Insects.
Neonates of Asian corn borer (Ostrinia furnacalis)and Rice stem borer (Chilo suppressalis) used in this study were obtained from the General Pest Company (Beijing, China) and were kept in a climate-controlled chamber at 26±h℃, 70± 7mber at 2s study were obtained from the General6:8 (L: D) h at Agro-biotechnology Research Institute, Jilin Academy of Agricultural Sciences, China.
Design and synthesis of novel Cry proteins.
The different Cry protein sequences, Cry1Ab1 (https://www.ncbi.nlm.nih.gov/protein/AEV45790.1), Cry1Ab5 (https://www.ncbi.nlm.nih.gov/protein/CAA28405.1), Cry1Ab14 (https://www.ncbi.nlm.nih.gov/protein/AAG16877), Cry1Ac (https://www.ncbi.nlm.nih.gov/protein/WP_000369821.1), Cry1Ah (https://www.ncbi.nlm.nih.gov/protein/AAQ14326.1), Cry1Ba (https://www.ncbi.nlm.nih.gov/protein/WP_000203376.1/), Cry1Be (https://www.ncbi.nlm.nih.gov/protein/O85805.1), Cry1Bb (https://www.ncbi.nlm.nih.gov/protein/WP_000203375.1), Cry1Ia (https://www.ncbi.nlm.nih.gov/protein/CAA44633.1), Cry1Fa (https://www.ncbi.nlm.nih.gov/protein/Q03746.1), Cry1Ja (https://www.ncbi.nlm.nih.gov/protein/WP_098369107.1), Cry1Jb (https://www.ncbi.nlm.nih.gov/protein/Q45716.1), Cry9Aa (https://www.ncbi.nlm.nih.gov/protein/WP_087997148.1), Cry9Ca (https://www.ncbi.nlm.nih.gov/protein/Q45733.1), Cry9Ec (https://www.ncbi.nlm.nih.gov/protein/AAS68357.1), Cry2Ab (https://www.ncbi.nlm.nih.gov/protein/WP_001089638.1), Cry2Ae (https://www.ncbi.nlm.nih.gov/protein/ABW49930.1), Cry2Ac (https://www.ncbi.nlm.nih.gov/protein/Q45743.1), Cry1Gc[35]were obtained from the National Center for Biotechnology Information (NCBI) database, and the genetic relationships were analyzed using DNAMAN software (Version 6). Construct domain mutants were created by swapping domain III of seven distantly related proteins for domain III of Cry1Ab protein. The selected new proteins were synthesized and expressed in Escherichia coli by Sheng Gong Company (China).
Dietary exposure screen.
E. coli clones that express seven Cry proteins, Cry1Ab-1Gc, Cry1Ab-1Ia, Cry1Ab-1Ac, Cry1Ab-9Ca, Cry1Ab-1Ba, Cry1Ab-2Ae, and Cry1Ab-1Jb were grown overnight. The expression vector pET28a without Cry protein was used as the negative control. 5ml overnight culture was sonicated and the amount of protein to be tested. Stock solutions of the seven Cry proteins were diluted with distilled water and incorporated into an artificial diet for Asian corn borer to the same concentration, 100μg/ml. Since the Asian corn borer diet must be heated during preparation, the Cry protein solutions were mixed into the diet when its temperature had decreased to <60°C to avoid degradation. Once the food was solid, it was cut into slices and individually placed in 10 cm diameter Petri dishes. Ten neonates of Asian corn borer were transferred into each Petri dish, which was then sealed with breathable film to prevent the larvae from escaping. The feeding assay was conducted in a growth chamber at 28°C, 80% relative humidity, and a 12 h photoperiod. Three replicates were used for each protein. Mortality was recorded after 5 days.
Construction of over-expression vector and genetic transformation.
Cry1Ab-1Gc, cry1Ab-1Ac, cry1Ab-1Ia were expressed in Escherichia coli, and the synthetic cry gene was designed as outlined through the codon usage of rice[48]. The pTF101.1-ubi vector and the plasmid containing cry genes were digested with SmaI and SacI, and two fragments were connected to the vector by T4 DNA ligase (Thermo Fisher Scientific, USA).
To test the toxicity of the new protein against insects in monocot crops, cry1Ab/Gc, cry1Ab/Ac,cry1Ab/Ia were transformed into rice (cv. Jijing 88) using Agrobacterium-mediated genetic transformation with the above vector, respectively. The same method was used to transform cry1Ab/Gc into maize (cv. HiII).
Enzyme-linked immunosorbent assay (ELISA).
