In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.

The most commonly used protocol of the RNA isolation, the guanidine thiocyanate method, was unsuitable for recalcitrant plant tissues containing a large amount of storage proteins and secondary metabolites. We demonstrated that RNA could bind to the silica particles, which have been used successfully in DNA isolation from various sources, under a high concentration of NaCl in the presence of ethanol and sodium acetate. Based on this observation, an efficient, inexpensive, and highly reproducible technique, the acid phenol silica method, was developed to isolate high-quality RNAs from various plant tissues recalcitrant to extraction in guanidine thiocyanate.

We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 50-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by in vitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region) 415/+54 containing Ethylene Responsive Element ERE (-355/-348) conferred threefold ethylene-induction of GUS expression, while-827/+54 which also contains ERE(-683/-676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-luc constructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at -795/-787 and -785/-777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other than Arabidopsis and that functional EEs and EREs are present in the 50-flanking region of a gene encoding cysteine proteinase.

In this paper, we report a useful protocol for cloning toxic protein genes. Use of the SOC medium, which isa glucose-containing rich medium, significantly improved the transformation efficiency of a recombinantplasmid containing a toxic plant subtilase SaSBT1 cDNA. Both glucose and rich nutrients present in the SOC medium prevented the unintended activation of the lac promoter carried on the cloning vector, and led to significantly improved transformation efficiency of recombinant plasmids containing toxicprotein genes and an increased rate of transformant growth.

Jatropha curcas, a monoecious perennial biofuel shrub belonging to the family Euphorbiaceae, has few female flowers, which is one of the most important reasons for its poor seed yield. This study was undertaken to determine the effects of the plant growth regulator 6-benzyladenine (BA) on floral development and floral sex determination of J. curcas. Exogenous application of BA significantly increased the total number of flowers per inflorescence, reaching a 3.6-fold increase (from 215 to 784) at 160 mg/l of BA. Furthermore, BA treatments induced bisexual flowers, which were not found in control inflorescences, and a substantial increase in the femaleto- male flower ratio. Consequently, a 4.5-fold increase in fruit number and a 3.3-fold increase in final seed yield were observed in inflorescences treated with 160 mg/L of BA, which resulted from the greater number of female flowers and the newly induced bisexual flowers in BA-treated inflorescences. This study indicates that the seed yield of J. curcas can be increased by manipulation of floral development and floral sex expression.