Application of gene WRKY24 in cooling induction of plant early flowering

Abstract

The invention provides an application of a gene WRKY24 in cooling induction of early flowering of plants, the gene WRKY24 is derived from myricaria laxiflora, and the CDS sequence of the gene WRKY24 is as shown in SEQ ID NO: 1. According to the invention, the CDS sequence of the gene WRKY24 derived from myricaria laxiflora is transferred into the plant, so that the transgenic WRKY24 plant capable of blooming early under the cooling induction condition is obtained, and the early blooming capability of the plant under the cooling induction condition is effectively improved.

Description

Technical Field

[0001] The invention relates to plant genetic engineering technology, and in particular to application of gene WRKY24 in inducing early flowering of plants by cooling. Background Art

[0002] During the long evolution, plants have formed mechanisms to perceive and respond to environmental changes. Faced with adverse environments, they can continuously monitor external changes and adjust growth and development strategies in real time. Among them, the regulation of flowering time is crucial for plants to adapt to adversity, and it is related to their adaptability and reproductive capacity. The main environmental factors affecting flowering include light intensity, light quality, photoperiod, high and low temperature, drought, nutrients and salt. In particular, when encountering low temperatures (such as 0℃-15℃), some plants will switch from vegetative growth to reproductive growth in advance, especially when seed yield is expected to be damaged, they will bloom as early as possible to ensure partial reproduction. This is called the phenomenon of early flowering induced by low temperature stress.

[0003] The goal and direction of researchers engaged in the research of early flowering induced by low temperature stress is to induce the expression of early flowering genes and clone them, and to breed and cultivate plant species, varieties and ecotypes that can flower early under low temperature stress. However, low temperature stress has high temperature requirements and high costs, so how to induce early flowering of plants under cooling-induced conditions has become a recent research goal. Summary of the invention

[0004] The present invention provides an application of a gene WRKY24 in inducing early flowering of plants by cooling, wherein the gene WRKY24 is derived from Echinops sparsely flowered, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO: 1. The present invention obtains a transgenic WRKY24 plant that blooms early under cooling induction conditions by transferring the CDS sequence of the gene WRKY24 derived from Echinops sparsely flowered into the plant, thereby effectively improving the early flowering ability of the plant under cooling induction conditions.

[0005] The present invention provides a method for improving the early flowering ability of plants under cooling induction conditions. The present invention effectively improves the early flowering ability of plants under cooling induction conditions by overexpressing the gene WRKY24 in the plants, wherein the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:1.

[0006] The present invention also provides an application of gene WRKY24 in plant breeding, wherein gene WRKY24 is derived from Cyperus sparsely flowered, and the CDS sequence of gene WRKY24 is shown in SEQ ID NO: 1. The present invention utilizes a recombinant expression vector to overexpress gene WRKY24 to breed temperature-lowering-induced early flowering plant varieties.

[0007] Cooling-induced early flowering of plants means that when plants experience cooling, they may switch from vegetative growth to reproductive growth in advance, thereby prompting the plants to bloom as early as possible. Cooling-induced early flowering of plants has important uses in agriculture. For example, by artificially controlling cooling treatment, the flowering time of certain crops can be adjusted to avoid adverse climatic conditions or meet market demand, which has important economic benefits.

[0008] Therefore, in order to promote early flowering of plants under cooling induction conditions, the first aspect of the present invention provides an application of a gene WRKY24 in cooling-induced early flowering of plants, wherein the gene WRKY24 is derived from Cyperus sparsely flowered, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO: 1.

[0009] In detail, the gene WRKY24 is derived from Myricaria laxiflora. Myricaria laxiflora is an upright shrub of the Tamarix family and the genus Myricaria, which can reach a height of 1.5 meters. It is distributed in Zigui, Badong, Hubei, and the Yangtze River in Wushan Gorge, Sichuan, China. It often grows on roadsides and riverbanks and is an excellent riverbank sand fixation and greening tree species. The gene WRKY24 belongs to the plant WRKY transcription factor family. The plant WRKY transcription factor family is a plant-specific zinc finger transcription regulatory factor, named because it contains a highly conserved 7-amino acid sequence consisting of WRKYGQK.

