[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.

[The inductive activation of the promoter of pib gene].

A 5.7 kb putative promoter region of pib gene was isolated from the pib genomic clone and substituted for the 35S promoter upstream of gus gene in plasmid pCAMBIA1301 to construct a new plant expression vector pNAR604 (putative pib promoter-GUS + 35S-hpt). From Agrobacterium-mediated transformation and hygromycin selective culture in vitro, hygromycin resistant calli and 36 transgenic rice (Oryza sativa L.) plants were obtained. Histochemical assays of GUS activity showed that no expression was observed in the resistant calli and roots from transgenic rice if cultured under light, but after 24 h dark treatment there was strong GUS staining. Fluorimetric quantitative analysis indicated that GUS expression was organ-specific in transgenic rice. Without the dark treatment, GUS activity in roots and stems were about 7 and 3 times higher than in leaves in which GUS activity was only trace detected. After 24 h dark treatment, GUS activity in roots, stems and leaves of transgenic plants were all promoted and the largest increase was observed in leaves. Twenty-four hours after spraying with 5 mmol/L SA (Salicylic Acid) or 0.3 mol/L NaCl, GUS activity in leaves of the transgenic plants was 2.7 or 3.6 times respectively higher than untreated control. It was confirmed that an inductive promoter was involved in this 5.7 kb upstream region of pib gene, and dark, SA and NaCl treatments were inductive factors for pib promoter.

[Inheritance, expression and armyworm resistance of protease inhibitor II gene (Pin II) driven by different promoters in transgenic rice].

The inheritance of rice lines transformed by protease inhibitor II gene under control of different promoters was investigated by analysis of hygromycin resistance, PCR and Southern blot. For segregation patterns of foreign gene, 68.4% of the transgenic rice plants were conformed to a Mendelian ratio and in which the rate of transgenic plants with single copy was 63.6%. Quantitative analysis of Pin II protein expressed in transgenic rice plants showed that Pin II protein in fresh leaves was 160 microg/g for Act-Pin II-2x, 176 microg/g for Ubi-Pin II-2x, and 104 microg/g for PIN5'-Pin II-4x separately while in control rice plants was only 20 microg/g. The inhibitory activity against tryspin of Pin II gene driven by Actl and Ubi promoter reached 37.7% and 43.1%, much higher than that driven by PIN5' (29.2%). Bioassay for insect resistance to armyworm (Pseudaletia separata Walker) revealed that transgenic plants had increased their resistance to the pest but there was not significantly different from controls, and also there was no correlation between insect resistance to armyworm and quantity of Pin II protein as well as promoters in transgenic rice.

[Cloning, Characterization and Expression Vector Construction of Potato Proteas-inhibitor II Gene (PIN II-2x) from Diploid Potato (Solanum phurejia).].

Based on the published gene sequence of tetraploid potato (Solanum.tuberosum) protease-inhibitor II, a genomic DNA and a cDNA sequence of potato protease-inhibitor II gene were obtained from the cDNA library and the genomic DNA of a diploid potato IVP101 (Solanum.phurejia) using PCR method and named PINII-2x. Nucleotide sequencing confirmed that the full-length DNA of PINII-2x was 580 bp, including an 115 bp intron and two exons. cDNA was 462 bp ( stop codon TGA not included) and had 88% similarity to the tetraploid potato protease-inhibitor II. The PINII-2x open reading frame encodes a 154-amino acid polypeptide with a predicated size of 16.6 KD and a calculated PI of 6.08. The deduced proteins from PINII-2x cDNA had 93% homology with other tetraploid potato protease-inhibitor II, which contain the intact signal peptide and two active site similar to the potato protease-inhibitor II family. Test of the RT-PCR indicated that PINII-2x mRNA is wound- induced expression in potato leaves. Binary vector of PINII-2x cDNA drove by either rice Actin I promoter (ActI) or maize Ubiquitin promoter (Ubi) was constructed.