Transcription factor Ntwrky99 for regulating plant aging, nucleic acid and application of transcription factor Ntwrky99

Abstract

The invention provides a transcription factor related to tobacco negative regulation senescence, namely tobacco Ntwrky99, a transcription factor related to tobacco negative regulation senescence and a transcription factor related to tobacco negative regulation senescence. Through transcriptome sequencing analysis, the tobacco gene Ntwrky99 of which the expression quantity is remarkably reduced in the aging period of tobacco leaves is screened, fluorescent quantitative PCR verifies that the expression quantity of the gene in the aging leaves is reduced by about 3 times, and after the transcription factor gene is cloned, an overexpression vector and an overexpression recombinant strain are constructed, it is proved that compared with a wild type, the transcription factor gene has the advantages that the expression quantity of the tobacco gene Ntwrky99 is reduced by about 3 times; the overexpressed plant can obviously delay senescence of arabidopsis thaliana leaves. Therefore, the Ntwrky99 gene is proved to be a negative senescence regulation gene. Therefore, functional identification of the tobacco Ntwrky99 gene not only enriches gene resources of plant aging breeding, but also is beneficial to analysis of diversity functions of tobacco WRKY transcription factors, and has important theoretical significance and application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a transcription factor Ntwrky99 for regulating plant senescence, a nucleic acid and an application thereof. Background Art

[0002] The mature harvest of tobacco leaves is an important link in the production of flue-cured tobacco. The mature harvest of tobacco leaves directly determines the availability value of tobacco leaves as raw materials for the cigarette industry. Tobacco leaves are harvested in five or six times according to the position and maturity, which is difficult to achieve full automatic mechanization. In addition, multiple manual harvests are labor-intensive, costly and inefficient. Reducing the number of harvests and promoting the "four-time harvest" mechanized agronomic technology requires suitable varieties with relatively concentrated maturity. The maturity characteristics of the existing main cultivated varieties are "layered yellowing". At present, there is an extreme lack of varieties and technologies suitable for mechanized "four-time harvesting" in tobacco production. Therefore, it is necessary to screen and cultivate new varieties with concentrated maturity in the process of promoting the mechanization of tobacco production.

[0003] Discovering the key genes that regulate the concentrated maturity of tobacco leaves is an effective means to research and develop new varieties of concentrated maturity of tobacco. However, at present, there are still few genes in tobacco that have been clearly identified to have the function of maturity and senescence. It is urgent to discover and identify genes that negatively regulate maturity and senescence. Summary of the invention

[0004] In order to regulate the aging of plants, especially tobacco, and cultivate new tobacco varieties with concentrated maturity, the present invention proposes a transcription factor Ntwrky99, a nucleic acid and its application for regulating plant aging. The present invention transforms the Ntwrky99 gene into Arabidopsis to obtain a transgenic strain that can significantly delay leaf aging compared with the wild type. The above purpose can be achieved by the following technical solutions:

[0005] The transcription factor Ntwrky99 for regulating plant senescence, wherein the amino acid sequence of the transcription factor Ntwrky99 for regulating plant senescence is shown in SEQ ID No.1.

[0006] The present invention also provides a nucleic acid encoding the transcription factor Ntwrky99 for regulating plant senescence.

[0007] The nucleotide sequence of the nucleic acid is shown in SEQ ID No.2.

[0008] The present invention also provides a vector, wherein the vector comprises the above nucleic acid.

[0009] The present invention also provides a primer for amplifying the above-mentioned nucleic acid, characterized in that it comprises:

[0010] Ntwrky99-upstream primer, the nucleotide sequence is shown in SEQ ID No.3;

[0011] Ntwrky99-downstream primer, the nucleotide sequence is shown in SEQ ID No.4.

[0012] The present invention also provides a kit, comprising at least one of the above-mentioned transcription factor Ntwrky99 for regulating plant senescence, nucleic acid, vector and primer.

[0013] The present invention also provides the use of the transcription factor Ntwrky99, nucleic acid, vector, primer and kit for regulating plant senescence in regulating plant senescence.

