| Triptolide has excellent insecticidal activity,mainly from the root of Celastraceae plant Tripterygium wilfordiii.However,the shortage of T.wilfordii plant resources limits its research and application in the field of pesticides.To study the transcription factors that regulate the expression of key enzymes in triptolide biosynthesis pathway and analyze their regulatory mechanism,it is one of the effective ways to solve the shortage of resources to induce transgenic hairy roots with high yield of triptolide by metabolic engineering technology.At present,the expression regulation mechanism of the key genes TwTPS27a/b downstream of the biosynthetic pathway of triptolide,an insecticidal active compound,is still unclear,and the transcription factors involved in regulating the biosynthesis of triptolide have not been reported.Therefore,in this study,two highly homologous genes named TwTPS27a and TwTPS27b were isolated from the hairy roots of Tripterygium wilfordii,and their sequence analysis and overexpression function were verified.After that,the promoters of TwTPS27a/b were cloned and analyzed.It was found that there were many potential transcription factor binding sites in their promoter sequences.On this basis,the regulation mechanism of TwTGA1 and TwMYC2a/b on TwTPS27a/b expression were analyzed by yeast one hybrid,GUS transactivation assays,RNAi and overexpression techniques.On this basis,through yeast one-hybrid and GUS transactivation assays as well as overexpression and RNA interference(RNAi)studies,the transcriptional regulation of Tripterygium wilfordii transcription factors TwTGA1 and TwMYC2a/b on TwTPS27a/b and triptolide biosynthesis in hairy roots were analyzed.The main results are as follows:(1)Two key genes TwTPS27a and TwTPS27b for triptolide biosynthesis were successfully isolated.Their nucleotide and amino acid sequences were highly homologous,with 98.36% and 98.13% identity,respectively.Meanwhile,the full-length CDS,5'/3'-UTR and g DNA sequences of these two gene were distinguished.The stable TwTPS27a and TwTPS27b overexpression transgenic hairy roots lines,including 27a-OE-1,27a-OE-2,27bOE-1,and 27b-OE-2,were successfully induced.RT-q PCR analysis showed that the relative expression of TwTPS27a/b in four independent hairy root lines was significantly higher than that in the control(p<0.01),which was 4.32-,3.61-,5.58-,and 5.22-fold higher than that in the control.The results of liquid phase analysis showed that the content of triptolide in four independent transgenic hairy roots lines were all significantly higher than that in the control(EV),with 58.05,49.43,61.26 and 58.93 ?g/g DW,respectively.Therefore,the relative expression of TwTPS27a/b is closely related to the accumulation of triptolide,which indicates that TwTPS27a/b can be used as two effective target to analyze the regulatory mechanism of triptolide biosynthesis.(2)The promoter sequences of the key genes TwTPS27a and TwTPS27b were successfully cloned with the lengths of 1496bp and 1862bp,respectively.In silico analysis revealed that both TwTPS27a and TwTPS27b promoters contained core elements,stress and hormone response elements.The analysis of the promoter activity showed that the region from-921 to-391 bp in the TwTPS27b promoter was the potential core region of the promoter activity and the main region responding to methyl jasmonate(MeJA).The discovery of the TGA transcription factor binding site TGACG-motif and the MYC2 transcription factor binding site E-box element in this potential core region laid a theoretical foundation for the study of TGA transcription factor and MYC2 transcription factor involved in the expression regulation of TwTPS27a/b in Tripterygium wilfordii.(3)The TGA transcription factor gene TwTGA1 was identified by gene cloning and bioinformatics analysis.Subcellular localization analysis showed that TwTGA1 was located in the nucleus.The results of yeast one hybridi (Y1H) and GUS transactivation assays showed that TwTGA1 specificly bound to the TGACG-motif element in the promoter of key genes TwTPS27a/b,and activated theirs promoter activity.The results showed that the relative expression level of TwTPS27a/b in the hairy roots of TwTGA1-overexpression was significantly increased,and the content of triptolide was also significantly increased(p <0.05),73.05?g/g DW,which was significantly higher than that of the control,1.93-fold of the control.Therefore,the regulatory mechanism of the transcription factor TwTGA1 positively regulating the biosynthesis of triptolide in the hairy roots of Tripterygium wilfordii was preliminarily analyzed: TwTGA1 specifically binds to the TGACG-motif element in the promoter of TwTPS27a/b,two key genes for triptolide biosynthesis,up-regulates the transcription level of these genes,and then positively regulates triptolide biosynthesis.(4)Two homologous MYC2 transcription factor genes,TwMYC2a and TwMYC2b,were isolated and identified for the first time through gene cloning and bioinformatics analysis.The results of subcellular localization showed that TwMYC2a and TwMYC2b were both located in the nucleus.Y1H and GUS transactivation assays proved that the transcription factors TwMYC2a/b bind to the E-box(CACATG)and T/G-box(CACGTT)elements of the key genes TwMYC2a/b promoters,and inhibit theirs promoter activity.The results of silencing TwMYC2a/b by RNA interference showed that the expression level of the key genes TwTPS27a/b was significantly up-regulated(p<0.01),and the content of triptolide in the hairy roots of TwMYC2a/b-RNAi(R1 line)was also significantly increased(p<0.05)to 65.38 ?g/g DW,which was 1.67-fold than that of the control.It is inferred that the transcription factor TwMYC2a/b as two negative regulator were involved in the regulation of triptolide biosynthesis by binding the E-box(CACATG)and T/G-box(CACGTT)in the key genes TwTPS27a/b promoters and inhibiting the expression of TwTPS27a/b gene,thereby negatively regulating the biosynthesis of triptolide.In conclusion,this study preliminarily analyzed the transcriptional regulation mechanism of TwTGA1 and TwMYC2a/b transcription factors on TwTPS27a/b,the key genes of triptolide biosynthesis pathway.At the same time,the transgenic hairy root lines with high content of triptolide established in this study provide alternative resources for solving the problem of insufficient resources in the development and utilization of triptolide in botanical pesticides,and lay an important theoretical foundation for the use of metabolic engineering strategies based on transcription factors to improve the content of triptolide. |
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