Development of Agrobacterium-mediated transient expression system in Caragana intermedia and characterization of CiDREB1C in stress response

Abstract

Background: The Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia.

Results: In this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment.

Conclusions: The optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia.

Keywords: Abiotic stress; Caragana intermedia; CiDREB1C; Transient expression.

Fig. 1

Phenotype of C. intermedia leaves after infiltrated with GV3101, EHA105, EHA101, LBA4404 and AGL1 1. Infiltration media + GV3101(pCambia1305.2) + 0.001% Silwet L-77 2. Infiltration media + EHA105(pCambia1305.2) + 0.001% Silwet L-77 3. Infiltration media + EHA101(pCambia1305.2) + 0.001% Silwet L-77 4. Infiltration media + LBA4404(pCambia1305.2) + 0.001% Silwet L-77 5. Infiltration media + AGL1(pCambia1305.2) + 0.001% Silwet L-77 bar = 1 cm

Fig. 2

GUS staining analysis of different surfactants, surfactant concentration and Agrobacterial strains in transiently expressed C. intermedia leaves a. 1. Infiltration media + GV3101 (pCambia1305.2) + 0.001% Silwet L-77; 2. Infiltration media + EHA105 (pCambia1305.2) + 0.001% Silwet L-77; 3. Infiltration media + EHA101 (pCambia1305.2) + 0.001% Silwet L-77; 4. Infiltration media + LBA4404(pCambia1305.2) + 0.001% Silwet L-77; 5. Infiltration media + AGL1(pCambia1305.2) + 0.001% Silwet L-77 bar = 1 cm. b. 1. Infiltration media + GV3101(negative control); 2. Infiltration media + GV3101 (pCambia1305.2); 3. Infiltration media + GV3101(pCambia1305.2) + 0.01% Silwet L-77; 4. Infiltration media + GV3101(pCambia1305.2) + 0.01% Tween-20; 5. Infiltration media + GV3101(pCambia1305.2) + 0.01% Trion X-100 bar = 1 cm. c. 1. Infiltration media + GV3101(negative control); 2. Infiltration media + GV3101(pCambia1305.2) + 0.001% Silwet L-77; 3. Infiltration media + GV3101(pCambia1305.2) + 0.005% Silwet L-77; 4. Infiltration media + GV3101(pCambia1305.2) + 0.01% Silwet L-77 bar = 1 cm

Fig. 3

Quantification analysis of the transiently expressed CiDREB1C in C. intermedia leaves by RT-PCR The CiDREB1C gene expression level in leaves of C. intermedia after infiltrating with pCanG-HA empty vector and CiDREB1C was detected by relative quantitative real-time PCR. The Expression values were calculated using the 2^-ΔΔCT method and CiEF1α was used as reference gene. The error bars represent the means of three technical replicates of each biological replicate ± SD. The experiments were performed two independent biological replicates with similar result. Statistical significance differences from control were determined by Student’s t test (**P < 0.01). The column was made by Graphpad prism 7. C: control

Fig. 4

Drought resistance detection of CiDREB1C transiently expressed C. intermedia seedlings a. Plain view of CiDREB1C transiently expressed C. intermedia seedlings grown in soil under drought treatment. Bar = 2 cm n = 16. b. The detached seedlings of both the control (up panel) and the CiDREB1C transgenic C. intermedia (lower panel) after re-watered for 8 days. Bar = 2 cm n = 16. c. The survival rate of both the control and the CiDREB1C transgenic C. intermedia seedlings after re-watered for 8 days. d. The chlorophyll content of both the control and the CiDREB1C transgenic C. intermedia seedlings after re-watered for 8 days. Days after infiltration was indicated in parentheses. Statistical significance differences from control were determined by Student’s t test (*P < 0.05). The column was made by Graphpad prism 7. C: control

Fig. 5

Salt resistance detection of CiDREB1C transiently expressed C. intermedia seedlings. a. Plain view of CiDREB1C transiently expressed C. intermedia seedlings grown in soil under salt treatment. Bar = 2 cm n = 16. b. The detached seedlings of both the control (up panel) and the CiDREB1C transgenic C. intermedia (lower panel) after 22 days of salt treatment. c. The survival rate of both the control and the CiDREB1C transgenic C. intermedia seedlings after 22 days of salt treatment. d. The chlorophyll content of both the control and the CiDREB1C transgenic C. intermedia seedlings after 22 days of salt treatment. Days after infiltration was indicated in parentheses. e. Seedlings growth rate of both the control and the CiDREB1C transgenic C. intermedia seedlings. The column was made by Graphpad prism 7. C: control

Fig. 6

ABA tolerance of CiDREB1C transiently expressed C. intermedia seedlings. a. Plain view of CiDREB1C transiently expressed C. intermedia seedlings grown in soil under ABA treatment. Bar = 2 cm n = 16. b. The survival rate of both the control and the CiDREB1C transgenic C. intermedia seedlings after ABA treatment for 10 days. c. The total chlorophyll content of both the control and the CiDREB1C transgenic C. intermedia seedlings after ABA treatment for 10 days. d. The leaf drop rate of both the control and the CiDREB1C transgenic C. intermedia seedlings after ABA treatment for 10 days. Days after infiltration was indicated in parentheses. Statistical significance differences from control were determined by Student’s t test (**P < 0.01). The column was made by Graphpad prism 7. C: control