Genetic and chemical validation 41 AtKAPAS from transgenic E coli

Total RNA isolated from leaf tissues of A. thaliana was used for preparation of poly(A)+mRNA. Double-stranded cDNA was constructed from 5 ^g of poly(A)+mRNA with the Time Saver cDNA synthesis kit (Pharmacia, Piscataway, NJ, USA), using Oligo(dT)18 as a primer. By performing PCR (polymerase chain reaction) with the two primers, the full-length AtKAPAS cDNA was amplified and isolated from A. thaliana cDNA library prepared. The primers encompassing the full-length cDNA of AtKAPAS, KAPAFB (5'-CAAAAAGAATTCGACGACGACGACAAGATGGCGGATCATTCGTGG GATAAA-3') and KAPARH (5'-GTGCACCTCGAGTTATAATTTGGGAAATAGAAAGGA-3'), were synthesized to include EcoRI and Xhol restriction site, respectively. Primers of KAPAFB and KAPARH were used in a PCR reaction to amplify the AtKAPAS-encoding region. The resulting PCR fragment was digested with EcoRI and XhoI, and cloned into MBP (maltose binding protein) fusion vector (Bioprogen Co., Ltd., Korea) to generate construct pEMBPek-KAPAS (Fig. 2). E. coli BL21-Gold(DE) (Stratagene, USA) was transformed with expression vector pEMBPek-KAPAS and than cultured in LB (Luria-Bertani broth, USB, USA) medium containing 100 ^g-mL-1 of ampicillin at 37oC (150 rpm) until the value of OD600 reached 0.6. In order to induce the expression of the target protein in E. coli cells, isopropyl-D-thiogalactoside was added to the suspension at a final concentration of 1 mM, and further cultured for 3 h. The culture cells were washed with 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA, after centrifugation at 9000g for 10 min. The cell pellets were resuspended and pooled in 50 mL of buffer solution (50 mM Tris-HCl, pH 8.0, 200 mM NaCl). The sample was sonicated for 30 s and cooled on ice for 3-5 min, and the procedure was repeated three times. After centrifugation at 1000g for 30 min, the supernatant was purified with MBP affinity chromatography and used as enzyme solution. Eluting fractions separated from E. coli transformed with pEMBPek-KAPAS recombinant vector and the control group was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. SDS-PAGE was performed on a 12% running gel and protein bands were visualized by staining with Coomassie Brilliant Blue G250.

The AtKAPAS cDNA was cloned into MBP fusion vector to generate the E. coli expression construct pEMBPek-KAPAS. SDS-PAGE analysis revealed that E. coli transformed with MBP fusion vector showed the expression of a very strongly induced fusion protein of ca. 98.2 kDa, which may be consisted of AtKAPAS protein of 51.3 kDa, and maltose binding peptide MBP affinity tag of 46.9 kDa. For the partial purification of AtKAPAS protein, the lysates from IPTG-induced E. coli containing pCKAPA as well as from E. coli harboring control vector MBP fusion vector were loaded onto maltose affinity column (1.1cm x 30cm, Millipore, USA). The AtKAPAS protein binding to MBP resin was eluted with 10 mM maltose solution. To confirm the purification of AtKAPAS protein, elutes with E. coli-expressed AtKAPAS protein and E. coli control in the various fractions of affinity chromatography were subjected to SDS-PAGE analysis (Fig. 4). Elutes of E. coli-expressed AtKAPAS protein contained the induced fusion protein of ca. 98.2 kDa while those of E. coli control didn't contain AtKAPAS protein.

Fig. 4. KAPAS over expression and purification from transgenic E. coli.

