Construction And Expressing Regulation Of Apical Tissue-specific And High Efficient Inducible Promoters

Based on the study of published cis-elements of tissue-specific and induciblepromoters. Salicylic acid (SA) inducible as-I element and the green tissue-specific 81 element, derived from the cauliflower mosaic virus 35S promoter,were selected. Because both of the two promoters have well-known researchbackground and more commonness than other cis-elements.Based on a truncation of?0 355 promoter (-90 to +1), nine different promoterswere constructed with artificially synthesized as-I sequence and BI sequence.Each one of them respectively has one copy, two copies or four copies of as-Isequence and 81 sequence, and were subsequently designated as IBIa promoter,182a promoter, lB4a promoter, 2BIa promoter, 2B2a promoter, 2B4a promoter,4Bta promoter, 4B2a promoter and 4B4a promoter. And all the constructs wereconfirmed by sequencing. Then nine plant expression vectors containing thesecorresponding promoters were constructed, in which the GFP gene wasdownstreamed as a reporter gene. These plant expression vectors weresubsequently designated as p811 B 1 a.GFP, p811 B I a.GFP, p811 B I a.GFP.pBI2Bla.GFP, pBl4BIa.GFP, pBIIB2a.GFP, pBl2B2a.GFP, pBl4B2a.GFP,pBIlB4a.GFP, pBI2B4a.GFP and pBl4B4a.GFP. Tobacco leaf discs weretransformed with Agrobacterium tumefaciences Conn LBA4404 containing theseexpression vectors, and 186 trangenic plants were obtained. Among of them, 106pass muster transgenic plants were firstly screened out by FluorescentMicroscope.Results of testing transgenic plants with PCR, Southern blot methods confirmedthe successful integration of exogenous gene into tobacco plants genome.The GFP expressing level of different tissues located on different parts ofrespective trangenic plants transformed by different promoters were observed bythe Fluorescent Microscopy, and the expressing level of different leaves located5on different parts of transgenic plants were quantitatively analyzed by FluorescentSpectrophotometer. The results of two methods were highly consistent with eachother and confirmed following conclusions:?The promoters containing B! element show a tissue-specific expressionprincipally in new leaves and the stem apex.?The B I element shows a accumulative characteristic when adding the its copynumber in promoter. The expression level of promoters containing fourcopies of B! sequence is 2.9 fold the level of promoters containing twocopies of B1 sequence, is 3.9 fold the level of promoters containing onecopy of 81 sequence. The expression level of promoters containing twocopies of B I sequence is I .3 fold the level of promoters containing one copyof 81 sequence.?No difference has been observed between the different transgenic plants ofthree kinds of promoters that containing same copy number of BI elementand different copy number of as-I element. The results mean that theexpression level of promoter is principally dependent on the number of 81sequence contained in the promoter, and the number of as-I sequence has noobvious influence on it under the general condition.After sprayed by salicylic acid solutions at various concentration, such as 2mM,20mM and 50mM, the transgenic tobacco plants carrying different promoters weretested for their response to the SA treatment by Fluorescent Microscopy andFluorescent Spectrophotometer both qualitatively and quantitatively. The resultsof two methods were highly consistent with each other and confirmed followingconclusions:?The as-I element shows a positive response to the SA signal transduction,and the promoter containing as-I element show a trait of transcriptionactivation by SA inducement.6?Inductive efficiency is closely dependent on concentration and pH value ofSA solution. The results suggest that the inducti...