Application of soybean gene GmSnRK2C-e in plant tolerance to cadmium stress

Abstract

The application of soybean gene GmSnRK2C-e in plant cadmium stress resistance belongs to the technical field of plant genetic engineering. The application of the soybean gene GmSnRK2C-e in plant cadmium stress resistance is provided to solve the technical problems that the cadmium tolerance of the soybean quality is poor in the prior art, and the research process of improving the cadmium tolerance of soybean by traditional breeding methods is slow, the nucleotide sequence of the soybean gene GmSnRK2C-e is shown as SEQ ID NO. 1, and the application of the expression vector and the recombinant bacteria containing the above-mentioned gene in plant cadmium stress resistance. The wild type and overexpression transgenic plants under cadmium stress are observed, it is found that compared with the wild type, the overexpression transgenic plants are less inhibited in growth, the shortening of the plant roots is lighter, and the cell damage of the plant leaves is smaller; the results prove that the soybean gene GmSnRK2C-e has the ability of positively regulating the cadmium stress resistance of soybean plants, and the provided GmSnRK2C-e gene has important theoretical significance and practical value for accelerating the breeding process of cadmium-resistant plants and improving the breeding efficiency.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a soybean gene GmSnRK2C-e in plant cadmium stress tolerance. BACKGROUND

[0002] Cadmium (Cd) is a heavy metal element with high toxicity, which has attracted much attention due to its accumulative nature in soil and organisms. Although the content of this element in nature is extremely small, with the intensification of human activities, especially the industrialization process and urbanization development, the emission of cadmium is increasing, leading to the accumulation of cadmium in the soil, thereby affecting the yield and quality of crops, and entering the food chain through crops, posing a potential threat to human health.

[0003] Soybean (Glycine max (Linn.) Merr.) is an important oil and protein source in the world, playing a key role in maintaining global food supply and nutritional security, and the yield and quality of soybean directly affect the global agricultural product market and consumer health. However, cadmium enters the plant body through the root system of soybean, affecting the growth and development of plants, reducing the efficiency of photosynthesis, and interfering with normal physiological metabolic processes, thereby inhibiting the growth of soybean and reducing key growth indicators such as plant height, leaf area, and root dry weight. Moreover, long-term exposure to cadmium stress can significantly reduce soybean yield, affecting yield parameters such as pod number and hundred-grain weight, so the impact of cadmium pollution on soybean production cannot be ignored.

[0004] Currently, the commonly available soybean varieties have poor tolerance to cadmium, resulting in excessive cadmium content in soybean crops grown in cadmium-contaminated soil. Meanwhile, the traditional breeding method for improving the cadmium tolerance of soybean is slow and difficult to achieve significant improvement in cadmium tolerance. Therefore, the technical personnel in the field have been eager to effectively improve the cadmium tolerance of soybean. SUMMARY

[0005] The application provides application of a soybean gene GmSnRK2C-e in plant cadmium stress tolerance to solve the technical problems of poor cadmium tolerance of soybean quality and slow research progress of traditional breeding method for improving the cadmium tolerance of soybean in the prior art.

[0006] One of the objectives of the application is to provide application of a soybean gene GmSnRK2C-e in plant cadmium stress tolerance, wherein the nucleotide sequence of the soybean gene GmSnRK2C-e is shown in SEQ ID NO. 1.

[0007] In a preferred embodiment of the present application, the application refers to the preparation of a transgenic plant overexpressing a soybean gene GmSnRK2C-e, which positively regulates the ability of the plant to tolerate cadmium stress, wherein the plant is soybean.

[0008] In a preferred embodiment of the present application, the method for preparing the transgenic plant comprises the following steps:

[0009] S1, cloning the soybean gene GmSnRK2C-e with the nucleotide sequence shown in SEQ ID NO. 1 using a primer pair, to obtain a gene cloning sequence;

[0010] S2, connecting the gene cloning sequence obtained in S1 to a vector by means of homologous recombination, performing LR reaction by means of Gateway to connect to a plasmid vector, to obtain an expression vector;

[0011] S3, introducing the expression vector obtained in S2 into Agrobacterium, to obtain a recombinant bacteria;

[0012] S4, infecting the plant with the recombinant bacteria obtained in S3, to obtain a transgenic plant overexpressing the soybean gene GmSnRK2C-e.

[0013] In a preferred embodiment of the present application, the upstream primer in the primer pair in S1 has the nucleotide sequence shown in SEQ ID NO. 2, and the downstream primer has the nucleotide sequence shown in SEQ ID NO. 3.