[0014] The present invention also provides a method for regulating plant senescence, characterized in that it comprises the following steps:

[0015] The above-mentioned transcription factor Ntwrky99 for regulating plant senescence is overexpressed in plants to delay plant senescence.

[0016] The above method for regulating plant senescence specifically comprises the following steps:

[0017] Step 1) amplifying the nucleic acid encoding the transcription factor Ntwrky99 for regulating plant senescence to obtain an amplified fragment of the Ntwrky99 gene;

[0018] Step 2) constructing an Ntwrky99 overexpression vector using the amplified fragment of the Ntwrky99 gene;

[0019] Step 3) The Ntwrky99 overexpression vector is introduced into plants to overexpress the transcription factor Ntwrky99 for regulating plant senescence in the plants.

[0020] Optionally, the Ntwrky99 overexpression vector in the step 2) is a pCHF3 vector comprising a 35S promoter and a NOS terminator;

[0021] Optionally, in the step 3), the Ntwrky99 overexpression vector is transferred into the plant by Agrobacterium-mediated method;

[0022] Preferably, the plant is Arabidopsis thaliana.

[0023] The present invention has the following technical effects

[0024] The present invention proposes a transcription factor related to the negative regulation of tobacco aging, namely, the tobacco Ntwrky99 gene, which can provide new gene resources and theoretical basis for molecular assisted breeding of crops. The present invention screens the tobacco gene Ntwrky99 whose expression level is significantly reduced during the tobacco leaf aging period by transcriptome sequencing analysis, verifies that the expression level of the gene in the aging leaves is reduced by about 3 times by fluorescent quantitative PCR, and then clones the gene, constructs an overexpression vector, and overexpresses the recombinant strain, and transforms it into Arabidopsis to obtain the transgenic Arabidopsis plant Ntwrky99-OX, and confirms that compared with the wild type, Ntwrky99-OX can significantly delay the aging of Arabidopsis leaves. This proves that the Ntwrky99 gene is a negative regulation aging gene. Therefore, the functional identification of the tobacco Ntwrky99 gene not only enriches the gene resources for plant aging breeding, but also helps to analyze the diverse functions of tobacco WRKY transcription factors, which has important theoretical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the expression level of Ntwrky99 gene, where the vertical axis is the relative expression level and the horizontal axis is the leaf development period.

[0026] Figure 2 Identification of senescence of Ntwrky99 transgenic Arabidopsis. In the figure: a is the delay of leaf yellowing by overexpressing NtWRKY99 in Arabidopsis, c is the relative expression of NtWRKY99 in three overexpression lines; d is the photosynthetic rate result of OX-6 / Col-0 detached leaves, e is the result of chlorophyll content of OX-6 / Col-0, f is the result of ion leakage of OX-6 / Col-0, g is the result of relative expression of OX-6 / Col-0 SAG12. DETAILED DESCRIPTION

[0027] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used can be purchased from biological or chemical reagent companies.

[0028] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0031] The present invention screened a tobacco gene Ntwrky99 that negatively regulates senescence genes and whose expression level is significantly reduced through transcriptome sequencing analysis. Its nucleotide sequence is shown in SEQ ID No. 2, and the encoded amino acid sequence is shown in SEQ ID No. 1. RT-qPCR verification found that compared with the mature stage, the expression level of the Ntwrky99 gene in tobacco senescent leaves was reduced by about 3 times, and it was preliminarily determined that the gene plays an important role in tobacco's participation in regulating tobacco leaf senescence.

[0032] SEQ ID No.1:

[0033] MESPLPEKSSADLKRAIDGLIRGQEFTRQLKEIIKKPLATIMAEDLVGKIMNSFSETLSVINSGESDEDTAEVKSPEDSSGSCKSTTTSFKDRRGCYKRRKTSETNTKESSDLVDDGHAWRKYGQKQILHSTYPRHYFRCTHKYDQNCQATKQVQKIQDNPP LFRTTYYGNHTCKPFPRVSQIILDTPIHGDSSILLSFDRNNNNNNYSSIQNVNYNYQPYNIPTFPSIKQETKEEVFQRSCTFYPKIEDQNQSSNSDNFLLANDDHLSTLTVFEASDGHMAATLSPDVISSGVYSSCTTSTDNLEIDFDFEESLWNFEGYNS.

