Application of gene WRKY24 in early flowering of plants induced by temperature reduction

Abstract

This invention provides the application of the gene WRKY24 in inducing early flowering in plants under cooling conditions. The gene WRKY24 is derived from *Myrica spp.*, and its CDS sequence is shown in SEQ ID NO:1. This invention obtains transgenic WRKY24 plants capable of early flowering under cooling-induced conditions by introducing the CDS sequence of the gene WRKY24 derived from *Myrica spp.* into plants, thereby effectively improving the plant's ability to flower early under cooling-induced conditions.

Description

Technical Field

[0001] This invention relates to plant genetic engineering technology, and more particularly to the application of the gene WRKY24 in inducing early flowering in plants by cooling. Background Technology

[0002] Over a long period of evolution, plants have developed mechanisms for sensing and responding to environmental changes. Faced with adverse environments, they can continuously monitor external changes and adjust their growth and development strategies in real time. Among these, the regulation of flowering time is crucial for plant adaptation to adversity, affecting their adaptability and reproductive capacity. The main environmental factors affecting flowering include light intensity, light quality, photoperiod, high and low temperatures, drought, nutrients, and salinity. In particular, when encountering low temperatures (e.g., 0℃-15℃), some plants will prematurely shift from vegetative growth to reproductive growth, especially when seed yield is expected to be damaged, and will flower early to ensure partial reproduction. This is called low-temperature stress-induced early flowering.

[0003] The goal and direction of researchers studying the induction of early flowering in plants by low-temperature stress is to induce the expression of genes for early flowering, clone them, and propagate and cultivate plant species, varieties, and ecotypes that exhibit low-temperature stress-induced early flowering. However, low-temperature stress has high temperature requirements and is costly; therefore, how to induce early flowering in plants under cooling conditions has become a recent research objective. Summary of the Invention

[0004] This invention provides the application of the gene WRKY24 in inducing early flowering in plants under cooling conditions. The gene WRKY24 is derived from *Myrica spp.*, and its CDS sequence is shown in SEQ ID NO:1. This invention obtains transgenic WRKY24 plants that flower early under cooling-induced conditions by introducing the CDS sequence of the gene WRKY24 derived from *Myrica spp.* into plants, thereby effectively improving the plant's ability to flower early under cooling-induced conditions.

[0005] This invention provides a method for improving the ability of plants to flower early under cooling-induced conditions. This invention effectively improves the ability of plants to flower early under cooling-induced 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] This invention also provides an application of the gene WRKY24 in plant breeding. The gene WRKY24 is derived from thinly flowering juniper, and its CDS sequence is shown in SEQ ID NO:1. This invention utilizes a recombinant expression vector to overexpress the gene WRKY24 for breeding early-flowering plant varieties induced by cooling.

[0007] Cooling-induced early flowering refers to the phenomenon where plants undergoing temperature drops may prematurely transition from vegetative to reproductive growth, thus prompting them to flower earlier. This method has important applications in agriculture; for example, by artificially controlling cooling, the flowering time of certain crops can be adjusted to avoid unfavorable climatic conditions or meet market demands, resulting in significant economic benefits.

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

[0009] In detail, the gene WRKY24 originates from *Myricaria laxiflora*. *Myricaria laxiflora* is an erect shrub belonging to the Tamarixaceae family and the *Myricaria* genus, reaching up to 1.5 meters in height. It is distributed in Zigui and Badong counties of Hubei Province and the Yangtze River estuary in Wushan County, Sichuan Province, China. It commonly grows along roadsides and riverbanks and is an excellent species for riverbank sand fixation and greening. The gene WRKY24 belongs to the plant WRKY transcription factor family. The plant WRKY transcription factor family consists of plant-specific zinc finger transcription factors, named for their highly conserved seven-amino acid sequence composed of WRKYGQK.

[0010] The CDS sequence of the WRKY24 gene is 1911 bp, as shown in SEQ ID NO:1. A CDS (Coding sequence) is a DNA sequence that corresponds one-to-one with a protein sequence, and contains no other sequences that do not correspond to that protein. The WRKY24 gene encodes 636 amino acids, and its amino acid sequence is shown in SEQ ID NO:2. Transforming the WRKY24 gene's CDS sequence into plants yields transgenic WRKY24 plants that flower earlier under cooling-induced conditions, thus effectively improving the plant's ability to flower earlier under these conditions.

