Construction And Functional Analysis Of Root-specific And Fungus-inducible Chimeric Promoters In Tobacco

Promoter is a fragment of DNA,which can be recognized and bound to by RNA polymerase to initiate transcription of the genes.Actually,promoters act as"SWITCHes" in the program of genes’expression,after all,resulting in cells’differentiation.Chimeric promoters are well known of many advantages,such as broad-spectrum elicitors,low background activity,and high promoter strength.A chimeric promoter is constructed with an array of cis-acting elements from various sources.So far,there are two common strategies to construct chimeric promoters,involving combination of a core promoter and several cis-elements,and construction of bidirectional promoters.Additionally,different cis-elements can also be combined to build a totally simple but efficient new promoter.Plant soil-borne diseases happen in roots or stem mostly and transmit with the soil,involving bacterial wilt,powdery mildew,botrytis and so on.With the wide application of fertilizers,soil microorganism groups are getting disordered and soil is becoming poorer and poorer.Consequently,plant soil-borne diseases seriously limited the development of agriculture and animal husbandry.Cultural control and chemical control do not function well because they are not environmental friendly while the newly emerging biological prevention stuck in several problems including singleness of the targeted diseases,large amounts of the required bacteria and easy degradation of the biocontrol bacteria.As a result,our researchs focus on construction of root-specific and fungus-inducible chimeric promoters to develop a new sight in prevention of plant soil-borne diseases.As reported,TobRB7 was characterized as a root-specific gene in tobacco and the upstream regulating sequence-636 bp to-299 bp with respect to transcription initiation site is sufficient to direct its root-specific expression,while the 700 bp of 5’ flanking sequence of gene win3.12 confers wound/fungus-regulated expression in transgenic tobacco.At the same time,Motif Ⅰ(GGTACGTGGCG)is known as a root-specificity related sequence whereas box-W1(TTGACC)has been identified as a fungal elicitor responsive element.Accordingly,four fragments TRWI(pTobR-pWIN),WITR(pWIN-pTobR),TC1W(pTobR-C1-pWIN),WC1T(pWIN-C1-pTobR)has been fused by overlapping extension PCR.Specifically,"C1”is a synthesized motif originated from Motif Ⅰ and box-W1.To evaluate the four chimeric promoters’activity,GUS report gene in binary expression vector pBI121 has been designed to take the responsibility when fused to the downstream of four chimeric sequences induvidually and transformed into tobacco.Under different treatments involving powdery mildew infection,single or repeated wounding,methyl jasmonate(MeJA,0.50 mg/mL),and salicylic acid(SA,50 mM,pH 6.8),GUS enzyme activity in different tissues originated from different transgenic tobacco lines will tell whether the chimeric sequences could finely direct root-specific and wound/fungus-inducible gene expression.In fact,Our results showed that TC1W and TRWI could drive GUS gene to express most in root,especially under powdery mildew infection,methyl jasmonate(MeJA,0.50 mg/mL)treatment,and salicylic acid(SA,50 mM,pH 6.8)treatment.