JIA-2018-09

1993 ZHANG Xiang et al. Journal of Integrative Agriculture 2018, 17(9): 1991–1998 the first or second node on the middle fruit branches. In July, white flowers from the first or second position in the middle of plants were labeled. On 30 July, 10 d after anthesis, the potted plants with labeled flowers were exposed to the stress in the greenhouse. In 2011 and 2012, boll samples were harvested after exposing them to 32, 34, 36, 38, and 40°C for 24 h. In 2012 and 2013, bolls were collected after exposing them to DH/NN treatment for 0, 4, 7, and 10 d, respectively. The controls (32/27°C day/night temperature regime) were sampled at the same time. Five bolls per treatment were collected, placed on ice, and brought to the laboratory where the boll shell, fiber, and seed were immediately separated. The boll shells were cleaned with distilled water and dried with paper towels. All samples were frozen in liquid nitrogen and stored at –80°C. These frozen tissues were used for assays of the Cry1Ac protein, free amino acid, soluble protein content, protease, and GPT activity. Determination of Cry1Ac protein concentration The concentrations of Cry1Ac protein in the boll shell and fiber extracts were determined by immunological analysis by means of ELISA (Chen et al . 1999). The tissue extracts were harvested by homogenizing the frozen tissue (1.5 g) in 2 mL of extraction buffer (Na 2 CO 3 1.33 g, DTT 0.192 g, NaCl 1.461 g, and Vc 0.5 g dissolved in 250 mL of distilled water). Then, the contents were transferred to a 10-mL centrifuge tube. The residue was washed with 3 mL of the buffer, and added to a centrifuge tube. The tubes were shaken by hand and stored at 4°C for 4 h. After centrifugation at 10 000×g, samples were held at 4°C for 20 min, the extracts were collected and then filtered through a C 18 Sep-Pak Cartridge (Waters, Milford, MA), and the filtered supernatants were collected for determination. Microtitration plates were coated with the standard Cry1Ac insecticidal proteins and samples and then incubated at 37°C for 4 h. The antibodies against the Cry1Ac insecticidal protein were added to each well and incubated for another 30 min at 37°C. After that, horseradish peroxidase-labelled goat anti-rabbit immunoglobulin was added to each well and samples were incubated for 30 min at 37°C. Finally, the buffered enzyme substrate (1,2-phenylenediamine) was added (Chen et al . 1999). The enzyme reaction was carried out in the dark at 37°C. After 15 min, the reaction was terminated using 50 μL of H 2 SO 4 (3 mol L –1 ). The results from absorbance measurements at 490 nm were recorded. ELISA data were calculated as described by Weiler et al . (1981). GPT activity assay GPT of boll shells was extracted and GPT activity was assayed according to Chen et al . (2005a) with slight modifications. The samples (1.0 g) were homogenized in 5 mL of 0.05 mmol L –1 Tris-HCl (pH 7.2), and the mixture was centrifuged at 26 100×g for 10 min. During the extraction process, the temperature was kept at 0°C. The supernatant solution was analyzed to detect GPT activity. First, 0.2 mL of the supernatant solution was added to a mixture containing 0.5 mL of 0.8 mol L –1 alanine in a 0.1 mol L –1 Tris-HCl (pH 7.5), 0.1mLof 2mmol L –1 pyriodoxal phosphate solution, and 0.2 mL of 0.1 mol L –1 2-oxoglutarate solution. Then, the reaction mixture was incubated at 37°C for 10 min, and 0.1 mL of trichloroacetic acid solution (0.2 mol L –1 ) was added to stop the reaction. Then, the pyruvate was converted to pyruvate hydrazine with chromogen. The color intensity of the hydrazine in waster saturated with toluene was red at 520 nm. The GPT activity, in terms of pyruvate production, was calculated from authentic pyruvate standards run simultaneously (Thomas 1975). Assay of free amino acid and soluble protein content The boll shells (0.8 g) were used for the extraction of amino acid and soluble protein according to Wei et al . (2016). Samples were stirred at 4°C in 3 mL of cold water (MilliQ reagent grade) and centrifuged at 800×g for 5 min. The supernatant was stored on ice. Then, the pellets were extracted twice, and the resulting supernatants were pooled for analysis. The absorbance was detected at 570 nm and the free amino acid content was expressed as g g −1 fresh weight (FW). The total soluble protein content was determined by the Coomassie blue dye-binding assay of Bradford (1976). First, 0.1 mL of extraction was pipetted into a test tube. Then 5 mL Coomassie brilliant blue G-250 solution was added to the test tubes before vortexing. The absorbance at 595 nm was measured. Bovine serum albumin was used to make a standard curve. Protease activity assay Protease was extracted and its activity was assayed according to Jessen et al . (1988). The boll samples (0.8 g) were homogenized at 4°C in 1 mL of β-mercaptoethanol extraction buffer (a mixture of ethylene glycol, sucrose, and phenylmethyl sulfonyl fluoride, pH=6.8). Cell debris was removed by centrifugation, and the supernatant was placed on ice and immediately used to estimate protease activity. Protease activity was determined spectrometrically at 400 nm using azocasein as a substrate (Vance et al . 1979) and expressed in mg pro g −1 FW h −1 . 2.3. Statistics analysis All samples were analyzed based on three replicates, and the data shown in Tables are their means. The statistical significance of differences between means was analyzed by analysis of variance using Proc ANOVA in SAS 6 (SAS institute, NC). Multiple mean comparisons were evaluated using the LSA test at P <0.05.