Method for improving seed germination rate of brassica napus under drought stress

Abstract

The invention relates to application of a brassica napus BnaA08. SNAT2 gene in improving the seed germination rate under drought stress, in particular to a method for improving the seed germination rate of brassica napus under drought stress. According to the invention, a BnaA08. SNAT2 gene is cloned from a brassica napus inbred line 'K407', an overexpression vector is constructed, and a transgenic line is obtained through agrobacterium transformation. Functional verification shows that the germination potential and germination rate of a transgenic line under drought stress (simulated by D-mannitol) are remarkably higher than those of a wild type, the content of hydrogen peroxide and superoxide anions is reduced, and the activity of antioxidant enzyme and the content of proline are increased. According to the breeding method provided by the invention, the drought tolerance of the plant seeds in the germination period can be improved by increasing the content and / or activity of the BnaA08. SNAT2 gene coding protein in the plant.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to a method for improving the seed germination rate of Brassica napus BnaA08.SNAT2 gene under drought stress. Background Art

[0002] Rapeseed is the largest source of edible vegetable oil in my country, accounting for over 55% of domestic oil crop production and playing a key role in maintaining national edible oil supply security. Drought is one of the major abiotic stresses that threaten agricultural production. Arid and semi-arid regions account for 41% of global land area, and climate change is leading to more frequent seasonal droughts. The rapeseed sowing period (seed germination) is likely to overlap with the dry season. Rapeseed cultivation in my country is primarily concentrated in the Yangtze River Basin and Northwest China. In the Yangtze River Basin, droughts are more frequent in autumn and winter, leading to unstable seedling emergence over large areas of direct-seeded rapeseed, hindering growth and reducing yield. Northwest China experiences drought and low rainfall, resulting in low soil moisture. Poor surface soil moisture during autumn sowing directly impacts rapeseed emergence and seedling growth, ultimately affecting yield. Cultivating varieties with strong drought resistance during the germination period can reduce or delay irrigation frequency, conserving water resources while ensuring adequate seed germination and seedling growth. Therefore, identifying drought-resistant genes in rapeseed and analyzing their functions is crucial for breeding drought-resistant rapeseed varieties and increasing rapeseed yield. Summary of the Invention

[0003] The present invention aims to provide a method for promoting Brassica napus seed germination under drought stress based on the Brassica napus melatonin synthesis gene BnaA08.SNAT2.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for improving the seed germination rate of Brassica napus under drought stress, which increases the content or activity of the protein encoded by the BnaA08.SNAT2 gene in Brassica napus.

[0006] Furthermore, the increase in the content or activity of the protein encoded by the BnaA08.SNAT2 gene in Brassica napus is achieved by connecting the full-length CDS sequence of the BnaA08.SNAT2 gene to an overexpression vector with a 35S promoter, and then transforming the Brassica napus with Agrobacterium to obtain transgenic Brassica napus plants.

[0007] Furthermore, the Brassica napus L. is the Brassica napus L. inbred line 'K407'.

[0008] Furthermore, the transgenic Brassica napus plants obtained by the method have an improved seed germination rate under drought stress.

[0009] Furthermore, in the transgenic Brassica napus plant, the drought stress is simulated by D-mannitol.

[0010] Furthermore, under drought stress, the transgenic Brassica napus plants have lower hydrogen peroxide and superoxide anion contents than wild-type Brassica napus plants, and higher antioxidant enzyme superoxide dismutase, peroxidase and catalase activities and proline content than the Brassica napus inbred line 'K407'.

[0011] Furthermore, the transgenic Brassica napus plants are used in breeding drought-resistant Brassica napus varieties.

[0012] A plant breeding method for increasing the content or activity of the protein encoded by the BnaA08.SNAT2 gene according to claim 1 in a plant, thereby increasing the drought resistance of plant seeds during the germination period.

[0013] Furthermore, the plant is a dicotyledonous plant, a cruciferous plant, Brassica napus or a Brassica napus inbred line 'K407'.