[0010] The CDS sequence of the gene WRKY24 is 1911 bp, as shown in SEQ ID NO:1; CDS (Coding sequence) refers to a DNA sequence that corresponds to a protein sequence one by one, and the sequence does not contain other sequences that do not correspond to the protein. The gene WRKY24 can encode 636 amino acids, and the amino acid sequence is shown in SEQ ID NO:2. By transferring the CDS sequence of the gene WRKY24 into the plant, a transgenic WRKY24 plant that blooms early under cooling induction conditions can be obtained, thereby effectively improving the plant's ability to bloom early under cooling induction conditions.

[0011] In a specific embodiment, the present invention overexpresses the gene WRKY24 in plants, thereby improving the early flowering ability of the plants under cooling induction conditions.

[0012] Among them, overexpressing a gene means cloning the CDS sequence of the target gene into the corresponding plasmid or viral vector, and using the regulatory elements constructed on the vector backbone to enable the gene to be transcribed and translated in large quantities under artificially controlled conditions.

[0013] Specifically, overexpressing the gene WRKY24 in a plant comprises the following steps:

[0014] 1) The CDS sequence of the gene WRKY24 was constructed on an expression vector to obtain a ligation product;

[0015] 2) Introducing the ligation product into transformants and screening to obtain transformants containing the recombinant expression vector;

[0016] 3) Using transformants containing the recombinant expression vector to infect plants, transgenic plants overexpressing the WRKY24 gene were obtained.

[0017] In the present invention, recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0018] In the present invention, transformants refer to bacterial cells or other recipient cells that have acquired new genetic markers after incorporation or introduction of exogenous DNA.

[0019] In the present invention, transgenic plants refer to plants that have genes from other species.

[0020] Through the construction of a recombinant expression vector and transgenic technology, a transgenic plant overexpressing the WRKY24 gene can be obtained. After the transgenic plant overexpresses the WRKY24 gene, the ability of early flowering under cooling induction conditions is improved.

[0021] Steps 1) to 3) of the present invention can be performed according to conventional techniques in the art, and the present invention does not impose any further limitation thereto.

[0022] Furthermore, the plant is Arabidopsis thaliana.

[0023] The second aspect of the present invention provides a method for improving the early flowering ability of a plant under cooling induction conditions, comprising: overexpressing a gene WRKY24 in a plant, wherein the gene WRKY24 is derived from Cyperus sparsely flowered, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO: 1. The present invention effectively improves the early flowering ability of a plant under cooling induction conditions by overexpressing the gene WRKY24 in the plant, and is verified in the model plant Arabidopsis thaliana.

[0024] In a specific embodiment, the method for improving the early flowering ability of plants under cooling induction conditions comprises the following steps:

[0025] The CDS sequence of the gene WRKY24 was constructed on an expression vector to obtain a ligation product;

[0026] The ligation product is introduced into transformants, and transformants containing the recombinant expression vector are screened;

[0027] The transformants containing the recombinant expression vector were used to infect plants to obtain transgenic plants overexpressing the gene WRKY24.

[0028] The transgenic plant overexpressing the gene WRKY24 obtained by the method of the present invention has the ability to bloom early under cooling induction conditions, specifically, the transgenic plant can bloom early after 7 days of stress at 18°C. In the prior art, promoting early flowering of plants under low temperature stress induction generally requires treating the plants with low temperature stress of 0°C-15°C to promote high expression of early flowering genes, thereby achieving the effect of early flowering of plants, but the cooling induction condition of the present invention only needs to reach 18°C, the temperature requirement of the present invention is simple and easy, and the cost is correspondingly reduced.

[0029] In addition, the gibberellin content in the leaves of transgenic plants overexpressing the WRKY24 gene was not less than 40 mg / L after the temperature was lowered to 18°C ​​for 7 days.

[0030] Furthermore, the plant is Arabidopsis thaliana.

[0031] A third aspect of the present invention provides an application of a gene WRKY24 in plant breeding, wherein the gene WRKY24 is derived from Echinops sparsely flowered, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:1.

[0032] In summary, the present invention provides an application of a gene WRKY24 in inducing early flowering of plants by cooling, which has the following advantages:

[0033] 1) The present invention discloses an application of gene WRKY24 in inducing early flowering of plants by cooling. By transferring the CDS sequence of gene WRKY24 derived from Cyperus sparsely flowered into plants, it is found that gene WRKY24 has the ability to improve the early flowering of plants under cooling-induced conditions.

