| A temperature-regulated low seed-setting rate mutant was discovered during the selection of MH 63 transgenic lines expressing Bt protein. The transgenic insertion site analysis of this mutant using TAIL-PCR revealed that the large fragment containing two copies of exogenous Bt gene was inserted in the short arm of chromosome 10 close to centromere. Further sequence alignment analysis confirmed that the exogenous Bt gene was inserted into the promoter regions of a function unknown gene. This mutant has been now named as tls (Temperature-regulated low seed setting rate) mutant and the gene corresponding to this mutant was named tls gene.Bioinformatics dissection found that the promoter sequence of the tls gene contains 19 heat shock elements (HSEs) and two drought, low temperature and salt stress responsive elements (DRE). The insertion of exogenous gene resulted in nine of the nineteen HSEs and one of two DREs were separated, which thus influence Bt mutant responsibility to the temperature and subsequently its seed setting rate decrease when encountered with temperature stress. In order to make clear of molecular and expressing characteristics, we amplified full length as well as different lengths of the tls promoter sequences according to the inclusion of HSE numbers (4, 10,11,15, and 19), respectively, via PCR using high fidelity Taq polymerase. And then we constructed 5 expression vectors in which reporter gene of gus driven by these different lengths of promoter fragments. These vectors were finally introduced into genome of japonica variety Nippobare. Adequate numbers of transformants positive to the transgene were used for expression activity and pattern analysis of the tls promoter.Basic expression analysis that was conducted using the semi-quantitative RT-PCR indicated that the wild and mutant promoters all drove GUS expression in the root, stem, leaf and flower. But for the pistil the wild had the expression while the mutant had not. The significant differences also appeared in the root but the tendency was opposite to that in the pistil.When stressed by the high temperature, the expression of the wild and mutant promoters was all up-regulated. Nevertheless the range of up-regulation were the latter larger than the former. Low temperature stress had limited up-regulation effect to both promoters. The effect on wild promoter could be observed 4 days later, which is different from that on mutant promoter observable after 2 days.Opposite to temperature stress, the salt and monitored drought stresses caused the overall expression of both wild and mutant promoters down-regulated. However the tendency of down-regulation on the wild and mutant as well as on the salt and monitored drought stresses was similar. The characteristics of its dynamic variation at exampling time points were remarkable down-regulation (6h), incomplete picking-up (12h), slightly down regulation (24h), followed by further slightly down regulation (48h). But the overall extent of these down regulations was the wild more severe than the mutant and the salt stress over the drought stress.Gus histochemical staining and fluorescence quantitative analysis results showed that:(1) the truncation promoters of H4 and H10 were unable to effectively drive the gus gene expressed in the transgenic plants, (2) the truncation and full length promoters of H11, H15, and H19 could drive gus expressed in roots, stem, leaf, leaf sheath, glume and rachis, but the expression level observed was highest in leaf tissue; (3) all promoters could drive gus gene expressed faintly in stamen but none in pistil, filament and mature seeds; (4) high temperature, drought, salt stresses were unable to up-regulate expression of gus gene driven by H4 and H10 truncation promoters in the transforments, but had influence on that driven by H11, H15, and H19.
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