[0014] In a preferred embodiment of the present application, the vector in S2 is Fu28, and the plasmid vector is pSoy1.

[0015] In a preferred embodiment of the present application, the Agrobacterium in S3 is EHA105.

[0016] In a preferred embodiment of the present application, the infection method in S3 is soybean cotyledon node transformation.

[0017] In a preferred embodiment of the present application, the plant in S4 is soybean.

[0018] The second object of the present application is to provide an application of an expression vector in the tolerance of a plant to cadmium stress, wherein the expression vector contains a soybean gene GmSnRK2C-e with the nucleotide sequence shown in SEQ ID NO. 1, and the plant is soybean.

[0019] The third object of the present application is to provide an application of a recombinant bacteria in the tolerance of a plant to cadmium stress, wherein the recombinant bacteria contains the above-mentioned expression vector, and the plant is soybean.

[0020] The application provides application of a soybean gene GmSnRK2C-e in plant cadmium stress tolerance, and expression of the soybean gene GmSnRK2C-e is analyzed, and the result shows that the expression amount of the soybean gene GmSnRK2C-e in a soybean root tissue is the highest, the expression amount in stem, flower and pod tissues is lower, and the expression amount in leaf and mature seed tissues is extremely low; compared with a soybean Dongnong 50 plant without treatment, the expression amount of the GmSnRK2C-e gene in the soybean plant treated by a 9mg / L CdCl2 solution is significantly increased, which indicates that the expression amount of the GmSnRK2C-e gene increases with the increase of time under the cadmium stress condition, and it is indicated that a sucrose non-fermentation related protein kinase coded by the soybean GmSnRK2C-e gene may participate in the cadmium stress response.

[0021] The transgenic plant material obtained by the application can become stable genetic material for studying cadmium stress through subculture propagation and identification; through observation of the overexpression transgenic plant and the wild type Dongnong 50 soybean plant under the cadmium stress, it is found that the above soybean leaves after cadmium treatment all appear wilting and yellowing symptoms in different degrees, the leaf veins appear red, the wild type plant is obviously inhibited in growth, and the overexpression transgenic plant is less inhibited in growth; after three days of cadmium stress treatment, the growth of the root of the wild type plant is significantly inhibited, and the root length is greatly shortened, while the growth of the root of the overexpression plant is less inhibited, and the lateral root growth trend is consistent with the main root, which indicates that the cadmium stress can inhibit the growth of the soybean root system. The above soybean leaves after the cadmium stress are subjected to NBT staining, and the result shows that the leaf cells of the overexpression plant are less damaged compared with the wild type under the cadmium stress.

[0022] It can be seen that the soybean gene GmSnRK2C-e has the ability of positively regulating the cadmium stress tolerance of the soybean plant, and the cadmium stress tolerance of the transgenic plant overexpressing the GmSnRK2C-e gene is significantly improved compared with the wild type. The application has important theoretical significance and practical value for accelerating the breeding process of the cadmium tolerance plant and improving the breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a GmSnRK2C-e gene expression amount detection graph in the soybean tissue in embodiment 1;

[0024] Figure 2 It is a GmSnRK2C-e gene expression amount detection graph under the cadmium stress condition in embodiment 1;

[0025] Figure 3 It is a recombinant bacteria liquid PCR detection graph in embodiment 2;

[0026] Figure 4 It is a transgenic plant cultivation process graph in embodiment 2;

[0027] Figure 5 Figure 2: BAR test strip detection chart of overexpression transgenic plants in Example 2;

[0028] Figure 6 Figure 3: Real-time fluorescent quantitative PCR chart of overexpression transgenic plants in Example 2;

[0029] Figure 7 Figure 4: Observation chart of wild type and transgenic plants under cadmium stress in Example 3; A is the observation chart of the whole plant and leaf, B is the observation chart of the plant root, and C is the NBT staining chart of the plant leaf. DETAILED DESCRIPTION

[0030] Those skilled in the art can improve the process parameters according to the content herein. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0031] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0032] Example 1: Expression analysis of soybean gene GmSnRK2C-e

[0033] In this embodiment, the different tissue parts (roots, stems, leaves, flowers, pods and mature seeds) of soybean Dongnong 50 plants growing to fully expanded true leaves were stored at -80°C, respectively. Meanwhile, the soybean Dongnong 50 plants growing to fully expanded true leaves were treated with cadmium stress, and 20 soybean Dongnong 50 plants with consistent growth were selected and soaked in a 9 mg / L CdCl2 solution. The samples were taken at 0 h and 72 h after treatment and stored at -80°C, respectively. The soybean Dongnong 50 plants without treatment were used as the control group.

