Cloning And Functional Analysis Of PNZIP Gene Promoter

In recent years, constitutive promoters, such as CaMV 35S, Actl and Ubi promoters , have been used in many transgenic plants. Although those promoters can drive exogenous gene to express efficiently, they could make energy waste unnecessarily and cause some negative effects. Tissue-specific promoters become more and more important for both research and industrialization of plant genetic engineering. Though many leaf-specific promoters have been investigated, we did not have property right in our country, so it is vital to construct our own leaf-specific expression vector.We have isolated PNZIP gene from Pharbitis nil choisy which can express strictly in leaf. The ISOObp promoter of PNZIP gene was cloned by adaptor PCR method.According to the character of the sequence of PNZIP gene promoter, we made a series of promoter deletion sequences by PCR. The length of deleted fragments are 1096 bp, 675 bp, 489 bp and 143 bp, respectively. Then we take the place of 35S promoter in pBI121 by the full-length PNZIP promoter and its deleted segments. Thus we got 5 plant expression vectors where PNZIP gene promoter can fuse GUS reporter gene . Through leaf disc transformation, some transgenic tobacco plants were obtained. The detail characteristics of PNZIP promoter were investigated by histochemical and quantitive analysis of GUS activities in leaves and roots from transgenic plants .1. PNZIP gene promoter is a promoter which has a character of strict leaf-specific expression . After GUS histochemical analysis, those leaves of tobacco who have PNZIP promoter turned blue, while those roots did not.2. The full-length promoter of PNZIP gene is a strong promoter whose GUS activity in leaves is 9 times as that of 35S.3. As a light regulated promoter, PNZIP gene promoter has some light responsive elements, such as G-box,GT-II box and AT-rich region .4. As some new elements which relate to leaf-specific expression , GAAATA and GATAC elements have been found in PNZIP gene promoter. GATAC element may determine the leaf-specific expression of gene , while GAAATA element is probably a enhancer which can enhance the gene expression in leaf.5. Position effect and multiple copies integration effect the exogenous gene expressiongreatly.