| Pib belongs to the family of NBS-LRR resistance gene,which was the first cloned rice blast resistance gene. Previous studies found that pathogens are not Pib expression inducing factors, on the contrary, darkness treatment, temperature, jasmonic acid, salicylic acid and so on were able to induce Pib expression effectively. Li Chanjuan confirmed the expression of Pib was the particular property of its promoter; Shao Keqiang used different lengths of 5'end deletion Pib promoter transforming into rice, studyed the relationship between copy number of cis-acting element YTCANTYY and the start activity and darkness inducing activity of Pib promoter, giving the results show that the YTCANTYY was a functional components, and its darkness inducing activity may be additive.Now generally considered promoter 3'end of the -25 TATA box and -75 CAAT box sequence is the key cis-element,which affecting the function of eukaryotic promoter start activity and determining the transcription start site and the start frequency, is necessary to the majority of eukaryotic genes to correct express. With the PLACE database scanning Pib gene promoter, found two motifs in it,and what is more were mutiply copies. Pib promoter carried by six YTCANTYY inducing elements, only two sections at -3042 to -3389, that most of its copy at the upstream of ATG 3'end, with the majority of TATA box and CAAT box in the same area.View of the importance of gene transcription of the 3'end core section, to understand efficiency and inducing activity of the Pib 3'end deletion promoter at the lack of TATA box and CAAT box? Darkness inducting element YTCANTYY in its 3'end downstream of TATA box and CAAT box whether being the darkness inducing activity, and their number whether being corresponded to inducing, was very important to study Pib promoter structure and function. This paper reports the Pib 3'end of deletion promoter fusing the reporter gene, by means of transgenic to study the relationships of YTCANTYY cis-acting element and the Pib promoter darkness inducing activity.1. By using PCR amplification, we constructed the vectors pNAR1001 (including two YTCANTYY components, three CAAT box, one TATA box), pNAR1002 (including four YTCANTYY components, ten CAAT box, two TATA box) and pNAR1003 (including six YTCANTYY components, thirteen CAAT box, seven TATA box), the recombinant plasmid after enzyme digestion, and then transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method.2. By Agrobacterium-mediated method, three recombined plasmids pNAR1001. pNAR1002 and pNAR1003 were transferred into japonica rice R109. pNAR1001 transformed plants was 28, pNAR1002 transformed plants was 38, pNAR1003 transformed plants was 32; PCR analysis with gus and hpt primers could amplify the specific bands of 441 bp and 509 bp, the positive transgenic plants had hybridization signals in Southern hybridization, no hybridization signal in wild-type plants, indicating that the gene was integrated into the rice genome.3. We used different length promoter of transgenic and control plant roots, stems, leaves for a quantitative analysis of GUS activity. After 24 h darkness treatment, the GUS activity of transgenic plants was higher than control, especially in roots are much higher than the stems, leaves, with organ-specific. Control plants after 24 h darkness treatment, no significant change; The transgenic plants of different plasmids pNAR1001, pNAR1002 and pNAR1003 before and after 24 h darkness inducing. GUS activity showing the same trend, which is highest in roots.and the lowest in leaves. The roots in transgenic plants after 24 h darkness treatment. GUS activity increased significantly, but in the stems and leaves only pNAR1003 have doubled. The results show that, Pib dark inducing activity mainly determined by the YTCANTYY dark elements, the TATA Box, CAAT motifs in the Pib gene sequence is not essential. In other words, YTCANTYY not only responsed to darkness inducing but also compensation for TATA, CAAT missing function. |
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