Arabidopsis thaliana ecotype Columbia-0 (wild type) was ordered from ABRC (Ohio State University) and the seeds were planted on agar plates containing Murashige and Skoog salts, 1% Suc (w/v), and 0.7% (w/v) agar, and adjusted to a pH of 5.7. After vernalization at 4°C for at least 2 d, seedlings were grown in an illuminated growth chamber at 23°C. Germinated seedlings were used for Agrobacterium-mediated transformation and the putative transgenic plants were then moved to pots containing soil of equal proportions of clay, sand, and peat moss (1:1:1). Finally, the plants were moved to a greenhouse and subjected to various molecular analyses and an insect bioassay.
Gossypium hirsutum (Xuzhou 142) seeds used in this study were acquired from the Institute of Cotton Research of the Chinese Academy of Agricultural Sciences (Anyang, China). The seeds were planted in containers of sand (one seedling per container) and grown under a 16-h light and 8-h dark cycle at 30°C in a climate-controlled greenhouse located at Shaanxi Normal University.
Synthesized ASGNA and transgenic plants
The original GNA sequence was obtained from National Center for Biotechnology Information (GenBank: M55556.1). The ASGNA gene was designed according to the dicot-bias of codon usage of the GNA gene. The 12 long fragments of ASGNA gene were synthesized by the Sangon Biotech company and ASGNA was obtained using the overlapping PCR method (Table S1) . Primers for ASGNA are listed in Table S1 in Supporting Information. Then the gene was cloned into the plant expression vector pART27. The constructs were introduced into Arabidopsis by the Agrobacterium tumefaciens floral-dip procedure , and transgenic plants were screened using 50 mg/mL kanamycin.
DNA isolation and PCR
The DNA was isolated using improved CTAB methods. Arabidopsis plant leaves (0.1 g) were ground into powder in liquid nitrogen. Then, 0.6 ml CTAB extraction buffer was added and the lysate was incubated at 65°C for 30 min. The DNA was purified by adding an equal volume of a mixture of chloroform: isoamyl alcohol (24:1) followed by centrifugation at 8000 × g for 10 min at 4°C. The supernatant was mixed with 0.6 volume of isopropanol and then subjected to centrifugation at 8000 × g. The precipitate was washed twice with 75% ethanol and then dissolved in 300 μl sterile water. Then we added NaAc (1/10 volume of 3 M, pH 5.2) and two volumes of ethanol to the dissolved precipitate and incubated each sample for 10 min at -20°C. The tube was centrifuged at 8000 × g for 5 min and the pellet was then washed twice with 75% ethanol and re-dissolved in sterile water.
The primer sequences used for this study are listed in Supplementary Table 1. The PCR thermal cycler program was as follows: 95°C for 3 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C elongation for 1 min. The PCR amplification products were electrophoresed on a 1% agarose gel and purified with the E.Z.N.ATM Gel Extraction Kit (OMEGA Bio-Tek, USA).
Determination of ASGNA expression levels in transgenic plants
Total protein was extracted from different fresh transgenic tissues (leaf, stem petal, root and silique). Ground leaves were put in 1.5 ml micro tubes with 400 μl of protein extraction buffer (150 mM NaCl, 5 mM MgCl2, 5 mM DTT, 0.1% NP40, 50 mM, Tris-HCl (pH 7.5), and EDTA-free complete protease inhibitor cocktail). The samples were vortexed to homogenize each one and incubated at 4°C for 2 hours and then centrifuged at 13,000 × g for 10 min. The supernatant was eluted and stored in new 1.5 ml tubes and Bradford reagent was used to quantify proteins. The expression of GNA protein was detected by an ELISA kit (Meuxuan biotechnology, Shanghai, China). In brief, 25 μl of either plant extract or purified GNA toxin standards (to obtain final concentrations from 5 to 60 ng of GNA sample in extract buffer) in ELISA plates coated with an anti-GNA antibody were incubated at 37°C for 2 h in an airtight container with 25 μl of alkaline phosphatase enzyme conjugate. Unbound protein was removed by washing with phosphate-buffered saline and 0.01% Tween 20 (v/v). Wells were again washed and then the assay was developed by the addition of 50 μl pnitrophenyl phosphate substrate solution and absorbances were read at 400 nm in a microtitre plate reader (Epoch, BioTek, Vermont). Levels of GNA were determined from the GNA calibration curve. Each sample was assayed in triplicate.
Aphid larvae and P. xylostella were collected from the Shaanxi Normal University field station (E, 108°93', N, 34°17', Shaanxi Province, Northwest China) in July and grown under laboratory conditions for a feeding bioassay. To investigate whether transgenic plants expressing ASGNA could confer enhanced resistance to P. xylostella, insect larvae either fed on control or transgenic plant tissue placed in petri-dishes, one larvae of each species per dish. To investigate aphid resistance of ASGNA plants, each whole plant was confined in an insect-proof fine-mesh nylon cage and 10 late-instar aphid nymphs were introduced with a hair brush to plant leaves of each plant on Day 0. Survival and growth rates of the insect populations were determined at 2-day intervals for a 14-day period. Cocoon proportion was calculated as follows: (number of larva with cocoon/number of total larva) × 100%. All the experiment was repeated three times (each independently derived transgenic line was micropropagated into three cloned plants).
Transient GNA expression in cotton
Cotyledon disks excised from 10–12-day-old cotton seedlings were used for the GNA transient expression analysis. Agrobacterium cultures were grown overnight at 28°C in LB medium containing the antibiotics 50 μg/ml kanamycin and 25 μg/ml gentamicin, as well as 10 mm MES and 20 μm acetosyringone. The cells were pelleted by centrifugation at 1500 g at room temperature for 5 min and re-suspended in infiltration culture containing 10 mm MgCl2, 10 mm MES and 200 μm acetosyringone. Cell suspensions were incubated at room temperature for at least 3 h. Agrobacterium cultures containing the GNA expression vector were infiltrated into two fully expanded cotyledons of 2-week-old plants using a needle-less syringe. To facilitate the infiltration, small holes were punched with a needle on the underside of the cotyledon. These experiments were repeated at least three times with more than six plants for each construct per repeat.