Use of glk genes to improve temperature tolerance in plants

Abstract

The application provides application of a GLK (Golden2-like transcription factor) gene in improving temperature tolerance of plants, and a method for improving temperature tolerance of plants, which comprises the step of overexpressing the GLK gene in the plants. The application discloses a new method for improving temperature tolerance of plants, by which the plants can survive and grow in a temperature range exceeding the original adaptive temperature range, and provides a new way for molecular breeding and genetic modification of plants.

Description

Technical Field

[0001] This invention relates to the study of plant temperature tolerance, and more particularly to the application of the GLK gene in improving plant temperature tolerance. Background Technology

[0002] Temperature is one of the most significant variables in a plant's growth environment. Because outdoor temperatures fluctuate greatly and are difficult to predict, plants growing in their natural environment can be threatened by extremely low or high temperatures. For example, when plants encounter unpredictable extreme temperatures, or are planted in environments exceeding their temperature tolerance range, their growth can be severely impacted, potentially even leading to death.

[0003] Golden2-like transcription factors (GLKs) are a class of transcription factors related to chloroplast development, belonging to the GARP family, and are widely found in higher plants. Current research shows that GLKs are closely related to plant growth and development, mainly participating in life processes such as cell differentiation, chloroplast development, plant responses to stress, and leaf senescence. To date, no research has shown a link between GLK genes and plant temperature tolerance. Summary of the Invention

[0004] In our study, we found that the GLK gene exists in two forms in Arabidopsis thaliana: AtGLK1 (SEQ ID NO:1) and AtGLK2, and in maize: ZmGLK1 (SEQ ID NO:2) and ZmG2 (SEQ ID NO:3).

[0005] In our research on Arabidopsis thaliana, we found that knocking out the GLK gene in Arabidopsis makes it more sensitive to both low and high temperatures, while overexpressing the GLK gene improves its tolerance to low temperatures and significantly enhances its tolerance to high temperatures. Overexpression of the maize-derived GLK gene in rice also showed an improvement in low-temperature tolerance.

[0006] Based on the above findings, this invention provides the application of the GLK (Golden2-like transcription factor) gene in improving the temperature tolerance of plants.

[0007] In one specific implementation, the temperature tolerance is either high temperature tolerance or low temperature tolerance.

[0008] In one specific implementation, the GLK gene is a gene derived from Arabidopsis thaliana or a gene derived from maize.

[0009] In one specific embodiment, the sequence of the GLK gene is selected from SEQ ID NO:1-3.

[0010] The present invention also provides a method for improving the temperature tolerance of plants, including the step of overexpressing the GLK gene in the plant.

[0011] In one specific implementation, the temperature tolerance is either high temperature tolerance or low temperature tolerance.

[0012] In one specific implementation, the GLK gene is a gene derived from Arabidopsis thaliana or a gene derived from maize.

[0013] In one specific embodiment, the sequence of the GLK gene is selected from SEQ ID NO:1-3.

[0014] In one specific implementation, the plant is Arabidopsis thaliana or rice.

[0015] This invention discloses a novel method for improving the temperature tolerance of plants, enabling them to survive and grow beyond their original temperature range of adaptation, thus providing a new approach for molecular breeding and genetic modification of plants. Attached Figure Description

[0016] Figure 1 Photos of Arabidopsis thaliana after 10 days of normal growth in a low-temperature experiment, and photos of Arabidopsis thaliana after being treated at -10℃ for 2 hours.

[0017] Figure 2 This is a statistical chart showing the survival rate of various Arabidopsis thaliana lines after being treated at -10℃ for 2 hours in a low-temperature experiment.

[0018] Figure 3 This is a photograph of rice that has been treated in a 4℃ incubator for 96 hours during a low-temperature experiment.

[0019] Figure 4 This is a statistical chart showing the survival rate of various rice lines after being treated in a 4℃ incubator for 96 hours in a low-temperature experiment.

[0020] Figure 5 The left image shows Arabidopsis thaliana after 12 days of normal growth in a high-temperature experiment, and the right image shows Arabidopsis thaliana after 5 days of normal growth, treated at 37℃ for 1.5 hours, recovered for 2 days, and then treated at 44℃ for 1.5 hours, recovered for 5 days.

