which 6 genes are differentially expressed genes,accounting for 1.03%of all differentially expressed genes.The thalianol gene cluster and marneral gene cluster are the selected molecular target of berberine in the study,and the thalianol gene cluster concluded thas,thah,thad and ACT;the marneral cluster contained MRN1,mro and CYP705A12.In the iTRAQ analysis,there are 646 differentially expressed proteins,including 236up-regulated proteins and 410 down-regulated proteins.In combination with gene function annotation,GO function analysis and KEGG Pathway functional analysis of differential expression genes,there were 12 significant differences metabolic pathways,and differential expression proteins are the most abundant in purine metabolic,starch and sucrose metabolism and pyrimidine metabolism.In order to further verify the earliest response concentration of berberine at gene level,wild-type A.thaliana seedlings growing for 7 d was treated with 0.25 mg·L-11 for 0 h,0.5 h,1 h,1.5 h,2 h,2.5 h,3 h,3.5 h and 4 h,respectively.The three genes of thas,thah and thad were all showed the lowest expression at 3 h,and the gene expression were 0.218,0.033and 0.130,respectively,which were consistent with the distribution of DR5-GFP auxin observed by confocal laser.The wild type A.thaliana,and several T-DNA insert A.thaliana mutants of thas,thah1-2,thad1-1,ACT,MRN1,MRO and CYP705A12,after growing for 7 days,were treated with 0.25 mg·L-11 for 3 h and 6 mg·L-11 1 h in berberine,and the result of RT-PCR with root showed that the expression levels of the seven genes:thas,thah,thad,ACT,MRN1,mro,and CYP705A12,in the eight kind of A.thaliana were all significantly decreased with 0.25 mg·L-11 berberine for 3 h,which showed that the molecular target of berberine is thalianol and marneral gene cluster.In addition,the wild type A.thaliana,and several T-DNA insert A.thaliana mutants of thas1-1,thah1-2,thad1-1,ACT,MRN1,MRO and CYP705A12 were treated with 0.25mg·L-11 and 6 mg·L-11 berberine for 7 days and 14 days,respectively.The result showed that the root of the thah1-2 and thad1-1 mutants were significantly longer compared with those of wild type A.thaliana when treated with 0.25 mg·L-11 berberine after 7 d;and after 14 d,the thah1-2 mutant also showed the longer root than the wild type A.thaliana at 0.25mg·L-11 berberine.Then,the upstream regulatory genes arp6 and hta9 of these two gene clusters were also studied by RT-PCR.It was found that compared with the control group,there was no change in the expression of the two genes with the treatment of berberine,indicating that berberine might not affect the upstream regulatory factors of the thalianol and marneral gene clusters.In conclusion,thah and thad genes are the main initial action sites of berberine in thalianol gene cluster.Therefore,one of the gene action sites on plants'root of berberine was thalianol and marneral gene cluster in sesquiterpenoid and triterpenoid biosynthesis pathway,which might interrupt the normal metabolism of the gene cluster mainly by inhibiting the expression of thah and thad genes,resulting in the accumulation of harmful intermediates,and then caused damage to plants,however,it might not affect the upstream regulatory factors of these two gene clusters. |
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