[0034] The soybean gene GmSnRK2C-e with the nucleotide sequence shown in SEQ ID NO. 1 was used as a template to design real-time fluorescent quantitative PCR primers. The nucleotide sequence of the upstream primer qRT-SnRK2C-e-F is shown in SEQ ID NO. 4, and the nucleotide sequence of the downstream primer qRT-SnRK2C-e-R is shown in SEQ ID NO. 5.

[0035] Total RNA was extracted from the roots, stems, leaves, flowers, pods and mature seeds of Dongnong 50 plants using the Plant Total RNA Isolation Kit (Foregene), and cDNA was synthesized by reverse transcription using a reverse transcription kit (Tiangen, China, code FP205-01) according to the manufacturer's instructions. The cDNA obtained above was used as a template for real-time fluorescent quantitative PCR primers, and qRT-SnRK2C-e-F and qRT-SnRK2C-e-R were used as primers for PCR amplification. The relative expression of the soybean gene GmSnRK2C-e was detected. GmUKN1 was used as an internal reference gene, and the nucleotide sequence of the upstream primer of the internal reference gene is shown in SEQ ID NO. 6, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 7.

[0036] The real-time fluorescent quantitative PCR reaction conditions were 94°C for 10 min→[94°C for 30 s→64°C for 30 s]×40→60°C for 30 s→72°C for 10 min→94°C for 2 min. The ΔCT method was used to calculate the gene expression, and each sample included 3 biological replicates and 3 technical replicates. The relative expression was 2-ΔCT = 2-(ΔCT treatment-ΔGmSnRK2C-e gene CT control) = 2-[(CT target gene in treatment-CT internal reference gene in treatment)-(CT target gene in control-CT internal reference gene in control)].

[0037] The results are shown in Figure 1 The expression of the soybean gene GmSnRK2C-e in the root tissue was the highest, and the expression in the stem, flower and pod tissues was lower, but the expression in the leaf and mature seed tissues was very low.

[0038] The results are shown in Figure 2 As shown in the results, compared with the control group, the expression of the GmSnRK2C-e gene in the soybean plants treated with 9 mg / L CdCl2 solution was significantly increased.

[0039] Example 2: Preparation of soybean gene GmSnRK2C-e overexpression plant

[0040] S1: using the soybean gene GmSnRK2C-e with the nucleotide sequence shown in SEQ ID NO. 1 as a template, and using the upstream primer with the nucleotide sequence shown in SEQ ID NO. 2 and the downstream primer with the nucleotide sequence shown in SEQ ID NO. 3 for PCR amplification to obtain a cloning sequence;

[0041] The PCR amplification system is as follows: 2xPhanta Max Buffer 25 μL, 10 mM dNTP 1 μL, 10 μM VaTPS9F 2 μL, 10 μM VaTPS9R 2 μL, cDNA template 2 μL, Phanta Max Super-Fidelity DNA Polymease (1 U / μl) 1 μL, and ddH2O to 50 μL;

[0042] The PCR amplification program is as follows: 95 °C pre-denaturation for 30 s, 95 °C denaturation for 10 s, 48 °C annealing for 15 s, 72 °C extension for 60 s, 39 cycles, and 72 °C thorough extension for 5 min;

[0043] S2: using homologous recombination to connect the cloning sequence obtained in S1 into the Fu28 vector, and then using Gateway means to perform LR reaction to connect into the pSoy1 plasmid vector to obtain an expression vector;

[0044] S3: introducing the expression vector obtained in S2 into EHA105 Agrobacterium to obtain a recombinant bacterium;

[0045] S4: using the soybean cotyledon node transformation method to infect the soybean Dongnong 50 plant with the recombinant bacterium obtained in S3 to obtain an overexpression transgenic soybean plant.

[0046] This example performs PCR detection on the bacterial liquid of the recombinant bacterium obtained in S3, and the results are shown in Figure 3 , and the 1024 bp target sequence is detected, indicating that the cloning sequence has been successfully connected to the pSoy1 plasmid vector and successfully expressed; the transgenic plant cultivation process is shown in Figure 4 .