[0021] Figure 6 This is a statistical chart showing the survival rate of various Arabidopsis thaliana strains during high-temperature experiments. Detailed Implementation

[0022] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] 1. Adversity management

[0024] 1.1 Low-temperature treatment of Arabidopsis thaliana

[0025] We used a non-acclimated (NA) method for low-temperature treatment and cultivation of Arabidopsis thaliana. The NA treatment was as follows: Arabidopsis seeds were inoculated onto MS medium and cultured under a 16 / 8 photoperiod (16 hours light / 8 hours dark cycle) at 22°C and a light intensity of 80 μmol / m². -2 .s -1 After culturing at 60% humidity for 10 days, the samples were directly transferred to a low-temperature incubator for low-temperature treatment.

[0026] After low-temperature treatment, the sample was first placed in a 4°C incubator in the dark for 12 hours, and then transferred to a photoperiod of 16 / 8, a temperature of 22°C, and a light intensity of 80 μmol / m. -2 .s -1 After the humidity was restored to 60% for three days, photos were taken to count the survival rate.

[0027] 1.2 Low-temperature treatment of rice

[0028] We used a non-acclimated (NA) method for low-temperature treatment and cultivation of rice. The NA treatment was as follows: seeds were first soaked to promote germination, then planted in a mixture of nutrient soil and vermiculite (8:1) for growth. The growth conditions were a photoperiod of 12 / 12, a temperature of 28℃ / 20℃, and a light intensity of 200 μmol / m. -2 .s -1 The humidity was 60%. When the plants reached the early four-leaf stage after two weeks of growth, they were transferred to a 4℃ incubator for 96 hours. Then they were transferred to normal conditions for recovery. During the recovery process, the phenotype and survival rate were observed.

[0029] 1.3 High-temperature treatment of Arabidopsis thaliana

[0030] We used a gradient high-temperature treatment method. The gradient high-temperature treatment was as follows: Arabidopsis seeds were inoculated on normal MS medium, with a photoperiod of 16 / 8, a temperature of 22℃, and a light intensity of 80 μmol / m. -2 .s -1 The organisms were grown at 60% humidity for 5 days, treated at 37℃ for 1.5 hours, and then grown under a photoperiod of 16 / 8, a temperature of 22℃, and a light intensity of 80 μmol / m. -2 .s -1 After two days of recovery at 60% humidity, the sample was treated at 44℃ for 1.5 hours, with a photoperiod of 16 / 8, a temperature of 22℃, and a light intensity of 80 μmol / m. -2 .s -1 Photos were taken after 5 days of growth at 60% humidity to determine the survival rate.

[0031] 2. Construction of Arabidopsis or rice mutants

[0032] Wild-type Arabidopsis thaliana was used as the starting plant. The GLK1 knockout mutant (glk1) and the GLK1 and GLK2 co-knockout mutant (glk1 / 2) were provided by Sichuan University. The Arabidopsis thaliana overexpression lines were constructed using the pCAMBIA1300 vector, started by 35S.

[0033] The method is as follows: The GLK1 overexpression line was obtained by constructing a 35S-initiated pCAMBIA1300 overexpression vector based on the CDS gene sequence of GLK1, and then obtaining the GLK1 overexpression line through Agrobacterium infection.

[0034] The rice overexpression lines were constructed by using the CDS sequences of maize homologous genes ZmGLK1 and ZmG2 to construct the pVec8-Gateway vector initiated by ZmUBI, and then transformed into the rice variety Oryza sativa spp.japonicacv.Kitaake via Agrobacterium tumefaciens. The resulting transgenic rice lines constitutively expressing the maize GLK1 or G2 genes were obtained. The seeds were provided by the Chinese Academy of Agricultural Sciences.

[0035] 3. Phenotypic statistics of Arabidopsis thaliana or rice and their mutants under different treatment conditions

[0036] 3.1 Statistical analysis of the results of low-temperature treatment in Arabidopsis thaliana

[0037] The low-temperature experiment used the NA treatment method. We divided the culture box into a grid consisting of four large squares. Starting from the top left, we inoculated wild type (col), GLK1 knockout mutant (glk1), GLK1 and GLK2 co-knockout mutant (glk1 / 2), and GLK1 overexpression (glk1OE) in a clockwise direction.