4.2 AtKAPAS inhibition in vitro treated with TPTA

For substrate synthesis and enzyme assay in vitro, substrate pimeloyl CoA was synthesized according to the method of Ploux and Marquet (1992). TPTA was purchased from Sigma (USA) and used as a KAPAS-inhibitor. KAPAS activity was determined according to the method of Webster et al. (2000) using a linked assay by monitoring the increase in absorption of NADH at 340 nm using a microplate spectrophotometer (Benchmark Plus, Bio-Rad, USA), thermostatically controlled at 37oC. The procedure was the same apart from the reaction volume of 250 pL instead of 1 mL. L-Alanine and pimeloyl-CoA were added to give the desired final concentrations. Prior to analysis, enzyme samples were dialyzed for 2 h at 4oC against 20 mM potassium phosphate (pH 7.5) containing 100 pM pyridoxal 5'-phosphate (PLP). The KAPAS concentration in all analysis was 10 pM in 20 mM potassium phosphate (pH 7.5) and the concentrations of TPTA were 3.125, 6.25, 12.5, 25, 50, and 100 pM. Reference cuvettes contained all other compounds except inhibitor.

Enzyme activity was assayed with the partially purified AtKAPAS protein extracted from transgenic E. coli. AtKAPAS protein was expressed in E. coli at a very high level, and a significant portion of these proteins was soluble, and their affinity-purified preparations contained a single major polypeptide. The dose-dependent in vitro inhibition of KAPAS activity by TPTA was noticeably examined and the IC50 was calculated as 19.85 pM (Fig. 5).

4.3 Herbicidal activity of TPTA under greenhouse condition

Seeds of A. thaliana were sown in plastic pots (24 cm2 surface area) filled with artificial nursery soil (Boo-Nong Soil, Seoul, Korea), and the plants were grown to the required iûû r iûû r

6.312.5 25 50 100

Fig. 5. KAPAS inhibition treated with triphenyltin acetate in vitro. Data was expressed as a mean ± S.D.

growth stage for application in a greenhouse maintained at 30~35oC during the day and 20~25oC at night. Application was conducted at 40 days after seeding for foliar application of 16, 32, 62.5, 125, 250, and 500 g-ha-1 with laboratory spray gun (spray volume of 1000 L-ha-!). The TPTA was used as a solution in acetone/water (60:40 by volume) containing 1.0 g-L-1 of Tween-20. The plants were photographed at 1 week after application. The herbicidal spectrum of TPTA was investigated to 10 weed species, Sorghum bicolor, Echinochloa crus-galli, Agropyron smithii, Digitaria sanguinalis, Panicum dichotomiflorum, Solanum nigrum, Aeschynomene indica, Abutilon avicennae, Xanthium strumarium, Calystegia japonica with foliar application. Foliar application of 0.25, 0.5, 1, 2, and 4 kg-ha-1 with laboratory spray gun (spray volume of 1000 L-ha-1) was conducted at 2 weeks after sowing each seeds in plastic pot (350 cm2 surface area) filled with upland soil. Visual injury was determined at 2 weeks after application with a scale of 0 (no injury) to 100 (complete death).

The foliar-treatment of 16, 32, 62.5, 125, 250, and 500 g-ha-1 TPTA to the 40-day old A. thaliana plants has caused herbicidal effects of 8.3, 20, 47, 90, 97, and 100%, respectively. The herbicidal activity was increased as time passed after application. The application rate of more than 125 g-ha-1 was shown almost complete death at 1 week after application (Fig. 6). The main symptoms were desiccation and burning effect. Symptoms begun to appear within several hours after application, and the applied region of the leaf was desiccated at 1 day after treatment of more than 250 g-ha-1.

Foliar application of TPTA to 10 weed species was showed good herbicidal activity. The most sensitive species was Xanthium strumarium which was completely dead at 250 g-ha-1 of TPTA foliar application. Abutilon avicennae, Calystegia japonica, and Aeschynomene indica were also controlled by 500 g-ha-1 of TPTA foliar application (Table 1). However, grass weed such as Sorghum bicolor, Echinochloa crus-galli, Agropyron smithii, Digitaria sanguinalis, and Panicum dichotomiflorum was tolerant to TPTA foliar application comparing to the broad-leaf weeds.

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