[0034] 2) At present, early flowering technology generally induces early differentiation of flower buds through agronomic measures, spraying hormones, etc. However, the present invention promotes high expression of the gene WRKY24 in plants under cooling induction conditions by means of transgenic means, thereby increasing the content of plant hormones such as gibberellins, causing the plants to bloom early, greatly reducing the waste of labor costs, and helping economic plants (such as vegetables, fruits, etc.) to bloom and bear fruit early, thereby providing early fruits for the market, which has important economic benefits.

[0035] 3) In the prior art, promoting early flowering of plants under low temperature stress induction generally requires treating the plants with low temperature stress at 0°C-15°C to promote high expression of early flowering genes, thereby achieving the effect of early flowering of plants. However, the cooling induction condition of the present invention only needs to reach 18°C. The temperature requirement of the present invention is simple and easy to implement, and the cost is correspondingly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0037] Figure 1 The electrophoresis diagram of the colony PCR product of Example 1;

[0038] Figure 2 This is a diagram of the relative expression of the gene WRKY24 in transgenic Arabidopsis plants in Example 4;

[0039] Figure 3 This is a diagram showing the relative expression of the gene WRKY24 in the stems and leaves of transgenic Arabidopsis plants in Example 5;

[0040] Figure 4 This is a diagram showing the early flowering phenomenon of Arabidopsis thaliana plants in the experimental group of Example 6;

[0041] Figure 5 This is a picture showing the phenomenon that the Arabidopsis plants in the blank control group of Example 6 did not bloom early;

[0042] Figure 6 This is a picture showing the phenomenon that the Arabidopsis plants in the negative control group of Example 6 did not bloom early. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific implementation methods listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The experimental methods in the following examples without specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The reagents and carriers used are commercially available or available from public channels unless otherwise specified.

[0045] The primer sequences involved in the following examples are shown in Table 1.

[0046] Table 1

[0047]

[0048] Example 1: Amplification of the CDS sequence of the gene WRKY24 from Echinops sparsely flowered

[0049] RNA was extracted from the leaves of Eupatoria sparsely flowered, and the RNA was reverse transcribed into cDNA. Primers 22OV100-F and primers 22OV100-R were designed according to the gene WRKY24 sequence, and the CDS sequence of gene WRKY24 was amplified by PCR technology using cDNA as a template. The PCR reaction conditions are shown in Table 2, including pre-denaturation at 94°C for 5 minutes, 32 cycles: denaturation at 98°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 68°C for 2 minutes, and finally extension at 68°C for 5 minutes, and then placed at 25°C for 1 minute. The PCR product was subjected to agarose gel electrophoresis and gel recovery, and the CDS sequence of gene WRKY24 from Eupatoria sparsely flowered was finally obtained. The CDS sequence is 1911 bp, as shown in SEQ ID NO:1; the gene can encode 636 amino acids, and the amino acid sequence is shown in SEQ ID NO:2.

[0050] Table 2

[0051]

[0052] Example 2: Construction of a recombinant expression vector

[0053] The CDS sequence of the gene WRKY24 derived from the truncatula sparsely flowered was connected with the purchased PYBA1132-GFP expression vector. The connection system was 3 μL of the CDS sequence of the gene WRKY24 derived from the truncatula sparsely flowered, 1 μL of the PYBA1132-GFP expression vector and 1 μL of pure water. After mixing, it was placed in a PCR instrument and connected at 32°C for 12 minutes to obtain a connection product.

[0054] Add the ligation product to the Agrobacterium competent cell suspension, heat shock in a 42°C water bath for 90 seconds (do not shake), heat shock for 16 seconds, cool on ice for 2 minutes, and then add 400 μL LB liquid culture medium. After mixing, shake and culture at 37°C and 180 rpm for 40 minutes to obtain the transformed bacterial solution. At this time, the bacteria in the transformed bacterial solution resume normal growth and express the antibiotic resistance gene (kanamycin) encoded by the expression vector. Mix the transformed bacterial solution and take 80 μL to evenly spread on the screening plate containing kanamycin. After the transformed bacterial solution is completely absorbed by the plate culture medium, invert the screening plate and culture for 12 hours. Select the monoclonal colony on the screening plate and culture it in LB liquid culture medium containing kanamycin to obtain a monoclonal bacterial solution. Use primers 22OV100-F and primers 22OV100-R to perform colony PCR and agarose gel electrophoresis to detect clone positivity. For details, see Figure 1 Positive clones were selected for sequencing verification. The sequencing primers were A580-seqR and PCB-seqE. The recombinant expression vector with correct sequencing results was successfully constructed.

