Plant material and experimental design
Field studies were conducted at Yangzhou University Farm, Jiangsu Province, China (32 ° 30 ′ N, 119 ° 25 ′ E) in 2017–2018. Bt transgenic cultivar Sikang3 (hybrid) was used as experimental material. The soil (sandy loam) contained about 19.8 g·kg− 1 organic matter, 105.0, 26.0, and 85.0 mg·kg− 1 available N-P-K in the studied duration, respectively. Other cultivation practices, including insecticides application, DPC (1,1-dimethyl piperidinium chloride, C7H16ClN) spraying, and irrigation, were conducted according to local cultivation practices. Each plot consisted of 6 rows, the plot size was 6 m in length with 5.4 m in width, row space was 0.90 m apart. Seeds were sown on April 15th (2017) and April 16th (2018) in a greenhouse. Seedlings were transplanted to the field on May 18th (2017) and May 19th (2018).
In 2017 and 2018, a randomized complete block design with three replications was conducted. Five plant densities (D1–D5: 15 000, 25 000, 45 000, 60 000, and 75 000 plants per hectare) were imposed on cotton plants.
In 2018, a flower-removal study was carried out with three treatments. A quarter (1/4) and a half (1/2), and none (CK) of white flowers were removed during the peak flowering stage, and the untreated plants were used as control (CK). The experiment was arranged according to a randomized complete block design with three replications. The planting density was 30 000 plants per hectare.
Preparation of plant material
Five bolls at 10 days after flowering were sampled and frozen with liquid nitrogen, stored in − 80 °C, and used for the measurements of Cry1Ac protein content, chemicals, and enzyme activities for protein metabolism in the boll wall.
Boll setting rate
The boll number was recorded on Jul 31th and Aug 10th (peak boll stage) for 20 plants in the two central rows in each plot, and the boll setting rate per plant was calculated by the difference of boll number within the two recording dates divided by the days.
The CryIAc protein concentration assay
The CryIAc protein concentrations of the boll wall for 10-day boll after anthesis were determined by immunological analysis ELISA (enzyme-linked immunosorbent assay) (Chen et al. 1997). Three subsamples of 0.5 g shell were prepared by homogenizing the frozen tissue in a 2 mL extraction buffer (Na2CO3 1.33 g, DTT 0.192 g, NaCl 1.461 g, Vitamin C 0.5 g dissolved in 250 mL distilled water), then transferred to a 10 mL centrifugation tube, the residue remaining on the wall of the mortar was washed again with 3 mL of the extraction buffer and this was also added to the centrifugation tube. The contents of this tube were shaken with hand and stored at 4 °C for 4 h. The supernatants were collected after centrifugation at 10 000×g at 4 °C for 20 min, passed through a C18 Sep-Pak Cartridge (Waters, Milford, MA), and three subsamples were pooled for CryIAc quantification. Quantification of the CryIAc in the combined samples was conducted using a commercially available kit (Scientific Service, Inc. China Agriculture University, Beijing). Microtitration plates were coated with the standard CryIAc insecticidal proteins and samples, incubated at 37 °C for 4 h. The antibodies were added to each well and incubated for another 30 min at 37 °C. The antibodies against the CryIAc insecticidal protein were obtained as described by Weiler and Conrad (1981). Then horseradish peroxidase-labeled goat anti-rabbit immunoglobulin was added to each well and incubated for 30 min at 37 °C. Finally, the enzyme-substrate buffer (orthopenylenediamino) was added, and the enzyme reaction was carried out in the dark at 37 °C for 15 min, then terminated using 3 mol·L−1 H2SO4. The absorbance was recorded at 490 nm. Calculation of the ELISA data was performed as described by Weiler and Conrad (1981).
