| WRKY genes are a family of transcriptional regulatory factors that have specific functions in plants. Plant WRKY gene-encoded transcrip -tional regulators appear to be involved in various physiological programs, including disease-resistance,senescence, stress responses of biotic and abiotic, growth and development processes. Promoters are important elements that regulated gene expression, activity of promoters indirectly reflect gene expression that they control. For mastering regulatory fuctions of OsWRKY19 and 0sWRKY89 in different responses, we used GUS reporter gene to analyse promoter fuctions of 0sWRKY19 and 0sWRKY89.The summary of our study as follows: 1 Analysis of rice 0sWRKY19 gene promoterA 1404bp promoter fragment of 0sWRKY19 gene was amplified by polymerase chain reaction(PCR) technique from rice Xiushuill.we called it 0sW19p.We forecast more twenty cis-elements in 0sW19p from PLACE website.The full length 0sW19p and 5' terminal deletions with a length of 105,378,678,977,1106,1205 and 1306bp were fused with GUS gene, and plant expression vectors were constructed respectively. All of vectors were transformed by agrobacterium-mediated method and gained some transgenic lines for each vector.Expression of only 0sWJ9p::GUS in all vectors was intensively restrained by 2,4-D in callus stage, we speculated there is 2,4-D responsive element in -1404—1306bp of 0sW19p and thought it as new element.GUS activity of all of deletions were detected in leaves oftransgenic seedlings, it suggested that some regulatory elements exist in -1404 — -1306bp and -1205~ -977bp, and fragments of -1306~-1205bp and -977— -378bp may contain negative elements.Expression of 0sW19p::GUS was observed in leaves, stems, and sheaths;pollens had't expression;mature seeds hadn't expression;the embryos of germinating seeds had expression; primary roots, adventitious roots and their lateral roots had expression but not in root tips of seedling stage; only lateral roots had expression in roots of maturation phase.We analyzed inducible expression pattern in T1 transgenic seedlings, the results showed:expression level of 0sW19p::GUS can be increased by ABA(100μM),SA(500μM), high temperature(42℃), low temperature (5℃) and wounding treatment;and can be restrained by 2, 4-D(1μM),NaCl(200mM)and PEG(25%);IAA(5μM),Me JA(100μM) and ultraviolet can't influence 0sW19p:: GUS expession. GUS activity of leaves of 0sW19p::GUS was lower than 35s::GUS in trangenic plants,but roots of it was higher one.In conclusion, we thought 0sWRKY19 gene promoter was an inducible promoter with tissue specific expression characteristic. 2 Analysis of rice 0sWRKY89 gene promoterA 1470bp promoter fragment of 0sWRKY89 gene was amplified by PCRtechnique from rice Xiushuill, we called it 0sW89p. We forecast more twentycis-elements in 0sW89p from PLACE website. The full length 0sW89pwa.s, fusedwith GUS gene, and plant expression vectors was constructed. Vector wastransformed by agrobacterium-mediated method and gained some transgeniclines. Expression of 0sW89p::GUS was intensively restrained by 2, 4-D incallus stage, we speculated there is 2,4-D responsive element in 0sW89p.The results of tissue specific expression of 0sW89p:: GUS and0sW19p::GUS were the same, we compared cis-elements between 0sW89p and0sW19p, and found they had the same tissue specific elements, and onlydifferences in number.We analyzed inducible expression pattern in T, transgenic seedlings,the results showed:expression level of 0sW89p::GUS can be increased byMeJA, IAA, ultraviolet, high temperature, low temperature and woundingtreatment, inducible effects of MeJA and ultraviolet were distinct ;and canbe restrained by 2, 4-D;ABA and SA can't influence 0sW89p:: GUS expession;NaCl and PEG can restrain expression level of 0sW89p:: GUS in roots butincrease expression level in leaves. GUS activity of 0sW89p:: GUS washigher than 35s:: GUS in trangenic plants. In conclusion, we thought0sWRKY89 gene promoter was an inducible promoter with tissue specificexpression characteristic. |
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