CaAMP1 synthesis and vector construction
The nucleotide sequence of CaAMP1 (GenBank ID: AAT35532.1) from C. annuum was sythesised added Xba I and Sac I recognition sites at the 5′ and 3′ ends, respectively (Sangon Biotech, Shanghai, China) for the construction of overexpression vector. The modified CaAMP1 gene was inserted into a pCambia3300 vector containing a modified CaMV 35S promoter [48](GenBank: GI3319906) to drive the constitutive expression of CaAMP1 in soybean. The gene sequence was PCR amplified using the primer pair CaAMP1-F/R at a final primer concentration of 0.4 μM (the primers used were listed in Table S1) under the following conditions: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 7 min. The purified fragment was then subcloned into a pCambia 3300 plasmid containing a phosphinothricin acetyl transferase (bar) resistance gene (encoding phosphinothricin N-acetyltransferase, PAT) as a plant selection marker driven by a modified CaMV 35S promoter [48](GenBank: GI3319906). The constructed pCambia3300-CaMV 35S-CaAMP1 plasmid was subsequently transformed into competent cells of Agrobacterium tumefaciens strain EHA101 through the freeze-thaw method [49, 50].
Regeneration and screening of transgenic plants
The Agrobacterium-mediated transformation method was used for the regeneration of transgenic soybean with the soybean cultivar Williams 82 as the recipient, which was kindly provided by Prof. Fudi Xie from Shenyang Agricultural University in China (ID:WDD00587, Chinese Crop Germplasm Information System, http://www.cgris.net). The details were described by Yang et al. (2018) and Zhang et al. (2014)[51, 52]. The PAT-tolerant regenerated plantlets were screened using the LibertyLink® strip test (cat #AS 013 LS; EnviroLogix Inc., Portland, ME, USA) and by PCR test. DNA was extracted from the leaves of transgenic and wild-type soybean using a simple homogenization and ethanol precipitation method for PCR test [53]. PCR was performed (the primers were listed in Table S1; CaAMP1-F1/R1, bar-F/R) with final primer concentrations of 0.2 μM under the conditions: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 10 min, in order to confirm the presence of CaAMP1 and bar. When the first trifoliate leaves of transformed plants fully expanded, leaf-spraying assay was performed to screen herbicide-tolerant T1–T3 transgenic lines with 500 mg∙L-1 glufosinate (Envirologix Inc., Portland, Maine, USA). Herbicide-tolerant lines were analysed by PCR using the primers (CaAMP1-F1/R1, bar-F/R) until we obtained the homozygous transgenic plants.
In order to confirm the intergration of T-DNA in transgenic soybean, T2 transgenic plants were selected for genomic DNA extraction with a modified high salt CTAB method (cetyl-trimethyl ammonium bromide) method [54]. The DIG High Prime DNA Labeling and Detection Starter Kit I (Labeling and Detection Starter Kit I, 11745832910; Roche Applied Science, Indianapolis, IN, USA) was used in Souther blot anslysis in accordance with the manufacturer’s instructions. Approximately 30 μg of the genomic DNA from transgenic soybean and control plant was digested completely with EcoR I (New England Biolabs Inc., Beverly, Massachusetts). And then the digested DNA was transferred onto positively charged nylon membranes (GE Amersham, RPN303B, USA). Hybridization was carried out at 42°C for 12–16 h using CaAMP1 labeled with digoxigenin-(DIG)11-dUTP as a probe. The washing conditions and signal detection were performed as described previously by Yang et al. (2018)[51].
Expression analysis of transgenic soybean
The RNA and protein were extracted for expression analysis. The 2-week-old leaves of T2 transgenic plants were collected individually for total RNA isolation. The RNA extraction was performed using an EasyPure PlantRNA Kit (TransGen Biotech, Beijing, China) and the elimination of genomic DNA contamination used DNase I. cDNA was then synthesized using the ThermoScript RT-PCR system (Invitrogen, USA) and the RT-PCR performed with the primers CaAMP-RF/RR (listed in Table S1; CaAMP-RF/RR). The GmActin (GeneBank ID: NM 001289231) was used as a control whcih amplified with the primers 5′-CACCGGAGTTTTCACCGATA-3′ and 5′-AGGAATGATGTTAA-3′.