[0014] Furthermore, the plant seeds have significantly improved drought resistance during the germination period, and the improved drought resistance of the plant seeds during the germination period is manifested as an increased germination rate, a decrease in at least one of hydrogen peroxide and superoxide anion content, and an increase in at least one of the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase, and a proline content.

[0015] Experiments have demonstrated that overexpressing the BnaA08.SNAT2 gene in the Brassica napus inbred line 'K407' can improve drought resistance in Brassica napus. This improved drought resistance is manifested by increased germination rate, decreased hydrogen peroxide and superoxide anion levels, and increased antioxidant enzyme activity and proline content under drought stress. This study demonstrates the importance of the BnaA08.SNAT2 gene in Brassica napus in breeding and research to improve drought resistance in rapeseed and can be used to develop drought-resistant plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Analysis of expression pattern of BnaA08.SNAT2 gene under drought stress.

[0017] Figure 2 : Analysis of germination phenotype of T3 generation BnaA08.SNAT2 transgenic rapeseed under drought stress.

[0018] Figure 3 :Effects of hydrogen peroxide (H2O2) and superoxide anion (O 2-) and proline contents, as well as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzyme activities. DETAILED DESCRIPTION

[0019] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] (1) The purpose of the present invention is to:

[0021] The invention provides an application method for promoting seed germination of Brassica napus under drought stress based on the Brassica napus melatonin synthesis gene BnaA08.SNAT2, which provides valuable gene resources and theoretical basis for Brassica napus breeding.

[0022] (2) Technical Solutions

[0023] Gene cloning and vector construction: The full-length CDS sequence of the BnaA08.SNAT2 gene was successfully cloned from the Brassica napus inbred line 'K407' and ligated into an overexpression vector with a 35S promoter.

[0024] Preparation of transgenic plants: The Brassica napus inbred line 'K407' was transformed with Agrobacterium to obtain transgenic lines.

[0025] Functional Verification: We further validated the role of the BnaA08.SNAT2 gene in regulating seed germination in Brassica napus under drought stress (simulated by D-mannitol). We observed that the germination rate and final germination percentage of the transgenic line under drought stress were significantly higher than those of the wild-type 'K407'. The transgenic line also exhibited reduced hydrogen peroxide and superoxide anion levels, increased antioxidant enzyme activity, and increased proline content. Therefore, we conclude that BnaA08.SNAT2 promotes seed germination in Brassica napus under drought stress.

[0026] (3) Specific measures

[0027] Phenotypic analysis of drought stress tolerance in transgenic rapeseed seeds during germination

[0028] Material selection: The two transgenic rapeseed lines OE#1 and OE#2 with the highest expression levels of the target gene BnaA08.SNAT2 among the BnaA08.SNAT2 transgenic rapeseed lines were selected as subsequent experimental materials.

[0029] Experimental methods: Transgenic rapeseed plants and wild-type 'K407' rapeseed seeds of uniform size were selected and tested under normal conditions (distilled water) and stress treatment (400 mM D-mannitol). Three replicates were set for each treatment, and 50 seeds were used for each replicate. The seeds were disinfected and washed with distilled water, then placed in a 90 mm diameter glass culture dish. 7.5 mL of D-mannitol solution was added, and the culture dish was placed in the dark at 4°C for 3 days. Subsequently, the dish was placed in a rapeseed growth chamber with a relative humidity of 54% and a temperature of 25°C (16 h light / 8 h dark) for normal culture. The germination was marked by the seed radicle breaking through the seed coat. The seed germination was observed at the same time point every day and the germination rate was calculated. The germination status was recorded by photographing on the 7th day after stratification.

[0030] Experimental results: Figure 2 As shown in the figure, the seed germination rates of BnaA08.SNAT2 transgenic rapeseed lines OE#1 and OE#2 under drought stress were significantly higher than that of the wild type 'K407'.