[0047] This example performs BAR test strip detection on the overexpression transgenic soybean plant obtained above, and when the third leaf of the overexpression transgenic soybean plant is fully unfolded, fresh leaves of the transgenic soybean plant are taken and placed in a 1.5 mL centrifuge tube, 500 μL of sterile water is then added, and the leaves are completely crushed using a grinding rod. The test strip is vertically inserted into the centrifuge tube according to the direction, and after waiting for 2-5 min, if two bands appear on the test strip, the plant is a positive transgenic soybean plant, and the results are shown in Figure 5 , and a total of 2 positive transgenic plants are identified.

[0048] The roots, stems and leaves of the two positive transgenic plants identified above were sampled, the DNA of the roots, stems and leaves samples was extracted by CTAB method, and real-time fluorescent quantitative PCR identification was carried out by using GmSnRK2C-e-F (as shown in SEQ ID NO. 4) and GFP-R (as shown in SEQ ID NO. 8) as primers, and the results are shown in Figure 6 The specific bands can be amplified in the roots, stems and leaves of the two positive transgenic plants identified above, further proving that the BAR test strip detection result is accurate.

[0049] Example 3: Application of soybean gene GmSnRK2C-e in plant cadmium stress resistance

[0050] In this example, wild type Dongnong 50 and homozygous transgenic soybean plants (overexpression plants) overexpressing GmSnRK2C-e gene obtained in Example 2 were planted in sterilized flower soil after sterilization in a fume hood. After the plants fully unfolded the true leaves, the roots were carefully washed clean and transferred to a hydroponic box containing Hoagland nutrient solution. Then 9 mg / L CdCl2 was added to the nutrient solution and the plants were observed.

[0051] The results are shown in Figure 7 As shown in Fig. A, the soybean leaves treated with cadmium showed different degrees of wilting and yellowing symptoms, and the leaf veins appeared red. The growth of wild type Dongnong 50 was significantly inhibited, but the overexpression plants were less affected by cadmium stress than the wild type soybean.

[0052] The results are shown in Figure 7 As shown in Fig. B, after 3 days of cadmium stress treatment, the second trifoliate leaves grew out, and the root morphology also changed significantly. The descending order of the main root length was wild type > overexpression plants, and the growth trend of lateral roots was consistent with that of the main roots. This result indicates that cadmium stress can inhibit the growth of soybean root system.

[0053] The results are shown in Figure 7 As shown in Fig. C, NBT staining was performed on the leaves after cadmium stress, and the results showed that the cell damage of the overexpression plants was smaller than that of the wild type under cadmium stress.

[0054] The content not described in detail in the specification of the present application is known to those skilled in the art. Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. Application of soybean gene GmSnRK2C-e in improving plant tolerance to cadmium stress, characterized in that, The nucleotide sequence of the soybean gene GmSnRK2C-e is shown as SEQ ID NO.

1. The application refers to the preparation of transgenic plants overexpressing the soybean gene GmSnRK2C-e, the ability of the soybean gene GmSnRK2C-e to positively regulate the tolerance of plants to cadmium stress, and the plants are soybeans.

2. Use according to claim 1, characterized in that, The preparation method of the transgenic plants comprises the following steps: S1, cloning the soybean gene GmSnRK2C-e with the nucleotide sequence shown as SEQ ID No. 1 using a primer pair, to obtain a gene clone sequence; S2, connecting the gene clone sequence obtained in S1 to a vector using a homologous recombination method, performing a LR reaction using a Gateway method to connect to a plasmid vector, to obtain an expression vector; S3, introducing the expression vector obtained in S2 into Agrobacterium, to obtain a recombinant bacterium; S4, infecting plants with the recombinant bacterium obtained in S3, to obtain transgenic plants overexpressing the soybean gene GmSnRK2C-e.

3. Use according to claim 2, characterized in that, The nucleotide sequence of the upstream primer in the primer pair in S1 is shown as SEQ ID No. 2, and the nucleotide sequence of the downstream primer is shown as SEQ ID No.

3.

4. Use according to claim 2, characterized in that, The vector in S2 is Fu28, and the plasmid vector is pSoy1.

5. Use according to claim 2, characterized in that, The Agrobacterium in S3 is EHA105.

6. Use according to claim 2, characterized in that, The infection method in S4 is a soybean cotyledon node transformation method.

7. Use of an expression vector for improving plant tolerance to cadmium stress, characterized in that, The expression vector contains the soybean gene GmSnRK2C-e with the nucleotide sequence shown as SEQ ID NO. 1, and the plants are soybeans.

8. Use of a recombinant bacterium in improving plant tolerance to cadmium stress, characterized in that, The recombinant bacterium contains the expression vector in claim 7, and the plants are soybeans.

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