Function Verifying Of Darkness Inducing Elements YTCANTYY In Pib Promoter Via Rice Transformation

As a important regulatory element of 5'upstream regions of structure gene, promoter controls gene expression characteristic of space and time in transcriptional level. Study on plant promoters of disease resistance and analysis of regulatory element's function in promoter region are the very significance to expression characteristic of disease resistance gene. The transformation analysis by 5'deletion promoter fragments is the important method to understand the biological function of these molecular elements.Pib gene is the first major blast resistant gene cloned from rice by mapping clone technique and which is belongs to NBS-LRR resistant disease gene family. Previous studies show that the expression of pib gene was induced by darkness and chemistry factors but not pathogen. Our experiment results also indicated that different length fragments of pib structure gene sequence under the drive of 35S promoter, their To transgenic rice plants showed different resistances to rice blast pathogen races. Our results indicated that the pib promoter has the activity of darkness inducing in the rice transgenic experiments with the constructs of pib promoter-gus fusion. In natural environment, only root of rice plant could enjoy full darkness but the above ground organs are in the condition of light or dark cycle alternative, which offer the expression of pib gene the special organ specificity and diurnal rhythm.For studying the molecular structure and mechanism of environmental condition inducing, we analysis this promoter region by using PLACE database. The results show that there are about hundred of molecular regulatory element, for activation in this region, except of TATA,CAAT commonly elements, there are six copies of YTCANTYY element defined for light-response of cis-regluatory element. To verifying whether the dark-inducing property of the pib promoter is relate to the YTCANTYY element and how this molecular element affect the dark-inducing property? By using transgenic rice plants of 5'deletion pib promoter fragment-gus constructions, the relationships between the copy number of molecular element YTCANTYY in pib promoter and activity of pib promoter and its darkness inducing attribute were systematically analyzed.1. Four different regions of pib gene promoter were obtained by PCR, the difference among each fragments only lengths in 5'deletion, the 3' downstream of four fragments all terminate to base 2(base G of signal ATG). The PCR products of the first 5'deletion promoter is 222bp which is without YTCANTYY element; the PCR product of the second 5'deletion promoter is 569bp which contains one YTCANTYY element (-220～-227bp); the PCR products of the third 5'deletion promoter is 1911bp which contain three YTCANTYY element (-220～-227,-567～-574,-728～-735bp); the PCR products of the full length promoter is 3575bp which contain six YTCANTYY element (-220～-227bp, -567～-574bp, -728～-735, -1909～-1916, -3042～-3049, -3382～-3389bp), these fragments were ligated into the binary vector pCAMBIA1301 to substitute the CaMV35S promoter which is upstream of gus. The recombined plasmids are pNAR801, pNAR802, pNAR803 and pNAR804. All recombined binary vectors were confirmed by restricted enzyme digestion and transferred into Agrobacterium tumefaciens strain EHA101 by freeze-thaw method.2. By Agrobacterium-mediated method, pNAR801, pNAR802, pNAR803, and pNAR804 were transferred into japonica rice R109. pCAMBIA1301 was also transferred at the same time. Total 136 independent transformed plants were got on regeneration medium. PCR analysis revealed that 96 plants were positive and the transform frequency is 70.6%. PCR and Southern blot tests confirmed the integration of foreign gene.3. By using transgenic rice plants of 5'deletion pib promoter fragment-gus constructions, the relationships between the copy number of molecular element YTCANTYY in pib promoter and activity of pib promoter and its darkness inducing attribute were systematically analyzed. The results from Gus histochemistry detection revealed, after darkness inducing the transgenic rice calli of pib promoter fragments carrying 6, 3 or 1 copy of YTCANTYY motif were appeared different degree Gus blue in X-gluc solution, but the 5'deletion pib promoter fragment with no YTCANTYY element was unable to drive gus gene to show any Gus blue in X-gluc solution. Fluorescence quantitative analysis results showed that pib promoter sequence had strong organ specificity, even though all 6 darkness inducing elements were knocked out, its transgenic rice plants still kept the tendency of promoter activity in root higher than that of above ground parts of plants. But the activity of promotion and darkness inducing were increased with the increase of promoter fragments in length and with increase of darkness inducing element copy. These results indicated that YTCANTYY in pib promoter is a functional motif for darkness inducing and in other words this motif confer the darkness inducing of pib promoter. At least within 6 copies, the activity of darkness inducing was positively correlated with the copy number of YTCANTYY element. And the activity for each individual YTCANTYY element seemed to additive.4. Using transgenic rice progenies of pib promoter-gus recombined plasmid, the inducing activities of pib promoter were analyzed. The fluorescence quantitative analysis of Gus activity in T₃ transgenic rice plants confirmed that 24h darkness treatment and temperature could influence promotion activity of pib promoter. Gus activity in root was always higher than leaf and they have strong organ specificity. Gus activity both in root and leaf of the transgenic rice plants all exhibited a clear diurnal rhythm, the Gus activity in daytime was at low level (15:00 was lowest) and high level was in night (3:00 was highest). These experimental results of Gus activity in the transgenic rice plants indicated the blast resistance production of pib structure gene was mainly synthesize in root and which was separated from infection position of rice blast. We infer that, the disease resistance product of pib structure gene is synthesized in root, and then transported to the target tissue of aboveground in the night to perform the function of blast resistance.