[0031] Determination of physiological indices of transgenic rapeseed seeds under drought stress during germination

[0032] Experimental method: Using the stress treatment method in the phenotypic analysis of drought stress tolerance of transgenic rapeseed seeds during germination, BnaA08.SNAT2 transgenic rapeseed line and wild-type 'K407' line were treated, and samples were taken 12 hours after stress. The content of hydrogen peroxide (H2O2) and superoxide anion (O 2- ) content, proline content, superoxide dismutase (SOD) activity, peroxidase (POD) activity and catalase (CAT) activity.

[0033] Experimental results: Figure 3 As shown in the figure, the BnaA08.SNAT2 transgenic rapeseed lines OE#1 and OE#2 showed significant changes in the expression of hydrogen peroxide (H2O2) and superoxide anion (O 2- ) contents were significantly lower than those in the wild type 'K407'; the superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities and proline content of BnaA08.SNAT2 transgenic rapeseed lines OE#1 and OE#2 under drought stress during the germination period were significantly higher than those in the wild type 'K407'.

[0034] (4) Scope of protection

[0035] The present invention provides a plant breeding method for increasing the content and / or activity of any of the aforementioned proteins, BnaA08.SNAT2, in plants, thereby increasing the drought tolerance of rapeseed seeds during germination. Any of the aforementioned plants may be any of the following: c1) to c4): c1) a dicot; c2) a cruciferous plant; c3) Brassica napus; c4) the Brassica napus inbred line 'K407'. The improved stress tolerance may be manifested as an increased germination rate, increased production of hydrogen peroxide (H2O2) and superoxide anions (O 2- ) content decreased, and at least one of the activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) and the content of proline increased.

[0036] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A method for improving the germination rate of Brassica napus seeds under drought stress, characterized by: Increase the content or activity of the protein encoded by the Brassica napus gene BnaA08.SNAT2.

2. The method for improving the seed germination rate of Brassica napus under drought stress according to claim 1, characterized in that: The increase in the content or activity of the protein encoded by the BnaA08.SNAT2 gene of Brassica napus is achieved by connecting the full-length CDS sequence of the gene BnaA08.SNAT2 to an overexpression vector with a 35S promoter, and then transforming the Brassica napus with Agrobacterium to obtain transgenic Brassica napus plants.

3. The method for improving the seed germination rate of Brassica napus under drought stress according to claim 1, characterized in that: The Brassica napus L. inbred line is Brassica napus L. inbred line 'K407'.

4. The transgenic Brassica napus plant obtained by the method according to any one of claims 1 to 3, characterized in that: The seed germination rate of the transgenic Brassica napus plant under drought stress is higher than that of the Brassica napus inbred line 'K407'.

5. The transgenic Brassica napus plant according to claim 4, characterized in that: The drought stress was simulated by D-mannitol.

6. The transgenic Brassica napus plant according to claim 4, wherein: Under drought stress, the transgenic Brassica napus plants have lower hydrogen peroxide and superoxide anion contents than wild-type Brassica napus plants, and higher superoxide dismutase, peroxidase, and catalase activities and proline content than wild-type Brassica napus plants.

7. Use of the transgenic Brassica napus plant according to claim 4 in cultivating drought-tolerant Brassica napus varieties.

8. A plant breeding method, characterized in that: The content or activity of the protein encoded by the BnaA08.SNAT2 gene according to claim 1 is increased in the plant, thereby increasing the drought resistance of the plant seeds during the germination period.

9. The plant breeding method according to claim 8, characterized in that: The plant is any one of a dicotyledonous plant, a cruciferous plant, Brassica napus or a Brassica napus inbred line 'K407'.

10. The plant obtained by the plant breeding method according to any one of claims 8 to 9, characterized in that: The drought resistance of the plant seeds during the germination period is improved, and the improved drought resistance of the plant seeds during the germination period is manifested in an increased germination rate, a decrease in at least one of hydrogen peroxide and superoxide anion content, and an increase in at least one of the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase, and a proline content.

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