[0011] In one specific embodiment, the present invention overexpresses the gene WRKY24 in plants, thereby improving the ability of plants to flower early under cooling-induced conditions.

[0012] Overexpression of genes refers to cloning the CDS sequence of a target gene into a corresponding plasmid or viral vector, and using regulatory elements constructed on the vector backbone to enable the gene to be transcribed and translated in large quantities under artificial control.

[0013] Specifically, overexpressing the WRKY24 gene in plants involves the following steps:

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

[0015] 2) The ligation product is introduced into the transformants, and transformants containing the recombinant expression vector are screened.

[0016] 3) Transgenic plants overexpressing the WRKY24 gene were obtained by infecting plants with transformants containing recombinant expression vectors.

[0017] In this invention, recombinant expression vectors refer 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 it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements.

[0018] In this invention, a transformant refers to a bacterial cell or other recipient cell that has acquired a new genetic marker after incorporation or introduction of exogenous DNA.

[0019] In this invention, a transgenic plant refers to a plant that possesses genes from other species.

[0020] By constructing recombinant expression vectors and using transgenic technology, transgenic plants overexpressing the WRKY24 gene can be obtained. After overexpressing the WRKY24 gene, these transgenic plants showed improved ability to flower early under cooling-induced conditions.

[0021] Steps 1)-3) of this invention can all be carried out using conventional techniques in the field, and this invention does not impose further limitations on them.

[0022] Furthermore, the plant is Arabidopsis thaliana.

[0023] A second aspect of this invention provides a method for improving the early flowering ability of plants under cooling-induced conditions, comprising: overexpressing the gene WRKY24 in the plant. The gene WRKY24 is derived from the thin-flowered juniper, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:1. This invention effectively improves the early flowering ability of plants under cooling-induced conditions by overexpressing the gene WRKY24 in the plant, and this has been verified in the model plant Arabidopsis thaliana.

[0024] In one specific implementation, a method for improving the ability of plants to flower earlier under cooling-induced conditions includes the following steps:

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

[0026] The ligation product was introduced into the transformants, and transformants containing the recombinant expression vector were screened.

[0027] Transgenic plants overexpressing the WRKY24 gene were obtained by infecting plants with transformants containing recombinant expression vectors.

[0028] Transgenic plants overexpressing the WRKY24 gene, obtained by the method of this invention, possess the ability to flower early under cooling-induced conditions. Specifically, these transgenic plants can flower early after being subjected to 18°C ​​stress for 7 days. In existing technologies, promoting early flowering under low-temperature stress generally requires treating plants with low-temperature stress of 0°C-15°C to induce high expression of the early-flowering gene, thereby achieving the effect of early flowering. However, the cooling-induced conditions of this invention only require reaching 18°C. The temperature requirements of this invention are simple and easy to implement, and the cost is correspondingly reduced.

[0029] In addition, transgenic plants that overexpress the WRKY24 gene had a gibberellin content of no less than 40 mg / L in their leaves 7 days after the temperature was lowered to 18°C.

[0030] Furthermore, the plant is Arabidopsis thaliana.

[0031] The third aspect of this invention provides the application of the gene WRKY24 in plant breeding, wherein the gene WRKY24 is derived from the thin-flowered juniper, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:1.

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

[0033] 1) This invention discloses the application of the gene WRKY24 in the early flowering of plants induced by cooling. By introducing the CDS sequence of the gene WRKY24 derived from the thin-flowered juniper into the plant, it was found that the gene WRKY24 has the ability to improve the plant's ability to flower early under cooling-induced conditions.

[0034] 2) Currently, early flowering techniques generally induce flower bud differentiation earlier through agronomic measures and hormone spraying. This invention, however, promotes high expression of the WRKY24 gene in plants under cooling-induced conditions via transgenic methods, thereby increasing the content of plant hormones such as gibberellin and causing early flowering. This significantly reduces the waste of labor costs and helps economically valuable plants (such as vegetables and fruits) to flower and bear fruit earlier, thus providing the market with early-ripening produce and yielding significant economic benefits.

