[Analysis of the molecular motif for inducing response to jasmonic acid and ethylene in Pib promoter via rice transformation].

The expression of Pib gene in rice was induced by hormone, such as jasmonic acid and ethylene. In order to determine the necessary regions of sequence or motifs for response to jasmonic acid and ethylene in Pib promoter, the full length promoter of Pib (-3,572 approximately 2 bp) and three different 5' deletion fragments of Pib promoter (-2,692 approximately 2 bp, -1,335 approximately 2 bp, -761 approximately 2 bp) were synthesized by PCR and then were substituted for 35S upstream gus in a binary plasmid to construct re-combined plasmids of Pib promoter-gus fusions. Transgenic rice plants of the four recombined plasmids were produced by Agrobacterium-mediated transformation. Quality and quantum analysis of gus activities in transgenic plants at both protein and mRNA levels were conducted. The promotion activity of the full length promoter of Pib (-3,572 approximately 2 bp, pNAR901) was the highest in the four recombinants and the gus activities in its transgenic plant organs were enhanced obviously at 6 h after treatment with jasmonic acid or ethylene. The promotion activity of the deleted Pib promoters was significantly decreased and the response to jasmonic acid or ethylene treatment was not present when the -3,572 approximately -2,692 bp sequence was knocked out from the Pib promoter. Although the disparity in the lengths of the deleted Pib promoter of pNAR902 (-2,692 approximately 2 bp), pNAR903 (-1,335 approximately 2 bp), and pNAR904 (-761 approximately 2 bp) was more than 2 or 3 times, the response to jasmonic acid or ethylene treatment was not different among their transgenic plants. All these results indicated that the common deleted sequences (-3,572 approximately -2,692 bp) in the three deleted Pib promoter constructs were the essential region to the response to jasmonic acid and ethylene treatment. The result of pib promoter sequence searching indicated that there was only one GCCGCC motif at -2,722 bp of this common deleted segment in the Pib promoter sequence. Our rice transgenic results showed that the GCCGCC may be a cis-motif for Pib gene conferring response to jasmonic acid and ethylene for Pib gene.

[Initial functional analysis of the promoter region and coding region of Pib gene in transgenic rice].

The promoter region and intact coding region of Pib gene (9.9 kb) was inserted into the downstream of CaMV 35S promoter in a binary vector pPZP2Ha3(+), resulting a plasmid pNAR701. And a fragment of Pib gene from 6 986 to 9 392 bp was placed into pPZP2Ha3(-) under the control of CaMV 35S promoter, producing an antisense expression vector pNAR703. These two recombined vectors were transferred into a blast medium susceptible rice cultivar R109 by an Agrobecterium-mediated method. Tests of PCR and Southern blotting for transgenic plants as well as the segregation of hygromycin resistance in T1 generation confirmed that the target DNA fragments were integrated into genome of R109 and hereditable. Northern blotting analysis showed the coding region of Pib gene double driven by 35S and its native promoter was able to transcript in T1 transgenic plants. Rice blast resistance test for T1 transgenic seedlings of 3-4 leaves stage and in vitro leaves in tillering stage showed that transgenic plants of pNAR701 were more resistant to blast race ZD1 and ZG1 than the wild type plants, but the resistance of antisense transgenic plants from pNAR703 was decreased compared to the controls.