[0055] Example 3: Infection of Arabidopsis thaliana

[0056] The monoclonal bacterial liquid with correct sequencing results was inoculated into 3 ml LB liquid medium (Kana 100 mg / L, Gent 25 mg / L) for culture, and then transferred to 500 ml LB liquid medium (Kana 100 mg / L, Gent 25 mg / L) at a volume ratio of 1:100, and cultured at 28°C, 250 rpm for 12 hours. When the OD value was 0.8, the culture was centrifuged at 4000 rpm for 10 min, and the precipitate was collected and resuspended in an equal volume of infiltration solution, which consisted of 5% (w / v) sucrose, 0.01% (v / v) Silwet surfactant and pure water. The infiltration solution was used to infect Arabidopsis thaliana, and transgenic plants overexpressing the gene WRKY24 were screened.

[0057] Example 4: Gene expression in transgenic Arabidopsis plants

[0058] RNA was extracted from transgenic plants overexpressing the gene WRKY24, and reverse transcribed into cDNA. Primers 22RT51-22OV100-F and 22RT51-22OV100-R were designed according to the sequence of the target gene WRKY24, and the target gene was amplified using qPCR technology using a 5-fold dilution of the above cDNA stock solution as a template. The actin gene of Arabidopsis thaliana was used as an internal reference gene, AT_UBQ5_qRT-F was used as a forward primer, and AT_UBQ5_qRT-R was used as a reverse primer. The amplification system is shown in Table 3, and the amplification conditions are 40 cycles: 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 20 seconds.

[0059] Table 3

[0060]

[0061] According to the Cq values ​​of the internal reference gene and the target gene amplification, the expression value of the transgenic plant sample with the overexpressed gene WRKY24 numbered 22ATH42-1 was 1, and the expression amount of the target gene in each sample was calculated. Figure 2 Among them, the expression level of gene WRKY24 in the transgenic Arabidopsis plant numbered 22ATH43-4 was the highest, and the transgenic WRKY24 Arabidopsis plant in subsequent experiments referred to this plant.

[0062] Example 5: Gene expression in stems and leaves of transgenic Arabidopsis plants

[0063] like Figure 3 As shown, the WRKY24 gene was highly expressed in the stems and leaves of the transgenic WRKY24 Arabidopsis plants, while the relative expression level of the WRKY24 gene was low in the wild-type Arabidopsis plants.

[0064] Example 6: Early flowering ability of transgenic Arabidopsis plants under cooling induction conditions

[0065] Transgenic WRKY24 Arabidopsis plants were sown, and when the seedlings grew to the sixth true leaf, they were placed in a low temperature (18°C) culture room for 7 days, which was set as the experimental group; wild-type Arabidopsis seedlings that had grown to the sixth true leaf were placed in a normal temperature (23-25°C) culture room for 7 days, which was set as the blank control group; wild-type Arabidopsis seedlings that had grown to the sixth true leaf were placed in a low temperature (18°C) culture room for 7 days, which was set as the negative control group. Subsequently, the experimental group, the blank control group, and the negative control group were uniformly moved to normal temperature (23-25°C) for 33 days, and the flowering of the Arabidopsis in the experimental group and the blank control group and the negative control group was observed. The flowering of the Arabidopsis in the experimental group was shown in Figure 2. Figure 4 The flowering of Arabidopsis thaliana in the blank control group is shown in Figure 5 The flowering of Arabidopsis thaliana in the negative control group is shown in Figure 6 .

[0066] like Figure 4 , Figure 5 and Figure 6 As shown, the Arabidopsis in the experimental group has already begun to flower, while the Arabidopsis in the blank control group and the negative control group have not begun to flower, indicating that the transgenic WRKY24 Arabidopsis plants can indeed flower early under cooling-induced conditions.

[0067] Example 7: Gibberellic acid content in leaves of transgenic Arabidopsis plants

[0068] Gibberellins (GAs) are an important class of plant hormones that participate in multiple biological processes such as plant growth and development. They play a key role in promoting plant cell elongation, seed germination, bud growth, flower development, and fruit ripening. They can break the dormancy of plants and promote flower bud differentiation, thereby causing plants to bloom early. The leaves of the experimental group plants in Example 6 that were placed in a culture room at 18°C ​​for 7 days were taken to detect the gibberellins content.