[0038] Low-temperature experiment: Arabidopsis thaliana was cultured for 10 days under a 16 / 8 photoperiod before low-temperature treatment, including a 2-hour treatment at -10℃. Photos of the resulting Arabidopsis thaliana seedlings are shown below. Figure 1 As shown. Statistical survival rate results are as follows. Figure 2 As shown in the figure. The results showed that the survival rate of Arabidopsis thaliana with GLK1 gene knockdown was significantly reduced after low temperature treatment, while the survival rate of Arabidopsis thaliana with GLK1 gene overexpression was significantly higher than that of wild type (col) after low temperature treatment. This indicates that in Arabidopsis thaliana, downregulation of GLK1 gene expression seriously affects its tolerance to low temperature, while overexpression of GLK1 gene can improve the low temperature resistance of Arabidopsis thaliana plants.

[0039] 3.2 Statistical analysis of low-temperature treatment results in rice

[0040] The rice low-temperature experiment used NA treatment. Before treatment, the order from left to right was wild type (WT), maize G2 gene overexpression line (ZmUBIpro:ZmG2), maize GLK1 gene overexpression line (ZmUBIpro:ZmGLK1). After treatment, the order from left to right was maize GLK1 overexpression line (ZmUBIpro:ZmGLK1), maize G2 overexpression line (ZmUBIpro:ZmG2), wild type (WT).

[0041] Low-temperature experiment: Before low-temperature treatment, rice was grown under conditions of a photoperiod of 12 / 12, a temperature of 28℃ / 20℃, and a light intensity of 200 μmol / m. -2 .s -1 Photos of rice seedlings grown for 14 days under 60% humidity conditions, including 96 hours of treatment at 4℃, followed by 6 days of growth under normal light and then low-temperature treatment, are shown below. Figure 3 As shown, the survival rate results after statistical processing are as follows: Figure 4 As shown in the figure, the results indicated that overexpression of both maize-derived GLK genes in rice plants significantly improved their survival rate after low-temperature treatment. This suggests that overexpression of the maize ZmG2 and ZmGLK1 genes in rice can enhance the low-temperature resistance of rice plants.

[0042] 3.3 Statistical analysis of the results of high-temperature treatment of Arabidopsis thaliana

[0043] The high-temperature experiment used a gradient high-temperature treatment method. We divided the culture box into a grid consisting of four large squares. Starting from the top left, we inoculated the GLK1 knockout mutant (glk1), wild type (col), GLK1 and GLK2 co-knockout mutant (glk1 / 2), and GLK1 overexpression strain (glk1OE) in a clockwise direction.

[0044] High-temperature experiment: Arabidopsis thaliana was cultured for 5 days under a 16 / 8 photoperiod before high-temperature treatment, including a 1.5-hour treatment at 37°C followed by a 2-day recovery period, then a 1.5-hour treatment at 44°C followed by a 5-day recovery period. Arabidopsis thaliana seedlings were obtained as follows: Figure 5 As shown, the statistical survival rate results are as follows: Figure 6 The results showed that the percentage of Arabidopsis plants with the GLK1 gene knocked out exhibiting a tolerance phenotype after high-temperature treatment was significantly lower than that of the wild type, and the plant mortality rate was significantly higher. In contrast, almost all plants overexpressing the GLK1 gene exhibited a tolerance phenotype after high-temperature treatment, far exceeding that of the wild type and knockout plants, and almost no plants died. This indicates that overexpression of the GLK1 gene can significantly improve the high-temperature tolerance of Arabidopsis plants.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. GLK ( Golden2-like Application of transcription factor gene in improving temperature tolerance in Arabidopsis thaliana, wherein the temperature tolerance is high temperature tolerance or low temperature tolerance, and the sequence of the GLK gene is shown in SEQ ID NO:

1.

2. A method for improving the temperature tolerance of Arabidopsis thaliana, characterized in that, The method includes the step of overexpressing the GLK gene in the Arabidopsis thaliana, wherein the temperature tolerance is either high-temperature tolerance or low-temperature tolerance, and the sequence of the GLK gene is shown in SEQ ID NO:1.

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