Isolation And Functional Analysis Of The Hyoscyamus Nigerputrescine N-methyltransferase (PMT) Gene Promoter

Hyoscyamus niger L., belonging to the family of Solanaceae, produces tropane alkaloids(TAs) including hyoscyamine and scopolamine widely used as anticholinergic agents.In the recent years, In recent years, the rise of metabolic engineering makes it possible to genetically modif ied TAs biosynthetic pathway, and the metabolic engineering depends on the study of molecular biology and biochemistry of the pathway. In the field of plant geneticengineer ing, functional gene expression and regulation in higher plants has been hotand cutting-edge. Study on plant promoters help us to understand the transcriptional regulation of gene expression patterns and regulatory mechanism, and applied in genetic engineer ing to improve or increase the expression of exogenous genes. Based on the transcriptional ways, the promoterscan be divided into constitutive promoters, tissue-specific promoters, and inducible promoters. Using the constitutive promoters to drive the expression of exogenous genes in genetic engineering often leads to a waste of resources, and excessive accumulation of heterologous proteins in plants, which will cause metabolic disordersand resulting the abnormallygrowing plants. Therefore, it is more valuable to study tissue specific promoter and inducible promoter.Jasmonate(JA) and methyl jasmonate(MeJ A) are important signal transduction molecules. JA, MeJA and other JA derivatives, collectively referred toas jasmonates(JAs). These signaling molecules affect a variety of plant processes, including wounding and abioticstresses, and defense against insects and necrotrophic pathogens. as well as the impact of plant secondary metabolites on transcriptionallevel.More and more attention has been paid to the synthesis of secondary metabolites by MeJ A as a elicitor. Until now, there are a large number of domestic and foreign reports on JAs inducting to produce plant secondary metabolites, and most of these reports are studiescombinating of genetic engineering, functional genomics and other molecular biology techniques. In our study, Hyoscyamus nigerwas used to study the promoter of putrescine N-methyltransferase gene, we aimed to clarify the function of the different regions of the promoter, and preliminary study on the response of the promoter to MeJA. The high-efficiency TAIL-PCR(hiTAIL-PCR) was used to clone the promoter of HnPMT gene.Then a series of 5’end deletion fragments obtained with PCR were fused with the GUS reporter gene to construct expression vectors, which were transformated to Arabidopsis thaliana and Hyoscyamus niger.Us ing 50μM MeJ A treatment Hyoscyamus nigerhairy root,we found that the promoter was induced by 50μM MeJA. GUS staining results on the positive Arabidopsis thaliana seedlings and Paraffin section resultson thepositive Hyoscyamus niger hairy roots showed that the promoter was a root-specific promoter. The main results are as follows:1.Specific primers were designedaccording to the 5’end regionof HnPMT gene, then using of hiTALL-PCR methods we cloned the 1207 bp length of HnPMT gene promoter(KU981110). Analyzedby online website tools,it was predicted that the ATG waslocated on the 79 bp downstream of the transcription start site. There is a TATA-box(TATAAA) located on-30 /-25 region upstream of the transcription start site, and there are several CAAT-box located on-254 /-359 region upstream of the transcription start site, it was the characteristic element of eukaryotic plant promoter. At the same time, it was found that the components involved in the response of methyl Jasmine, such as CGTCA-motif, TGACG-mitif and G-box; hormone-responsive elements such as AuxRR-core; SA responsive elements such as TCA-element; activation of defense genes involved in regulatory elements, such as MBS, TC-rich repeats, W- box, HSE, LTR; light-responsive elements such as Box 4, Box I site, ATCC-motif,GAG-motif, Gap-box, Sp1, etc.; OSE2 ROOTNODULE, OSE1 ROOTNODULE and NTBBF1 ARROLB is root specific elements.2.The HnPMT longest promoter(-1128 / + 79) 、deleted promoter(-701 / + 79-451 / + 79-217 / + 79) and G-box mutation ptomoter were built into the plant binary expression vector pCAMBI A1391 Zvector,productionnamed pCAM-HnPMT217, pCAM-HnPMT451, pCAM-HnPMT701, pCAM-HnPMT1128 and pCAM-HnPMT217-muG-box, and then the Agrobacterium-mediated transformation was done to obtainHyoscyamus niger hairy roots and Arabidopsis thaliana. Success for obtaining positive transformation of Arabidopsis thalianaplants AP217, AP451, AP701, AP1128, positive Hyoscyamus niger hairy root HR217, HR451, HR701, HR1128 and HR217-muG-box, those plants help us to provide the test material to carry out the follow-up promoter function test.3.Real-time quantitative PCR results showed that HnPMT gene expression mainly exists in roots, almost no expression in leaves. The GUS staining of transgenic Arabidopsis thaliana showed that: the promoter AP217, AP451 and AP701 drived GUS reporter geneexpressed in Arabidopsis thaliana leaves and roots, showed no tissue-specific. But the staining resultsof the promoter AP1128 showed that the-1128 / + 79 segments can drive GUS reporter gene specifically expressed in roots, but not in leaves. Thus, Hn PMT promoterexpressed specifically in the roots, and the root-specific elements are present between the promoter-1128 /-701 region. Analysis of GUS staining of paraffin sections of Hyoscyamus niger hairy roots showed: In all of the truncated different length of promoters, the root tipand the epidermis have no expression of GUS, in the HR217, HR451 and HR701 hairy rootsthe promoterdrived the reporter gene in the root cortex, endodermis and dimensional tubular column, but results of HR1128 slices showed 11828 / + 79 segments of promoter drived GUS reporter gene mainly in the inner cortex and dimensional tubular column, but not in the cortex. Therefore it was concluded that, in the HnPMT promoter-1128 /-701 segments have some role in the regulation element of this phenomenon.4.The Hyoscyamus niger hairy roots which culturedin the triangular flask for 28 days were treated with 50μM MeJA for 0h, 1h, 6h, 12 h, 24 h, and then real-time quantitative PCR method was used to analyze the relative expression of HnPMTin different processing time, the results show in the 24 h, genes have the most powerful response to 50μM MeJA treatment, the highest expression of the gene at this point. The same approach to the transgenic hairy root of different lengths promoter s, the relative activity assay of GUS was measured after 24 h, results showed: HR217, HR451, HR701, HR1128 transgenic hairy roots have been induced by MeJA,So the HnPMT promoter is a promoter that is induced by MeJA.Further analysis of G-box site-directed mutagenesis in promoter-217 / + 79 section, GUS activity results showed that: 50μM MeJA induced, GUS activity was significantly reduced after G-box site-directed mutagenesis,the ratio up to 1.24, so G-box is an important element in response to MeJAin HnPMT promoter.