[0035] 3) In the prior art, promoting early flowering of plants under low temperature stress generally requires treating the plants with low temperature stress of 0℃-15℃ to promote high expression of early flowering genes, thereby achieving the effect of early flowering. However, the cooling induction conditions of the present invention only need to reach 18℃. The temperature requirements of the present invention are simple and easy to implement, and the cost is also reduced accordingly. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is an electrophoresis image of the colony PCR products from Example 1;

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

[0039] Figure 3 This is a diagram showing the relative expression levels of gene WRKY24 in the stems and leaves of transgenic Arabidopsis thaliana 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 The image shows that Arabidopsis thaliana plants in the blank control group of Example 6 did not exhibit early flowering.

[0042] Figure 6 The image shows the Arabidopsis thaliana plants in the negative control group of Example 6, which did not exhibit early flowering. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining 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 embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents and carriers used are commercially available or publicly available.

[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 derived from sparsely-flowered juniper.

[0049] RNA was extracted from the leaves of *Myricaria sparsely-flowered* and reverse transcribed into cDNA. Primers 22OV100-F and 22OV100-R were designed based on the WRKY24 gene sequence. The CDS sequence of the WRKY24 gene was amplified using PCR with cDNA as a template. The PCR reaction conditions are shown in Table 2, specifically including 94℃ pre-denaturation for 5 minutes, 32 cycles: 98℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 68℃ extension for 2 minutes, final extension at 68℃ for 5 minutes, and then incubation at 25℃ for 1 minute. The PCR product was subjected to agarose gel electrophoresis and gel recovery to obtain the CDS sequence of the WRKY24 gene derived from *Myricaria sparsely-flowered*. This CDS sequence is 1911 bp, as shown in SEQ ID NO:1; the gene encodes 636 amino acids, the amino acid sequence of which is shown in SEQ ID NO:2.

[0050] Table 2

[0051]

[0052] Example 2: Construction of recombinant expression vector

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

[0054] The ligation product was added to the Agrobacterium competent cell suspension, and the mixture was heat-shocked at 42°C for 90 seconds (without shaking), followed by another heat shock for 16 seconds, and then cooled on ice for 2 minutes. 400 μL of LB broth was then added, and the mixture was incubated at 37°C with shaking at 180 rpm for 40 minutes to obtain the transformed bacterial culture. At this point, the bacteria in the transformed culture had returned to normal growth and expressed the antibiotic resistance gene (kanamycin) encoded by the expression vector. 80 μL of the transformed bacterial culture was evenly spread onto a selection plate containing kanamycin. After the transformed bacterial culture was completely absorbed by the plate, the selection plate was inverted and incubated for 12 hours. Single colonies from the selection plate were selected and cultured in LB broth containing kanamycin to obtain single-clone bacterial cultures. Colony PCR and agarose gel electrophoresis were performed using primers 22OV100-F and 22OV100-R to detect positive clones. (See details...) Figure 1 Positive clones were selected for sequencing verification. The sequencing primers were A580-seqR and PCB-seqE. The recombinant expression vectors that produced correct sequencing results were successfully constructed.

[0055] Example 3: Infection of Arabidopsis thaliana

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

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

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

[0059] Table 3

[0060]

[0061] Based on the Cq values ​​of the amplified internal reference gene and the target gene, and using the expression value of WRKY24 overexpressed in the transgenic plant sample numbered 22ATH42-1 as 1, the expression level of the target gene in each sample was calculated. (See details at [link to relevant documentation]). Figure 2 Among them, the transgenic Arabidopsis plant numbered 22ATH43-4 showed the highest expression level of the WRKY24 gene, and the transgenic Arabidopsis plant of WRKY24 used in subsequent experiments refers to this plant.

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

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

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

[0065] Transgenic WRKY24 Arabidopsis thaliana plants were sown. When seedlings reached the sixth true leaf, they were placed in a low-temperature (18℃) incubator for 7 days, serving as the experimental group. Wild-type Arabidopsis thaliana seedlings with the sixth true leaf were placed in a normal-temperature (23-25℃) incubator for 7 days, serving as the blank control group. Wild-type Arabidopsis thaliana seedlings with the sixth true leaf were placed in a low-temperature (18℃) incubator for 7 days, serving as the negative control group. Subsequently, the experimental group, blank control group, and negative control group were all moved to a normal-temperature (23-25℃) environment for 33 days. The flowering status of the Arabidopsis thaliana in the experimental group, blank control group, and negative control group was observed. The flowering status of the experimental group is shown in [details omitted]. Figure 4 The flowering status of Arabidopsis thaliana in the blank control group is shown in the figure. Figure 5 The flowering status of Arabidopsis thaliana in the negative control group is shown in the figure. Figure 6 .

