Isolation and functional assessment of a tomato proteinase inhibitor II gene.

A genomic clone encoding a serine proteinase inhibitor II, designated as TPI-2, was isolated from tomato (Lycopersicon esculentum Mill.) seedling. It consisted of a 990 bp upstream regulatory region and a 680 bp transcription region containing an intron. As shown by northern hybridization, mechanical injury activated its expression in roots, stems and leaves, and so did exogenous hormones jasmonic acid (JA) and alpha-Linolenic acid (LA), though abscisic acid (ABA) and NaCl failed to induce its expression. Salicylic acid (SA) was found to inhibit the inducing effect of LA but not those of mechanical injury and JA. As demonstrated experimentally, TPI-2 could be expressed effectively in tobacco cells and the protein products showed insecticidal activity.

[Prokaryotic expression and characterization of rice Rac protein osRACB].

As the sole ubiquitous signal GTP-binding protein family in higher plants, Rac genes act as pivotal molecular switches and participate in many regulations of life activities. In order to study the biochemical characteristics of rice Rac protein osRACB, the complete coding sequence of osRACB was cloned into expression vector pET28a and expressed in E. coli BL21(DE3). After induced by 1 mmol/L IPTG at 37 degrees C for 4 h, the fusion protein His-osRACB was produced in a large amount. The fusion protein was purified by Ni(2+)-NTA column and digested by thrombin. After a series of processes including separation and recovery by electrophoresis, renaturation by glutathione and concentration by ultrafiltration, pure osRACB protein in an active form was obtained. The GTP-binding and hydrolyzing assay showed that osRACB has strong GTP special binding and hydrolysis activity regulated by Mg(2+). By comparing it with another rice Rac protein osRACD, it can be concluded that osRACB has stronger GTP-binding activity and weaker hydrolysis activity than osRACD.

Molecular characterization of poly(A)-binding protein from Daucus carota.

Poly(A)-binding proteins (PABs) bind to the poly(A) tails of most eukaryotic mRNAs and thereby influence the translational efficiency as well as the stability of the mRNAs thus bound. Compared with the data on yeast PAB, relatively little is known about the functions of PABs in higher plants. The cDNA encoding PAB was cloned by the method of "virtual subtraction" from carrot somatic embryos cultured with different sucrose concentrations. Sequence alignment reveals a significant homology between the deduced amino acid sequence of DcPAB and those of other PABs. The deduced sequence consists of 658 amino acids with a calculated molecular weight of 71.9 kDa. The cDNA has been expressed as a recombinant protein in Escherichia coli. The differential expression of DcPAB under different conditions suggests a potently unique role in the development of carrot somatic embryo.

Rice GTPase OsRacB: potential accessory factor in plant salt-stress signaling.

As the sole ubiquitous signal small guanosine triphosphate-binding protein in plants, Rop gene plays an important role in plant growth and development. In this study, we focus on the relationship between the novel rice Rop gene OsRacB and plant salt tolerance. Results show that OsRacB transcription is highly accumulated in roots after treatment with salinity, but only slightly accumulated in stems and leaves under the same treatment. Promoter analysis showed that OsRacB promoter is induced by salinity and exogenous salicylic acid, not abscisic acid. To elucidate its physiological function, we generated OsRacB sense and antisense transgenic tobacco and rice. Under proper salinity treatment, sense transgenic plants grew much better than the control. This suggests that overexpression of OsRacB in tobacco and rice can improve plant salt tolerance. But under the same treatment, no difference could be observed between OsRacB antisense plants and the control. The results indicated that OsRacB is only an accessory factor in plant salt tolerance.

Structural analysis and identification of cis-elements of rice osRACD gene.

The osRACD gene correlated with fertility transformation in the photoperiod sensitive genic male sterile rice (PGMR), Nongken 58S, encoded a rice (Oryza sativa L. ssp. japonica) small GTPase belonging to the Rac/Rho family. Inverse PCR was performed to amplify a fragment about 1.4 kb in 5' upstream region of the osRACD promoter. Deletion mutation and gel mobility shift assay characterized two fragments (-799 to -686 nt, and -686 to -431 nt) in the osRACD promoter that could be involved in its transcriptional regulation. When these two deletion fragments were used as probe respectively, a retarded band appeared in the nuclear extracts of fertile 58S rice under short day (58S-SD). Whereas no retarded band was shown in the nuclear extracts of sterile 58S rice under long day (58S-LD). Competition assay indicated that the factors in the retarded bands binding to these two fragments were the same trans-acting factor (termed rice factor, RF). The binding affinity of RF was affected by phosphorylation and was higher in SD-growth rice than that of LD-growth rice.