Crude protein of the transgenic soybean and control was extracted from ~100 mg fresh leaves of T3 transgenic soybean, separated on a 12% (w/v) SDS-PAGE gel, and transferred electrophoretically onto a PVDF membrane (AmershamTM HybondTM, GE Healthcare, USA)[41]. After blocking with 3% dried skimmed milk diluted in PBST (1× PBS, 0.1% Tween-20), the membrane was blotted with a rabbit polyclonal antibody (1:500 dilution), which was raised against recombinant CaAMP1 protein (GenScript Co., Ltd. Nanjing, China), and horseradish peroxidase (HRP)-labeled goat-anti-rabbit IgG (1:5000 dilution, Abcam, UK) at 25°C for 4 h. The bands of Weatern blot were visualized using BiodlightTM Western Chemiluminescent HRP substrate (Bioworld Technology, Inc., St. Louis, MN, USA) after extensicve washing.
Evaluation of tolerance to PRR under greenhouse conditions
In order to evaluate the tolerance to P. sojae race 1 of trangenic soybean, three consecutive generations of transgenic lines, that was T2-T4, were selected to be infected with P. sojae race 1 according to method discribed by Schmitthenner et al. (1994)[55]. P. sojae race 1's isolation and cultivation were referenced to Akamatsu et al. 2010 and Du et al. 2018 [56, 28]. Transgenic soybean, wild-type Williams 82, and PRR-sensitive cultivar Jiunong 21 (ID:ZDD22796) which was cultivated and provided by Soybean Research Institude of Jilin Academy of Agricultural Sciences, were grown in a greenhouse, and the hypocotyls of 15-day-old seedlings were inoculated with macerated mycelia of P. sojae. After that, the plants were maintained in a humid environment for 15–24 h, and subsequently transferred back to the greenhouse for symptom development at 25°C under an 18 h light/6 h dark condition [28]. After 5 to 10 days inoculation, data were collected for plant infection and mortality rates [57]. All of the experiments were performed three replicates with 20 inoculated plants for each replicate.
A t-test at a significance level P = 0.05 or 0.01 via the Microsoft Analysis Tool was used for quantitative analysis in order to compare with the dofferences in the survival rate of each transgenic line and control.
Quantitative RT-PCR analysis of the disease-responsive genes
The leaves from the T3 transgenic and wild-type plants were collected for quantitative PCR at 0, 1, 2, 4, 8, 12, and 24 h after inoculation with P. sojae mycelia. Total RNA extraction and cDNA synthesis were prepared as described in the section Expression analysis of transgenic soybean. 12 genes which involved in multiple responding pathway to stress were selected for qRT-PCR to analyze their relative expression levels,which were PR1 (AF136636), PR2 (M37753), PR3 (AF202731), PR5 (BU765509), PR12 (BU964598), PAL (X52953), PPO (EF158428), AOS (DQ288260), SGT1 (NM_001249656), GmNPR1-1 (FJ418594), GmNPR1-2 (FJ418596), and GmRAR1 (FJ222386), with GmACT (U60500) as the internal control. Amplification was performed using a SYBR Green-based One-Step qRT-PCR kit (TransGen Biotech, China) in a final reaction volume of 20 µL with ~80 ng cDNA, and 0.4 µL each of forward and reverse primers (the primers were listed in Table S1). The qRT reaction was under the condition: 50°C for 2 min; 95°C for 10 min; and 45 cycles of 95°C for 2 min, 62°C for 30 s, and 72°C for 30 s. Relative quantitative expression was determined using The 2-ΔΔCt method was used to determined the relative expression levels of each gene [58]. To get accurate data, three biological and three technical replicates were performed for each expremient.
Agronomic traits of the transgenic lines
Seven agronomic traits of T3 transgenic lines and wild type soybean were selected for identification of transgenic soybean, including plant height, branch number, node number, pod number, and seed numbers, total seed weight, and 100-seed weight. And the t-test (mentioned above) was also used for quantitative analysis.