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

[0067] Example 7: Gibberellin content in leaves of transgenic Arabidopsis thaliana plants

[0068] Gibberellins (GAs) are an important class of plant hormones involved in multiple biological processes, including plant growth and development. They play a crucial role in promoting plant cell elongation, seed germination, bud growth, flower development, and fruit ripening. GAs can break plant dormancy, promote flower bud differentiation, and thus induce earlier flowering. Leaves from the experimental group plants in Example 6, cultured at 18°C ​​for 7 days, were used to determine the gibberellin content.

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

[0070] In summary, this invention involves introducing the CDS sequence of the gene WRKY24, derived from *Myrica spp.*, into *Arabidopsis thaliana* plants, resulting in transgenic *Arabidopsis thaliana* plants with the gene WRKY24. The gene WRKY24 is highly expressed in the stems and leaves of these transgenic plants, and due to the introduction of WRKY24, the transgenic plants acquire the ability to flower earlier under cooling-induced conditions. Furthermore, the gibberellin content in their leaves is significantly higher than that of wild-type *Arabidopsis thaliana* plants. It is anticipated that this gene can also be used to enhance the ability of other plants to flower earlier under cooling-induced conditions. Therefore, this invention utilizes a recombinant expression vector to overexpress the gene WRKY24 for plant breeding, ultimately obtaining plant varieties that flower earlier under cooling-induced conditions.

[0071] Currently, early flowering techniques generally involve inducing flower bud differentiation through agronomic measures and hormone spraying. This invention, however, utilizes transgenic methods and, under cooling-induced conditions, promotes high expression of the WRKY24 gene in plants, thereby increasing the content of plant hormones such as gibberellin and causing early flowering. This significantly reduces labor costs and helps economically valuable plants (such as vegetables and fruits) flower and bear fruit earlier, ultimately providing the market with early-ripening produce and offering substantial economic benefits.

[0072] Compared with other methods for inducing early flowering in plants by low temperature stress, this invention has lower requirements for induction conditions. In the prior art, plants are generally treated with low temperature of 0℃-15℃ to promote high expression of early flowering genes, thereby achieving the effect of early flowering. However, the cooling induction conditions of this invention only need to reach 18℃, which has lower temperature requirements and correspondingly lower costs.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of gene WRKY24 in inducing early flowering in plants by cooling, characterized in that, The gene WRKY24 is derived from sparsely flowering juniper, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:1; Overexpression of the WRKY24 gene in plants enhances their ability to flower early under cooling-induced conditions. The plant in question is Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, Overexpressing the WRKY24 gene in plants involves the following steps: The CDS sequence of the WRKY24 gene was constructed into an expression vector to obtain the ligation product; The ligation product was introduced into the transformant, and transformants containing the recombinant expression vector were obtained by screening. Transgenic plants overexpressing the WRKY24 gene were obtained by infecting plants with the transformants containing the recombinant expression vector.

3. A method for improving the ability of plants to flower earlier under cooling-induced conditions, characterized in that, include: The gene WRKY24, derived from sparsely flowering juniper, was overexpressed in the plant. The CDS sequence of the gene WRKY24 is shown in SEQ ID NO:

1. The plant in question is Arabidopsis thaliana.

4. The method according to claim 3, characterized in that, Includes the following steps: The CDS sequence of the WRKY24 gene was constructed into an expression vector to obtain the ligation product; The ligation product was introduced into the transformant, and transformants containing the recombinant expression vector were obtained by screening. Transgenic plants overexpressing the WRKY24 gene were obtained by infecting plants with the transformants containing the recombinant expression vector.

5. The method according to claim 4, characterized in that, The transgenic plants were able to flower earlier after being subjected to 18°C ​​stress for 7 days.

6. The method according to claim 5, characterized in that, After being subjected to 18°C ​​stress for 7 days, the gibberellin content in the leaves of the transgenic plants was not less than 40 mg / L.

7. The application of gene WRKY24 in plant breeding, characterized in that, The gene WRKY24 is derived from the thin-flowered juniper, and the CDS sequence of the gene WRKY24 is shown in SEQ ID NO:

1. Overexpression of the gene WRKY24 in the plant enhances the plant's ability to flower early under cooling-induced conditions. The plant is Arabidopsis thaliana.

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