Comparative Evaluation of Transient Protein Expression Efficiency in Tissues across Soybean Varieties Using the Tsukuba System

Abstract

Transient protein expression is a versatile tool with diverse applications and can be used in soybeans to study gene function, obtain mutants, and produce proteins for commercial use. However, soybeans are considered recalcitrant for agroinfiltration. Subsequent studies on soybeans have demonstrated a green fluorescent protein (GFP) expression in seedpods, but not in leaves, using syringe agroinfiltration. To evaluate agroinfiltration-based transient protein expression levels in plant cells, we used the transient expression vector pTKB3 harboring the GFP gene. Using Agrobacterium tumefaciens, vacuum agroinfiltration of the leaves and needle agroinfiltration of the seedlings of different soybean varieties were performed. GFP was transiently expressed in all of the samples. However, the Enrei and Williams 82 varieties presented better results than the other varieties in the leaf tissue, with results confirmed by immunoblot analysis, demonstrating that both varieties are good candidates for molecular biological studies. GFP expression in the seedlings was less extensive than that in the leaves, which may be due to the tissue characteristics, with Enrei showing the best results. Based on this observation, we conclude that the Tsukuba system is an effective tool that can be used for different tissues and soybean varieties.

Keywords: GFP; Glycine max; agroinfiltration.

Figure 1

Green fluorescent protein (GFP) expression in plant leaves. Images depict GFP expression in leaves, showing the fluorescence captured by the filter (Fujifilm SC-52) and camera, followed by the detailed GFP expression observed through the microscope. The last image compares the filtered photos of the control samples, emphasizing the specificity of the fluorescence in relation to the experiments. The white bar in the filter picture indicates 1 cm in length. The bar in the microscope picture represents 0.1 cm in length.

Figure 2

Green fluorescent protein (GFP) expression in seedlings. Images depict the GFP expression in seedlings, including detailed micrographs obtained using a bright field microscope. The last image compares the photos of the control samples, emphasizing the specificity of the fluorescence in relation to the experiments. The white line represents 0.1 cm in length.

Figure 3

Green fluorescent protein (GFP) expression in seeds. Images depict GFP expression in seedlings, including detailed micrographs obtained using a bright field microscope. The first and second image demonstrate different tissues in soybean seeds. The last image compares the photos of the control samples, emphasizing the specificity of the fluorescence in relation to the experiments. The white line represents 0.1 cm in length.

Figure 4

Analysis of the green fluorescent protein (GFP) expression in the leaf samples of different soybean varieties. (A) Graphical representation of the relative fluorescence signal expression, highlighting the variations in the GFP signal intensity between the Enrei, Fukuyutaka, Williams 82, Doko, and Cristalina samples, and a comparison with control samples. Mean values ± SD for n = 3. Different lowercase means significant differences in the Tukey’s test (p < 0.05). (B) Western blot analysis of the soybean samples including a positive control (lettuce samples expressing pTKB3-eGFP plasmid) with an anti-GFP antibody and a marker for reference. All of the samples were diluted 40 times the initial concentration. (C) Gel stained with Coomassie Brilliant Blue (CBB) to compare the total amount of protein in the Enrei leaf samples and different bovine serum albumin (BSA) concentrations for comparison. Red arrow indicates the GFP protein size.

Figure 5

Agroinfiltration procedure. (A) Vacuum procedure for leaf infiltration involves positioning the plant upside down and with the leaves in contact with the infiltration buffer. (B) The syringe procedure for seedling infiltration includes removing one cotyledon and in the remaining meristem, after the needle is inserted in the stem and the Agrobacterium tumefaciens with infiltration buffer is pushed inside the seedling stem.