[0069] The results showed that the average gibberellin content in the leaves of the experimental group Arabidopsis (transgenic WRKY24 Arabidopsis) was 45.86 mg / L, the average gibberellin content in the leaves of the blank control group Arabidopsis (wild-type Arabidopsis) was 20.32 mg / L, and the average gibberellin content in the leaves of the negative control group Arabidopsis (wild-type Arabidopsis) was 21.15 mg / L. The average gibberellin content in the leaves of the experimental group was higher than that of the blank control group and the negative control group Arabidopsis. The above results show that the present invention can increase the gibberellin content of the leaves of Arabidopsis plants under cooling induction conditions by promoting the overexpression of the gene WRKY24 in Arabidopsis plants, thereby promoting the early flowering of Arabidopsis plants under cooling induction conditions.

[0070] In summary, the present invention transfers the CDS sequence of the gene WRKY24 derived from the sparse flowered cyperus into the Arabidopsis plant, and can obtain the transgenic WRKY24 Arabidopsis plant. The gene WRKY24 is highly expressed in the stems and leaves of the transgenic Arabidopsis plant, and due to the transfer of the gene WRKY24, the transgenic Arabidopsis plant acquires the ability to bloom early under cooling induction conditions, and the gibberellin content in its leaves is also significantly higher than that of the wild-type Arabidopsis plant. It can be expected that the gene can also be used to improve the early flowering ability of other plants under cooling induction conditions. Therefore, the present invention uses a recombinant expression vector to overexpress the gene WRKY24 for plant breeding, and finally obtains a plant variety that blooms early under cooling induction conditions.

[0071] At present, early flowering technology generally induces early differentiation of flower buds through agronomic measures, spraying hormones, etc. However, the present invention promotes high expression of the gene WRKY24 in plants through transgenic means and under cooling induction conditions, thereby increasing the content of plant hormones such as gibberellins, causing early flowering of plants, greatly reducing the waste of labor costs, and helping economic plants (such as vegetables, fruits, etc.) to bloom and bear fruit early, thereby providing early fruits for the market, which has important economic benefits.

[0072] Compared with other methods of inducing early flowering of plants under low temperature stress, the present invention has low requirements for induction conditions. In the prior art, plants are generally treated at low temperatures of 0°C-15°C to promote high expression of early flowering genes, thereby achieving the effect of early flowering of plants. The cooling induction conditions of the present invention only need to reach 18°C, which has low temperature requirements and reduces costs accordingly.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of gene WRKY24 in inducing early flowering of plants by cooling, characterized in that: The gene WRKY24 is derived from Echinops sparsely flowered branches, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Overexpression of the WRKY24 gene in the plant improves the plant's ability to flower early under cooling-induced conditions.

3. The use according to any one of claims 1-2, characterized in that: Overexpressing the WRKY24 gene in a plant comprises the following steps: The CDS sequence of the gene WRKY24 was constructed on an expression vector to obtain a ligation product; Introducing the ligation product into transformants, and screening to obtain transformants containing the recombinant expression vector; The transformant containing the recombinant expression vector is used to infect plants to obtain transgenic plants overexpressing the gene WRKY24.

4. The use according to any one of claims 1 to 3, characterized in that: The plant is Arabidopsis thaliana.

5. A method for improving the early flowering ability of plants under cooling induction conditions, characterized in that: include: The gene WRKY24 is overexpressed in the plant, wherein the gene WRKY24 is derived from the branch of Echinops sparsely flowered, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:

1.

6. The method according to claim 5, characterized in that The steps include: The CDS sequence of the gene WRKY24 was constructed on an expression vector to obtain a ligation product; Introducing the ligation product into transformants, and screening to obtain transformants containing the recombinant expression vector; The transformant containing the recombinant expression vector is used to infect plants to obtain transgenic plants overexpressing the gene WRKY24.

7. The method according to claim 6, characterized in that The transgenic plants can bloom early after being stressed at 18°C ​​for 7 days.

8. The method according to claim 7, characterized in that After the transgenic plant is stressed at 18° C. for 7 days, the gibberellin content in the leaves of the transgenic plant is not less than 40 mg / L.

9. The method according to any one of claims 5 to 8, characterized in that: The plant is Arabidopsis thaliana.

10. Application of gene WRKY24 in plant breeding, characterized in that: The gene WRKY24 is derived from Echinops sparsely flowered branches, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:1.

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