The expression of Cry1Ab/Gc protein in transgenic plants was quantitatively analyzed by Enzyme-linked immunosorbent assay (ELISA) using the Cry1Ab/Cry1Ac plate kit (Agida, USA) according to its instructions. Leaves of transgenic rice at the tillering stage were ground into powder using liquid nitrogen, and 0.02 g powder was weighed into 1.5 ml centrifuge tubes with 500 μl extraction buffer. Samples were diluted 400-fold after mixing, and 50 μl of each sample was used for detection. The non-transgenic rice (cv. Jijing 88) was used as a negative control. The standard curve was constructed using a concentration gradient (0.02, 0.016, 0.012, 0.008, 0.004, and 0 ng) for the quantitative analysis of samples. We selected one transgenic line (C-3) with high Cry1Ab/Gc protein levels for insect bioassay. The same method was used to confirm the presence of the transgene in maize.
Evaluation of insect resistance of transgenic rice.
The insect bioassay was conducted using the test tube method in the laboratory. Rice stems of these three transgenic rice with cry1Ab/Gc, cry1Ab/Ac and cry1Ab/Ia at tillering stage were cut into 6-cm sections. Five stem sections were each placed into separate test tubes, filled with 1% agar to a depth of 2 cm. Ten rice stem borer larvae were released into each test tube with 5 repetitions. Test tubes were sealed with cotton balls to prevent the larvae from escaping. The feeding assay was conducted in a growth chamber at 28°C, 80% relative humidity, and 12-h photoperiod. Damage to stem tissues and larval mortality were observed and analyzed by t-test after infestated for five days. Non-transgenic plants were used as a negative control. All data were analyzed by t-test (*P<0.05; **P< 0.01).
Transgenic rice event C-3 with cry1Ab/Gc gene was planted in a greenhouse with normal field treatment and surrounded with wild-type Jijing88 as a negative control. No pesticide was applied during the growth period. Twenty neonates of rice stem borer were placed into the hearts of tillers of every rice plant at tillering stage. Insect occupancy was observed every 12 days. Rice tiller performance and the number of tillers damaged were compared with the controlled rice by t-test (*P<0.05; **P< 0.01).
Evaluation of insect resistance of transgenic maize.
Cry1Ab/Gc was transformed into maize (cv. HiII) using Agrobacterium-mediated genetic transformation with pTF101.1-ubi-cry1Ab/Gc vector. Twenty neonates of Asian corn borer were placed into the heart leaf of each transgenic plant and the control plant at the silking stage in a function identification pool. The insect occupancy was observed after 14 days. Agronomic performance, length of the tunnels produced by larvae, and the number of survival larvae were observed and analyzed by t-test (*P<0.05; **P< 0.01).
The best insect resistance performance line was bred by backcross breeding. To the further, Cry1Ab/Gc-expressing maize inbred lines cv. Ji853 and Y822 were assessed by the same method above. Agronomic performance and length of the tunnels were compared with the control by t-test (*P<0.05; **P< 0.01).
Hybrid yield test and statistical analyses
Xiangyu 998 is a commercial hybrid that uses Y822 as the female parent and X923-1 as the male parent. We crossed the Y822 transgenic inbred line with X923-1 to obtain Xiangyu998 transgenic hybrid combinations. We select three lines of transgenic hybrids xiangyu-1(+),xiangyu-2(+),xiangyu-3(+), and the corresponding negative materials, xiangyu-1(-),xiangyu-2(-),xiangyu-3(-). Each material plant 10 rows, each row plant 20 plants under natural rainfall conditions. All material plants were open pollination, and collected at maturity period. Measure the weight of each row corn ears, record the number of ears in each row, calculate the weight of each ear, and perform statistical analysis by t-test.
Biodiversity investigation and statistical analyses
Cry1Ab/Gc maize and the non-BT maize (CK) were grown in the GM fields of Jilin Academy of Agricultural Sciences located in Jilin province (2020; China; E125°, N44°). Cry1Ab/Gc maize was divided into two groups, spray herbicide glufosinate (F-P) and none glufosinate application (F-BP). The number of arthropods was investigated and analyzed by direct observation and pitfall traps method[46]. Six species of arthropods (Harmonia axyridis, ladybird larvae, aphids, Monolepta hieroglyphica, spiders, and Propylaea japonica) were counted and analyzed by the method of direct observation; Four species of arthropods (Teleogryllus infernalis, Earwig furficulidae, Opiliones, and Carabidae sp.4) were counted and analyazed by the method of pitfall traps. Three indices were used to analyze the dynamics of the arthropod community: Shannon index[49], Pielou index [50], and Simpson index[51].