Assay of free amino acid and soluble protein content
The boll wall samples (0.5 g) from different treatments were used for the extraction and analysis of amino acid concentration and soluble protein content. The sample was homogenized at 4 °C in 5 mL cold water (Milli-Q reagent grade) and centrifuged at 800×g for 5 min. The supernatant was stored on ice, and the pellet was resuspended in 3 mL cold water prior to re-centrifugation (800×g) for another 5 min, The supernatant from both centrifugations were pooled and stored on ice, the pellet was re-suspended in 2 mL cold water, and centrifuged at 800×g again. The supernatant was pooled for analysis. The total free amino acid content was determined by ninhydrin assay (Yemm et al. 1955). The absorbance readings (in amino acid, fresh weight (FW)) were converted to μg·g− 1 using the glycine standard curve. The total soluble protein content was determined by the Coomassie Blue dye-binding assay of Bradford (1976). The absorbance readings were converted to protein concentration using BSA standard curve.
Glutamic-pyruvic transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) assay
The boll wall samples of the different treatments were used as an analysis of GPT and GOT activities. The samples (0.5 g) were homogenized in a buffered medium (0.05 mmol · L − 1 Tris-HCl, pH 7.2), and the homogenate was centrifuged at 26 100×g for 10 min at 0 °C. The supernatant was analyzed for GOT activity. A mixture of 0.5 mL of a 0.8 mol·L−1 alanine in 0.1 mol·L Tris-HCl (pH 7.5) together with 0.1 mL of 2 mmol·L− 1 pyridoxal phosphate solution was used, and to this mixture 0.2 mL of 0.1 mol · L − 1 2-oxoglutarate solution and 0.2 mL the prepared enzyme were added. The reaction mixture was incubated at 37 °C for 10 min followed by termination of reaction with 0.1 mL of 0.2 mol · L − 1 trichloroacetic acid solution, then the pyruvate with chromogen was converted to pyruvate hydrazone. The color intensity of the hydrazone in saturated water toluene was measured at 520 nm. The GOT activity, in terms of pyruvate production, was calculated from authentic pyruvate standards simultaneously. The procedure used for assaying the activity of GPT was identical to the GOT assay, except that in the GPT assay, 0.5 mL of 0.8 mol alanine in 0.1 mol·L− 1 Tris-HCl (pH 7.5) were substituted for 0.5 mL of 0.1 mol·L−1 buffered aspartate solution in the reaction mixture and aniline citrate addition was omitted (Tonhazy et al. 1950).
Assay of protease and peptidase activity
The boll wall samples were also used as an analysis of protease and peptidase activities. The samples (0.8 g) were homogenized at 4 °C in 1 mL of β-mercaptoethanol extraction buffer (a mixture of ethylene glycol, sucrose, and phenylmethylsulfonyl fluoride, pH 6.8). Cell debris was removed by centrifugation, and the supernatant was placed on ice and immediately used to estimate the square protease. Protease activity was determined using azocasein as substrate (Vance and Johnson 1979) and expressed as the change of absorbance (400 nm) in mg (protein) ·g− 1 (fresh weight).
The boll wall samples (0.5 g) were homogenized at 4 °C in 8 mL of Tris-HCl extraction buffer (a mixture of 4 mmol·L− 1DTT, 4 mmol·L− 1 EDTA, 1% pH 7.5), and then centrifuged at 15 000 ×g for 30 min at 0 °C. The supernatant was used to estimate the peptidase activity (Vermon 1979). A mixture of 0.4 mL acetate buffer (pH 4.8), 1% bovine hemoglobin compounded with 0.2 mL acetate buffer (pH 4.8) was incubated at 37 °C for 10 min. Then 0.4 mL of the enzyme sample was added to the mixture and was incubated at 38 °C for 60 min followed by termination of reaction with 1 mL of 10% trichloroacetic acid solution. For the control treatment, the termination solution was added before the reaction. The solution was then centrifuged at 4 000×g for 5 min again after incubation at 4 °C for 30 min. The supernatant was pooled for analysis of the amino acid content which was determined by ninhydrin assay (Yemm et al. 1955), and expressed as the change in absorbance ‘μmol (amino acid) ·g− 1 (fresh weight)’.
Analysis of variance (ANOVA) was conducted for insecticidal content etc. using Proc ANOVA in SAS (SAS Institute 1989). The differences between treatments were tested for significance using LSD (P < 0.05). The correlation study was computed as the Pearson correlation coefficient.