[0034] SEQ ID No.2

[0035] ATGGAGTCTCCGTTGCCGGAAAAGTCATCAGCGGATCTGAAAAGGGCAATCGACGGGTTAATTCGTGGCCAGGAATTTACACGACAATTAAAAGAGATAATCAAGAAACCTCTTGCAACCATTATGGCTGAGGATTTAGTTGGAAAAATTATGAATTCATTTTCTGAGACTCTTTCCGTCATAAACTCCGGCGAGTCCGATGAGGATACCGCCGAAGTTAAGTCGCCGGAAGATTCTAGTGGAAGTTGCAAGAGTACTACTACATCATTCAAAGATCGAAGAGGATGCTACAAGAGAAGGAAAACTTCAGAAACAAACACAAAAGAATCCTCAGATTTGGTGGATGATGGCCATGCTTGGAGAAAATATGGACAAAAACAGATCCTCCATTCCACTTATCCAAGGCACTATTTTAGGTGCACCCATAAATATGATCAAAATTGTCAAGCAACCAAACAGGTGCAGAAAATTCAAGACAATCCACCACTGTTCCGGACAACATACTATGGAAATCACACATGCAAACCTTTCCCTAGAGTTTCTCAAATAATTTTGGATACTCCTATTCATGGCGATTCCTCTATTCTACTTAGTTTTGATCGTAACAACAACAATAATAACTACTCTTCAATCCAAAACGTTAATTATAATTACCAGCCTTATAATATTCCTACATTTCCCTCAATAAAACAGGAAACCAAAGAGGAAGTATTCCAAAGATCATGTACTTTCTATCCAAAAATAGAAGATCAAAACCAATCATCAAACTCTGATAATTTTCTACTAGCCAATGATGATCATCTGAGTACTCTTACAGTATTTGAAGCCTCCGACGGCCATATGGCGGCGACATTATCGCCGGATGTCATATCATCTGGGGTCTATTCTTCTTGTACGACTAGTACAGATAATCTTGAGATTGATTTTGATTTTGAGGAGAGTCTTTGGAACTTTGAAGGGTACAATTCTTGA。

[0036] The present invention also provides a vector, wherein the vector comprises the above nucleic acid.

[0037] The present invention also provides a primer for amplifying the above-mentioned nucleic acid, comprising:

[0038] Ntwrky99-upstream primer, the nucleotide sequence is: GGATCCATGGAGTCTCCGTTGCCG; Ntwrky99-downstream primer, the nucleotide sequence is: GGATCCAGAATTGTACCCTTCAAAGTTCCA.

[0039] The present invention also provides a kit, comprising at least one of the above-mentioned transcription factor Ntwrky99 for regulating plant senescence, nucleic acid, vector and primer.

[0040] The present invention also provides the use of the transcription factor Ntwrky99, nucleic acid, vector, primer and kit for regulating plant senescence in regulating plant senescence.

[0041] The present invention also provides a method for regulating plant senescence, comprising the following steps:

[0042] The above-mentioned transcription factor Ntwrky99 for regulating plant senescence is overexpressed in plants to delay plant senescence.

[0043] The above method for regulating plant senescence specifically comprises the following steps:

[0044] (1) Amplify the tobacco negative regulator of aging gene Ntwrky99;

[0045] (2) The tobacco negative regulator of senescence gene Ntwrky99 was cloned, and after enzyme digestion and purification, it was connected to the expression vector to obtain an overexpression recombinant vector;

[0046] (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0047] (4) Transforming the overexpression recombinant strain into plants, screening and obtaining the Ntwrky99 transgenic Arabidopsis strain, so as to improve the plant's anti-aging ability.

[0048] The reaction conditions of the amplification were as follows: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 57°C for 10s, and 72°C for 50s, for 32 cycles; and post-extension at 72°C for 10min.

[0049] The expression vector in step (2) is a pCHF3 vector, which contains a 35S promoter and a NOS terminator.

[0050] The plant is Arabidopsis thaliana, but is not limited to Arabidopsis thaliana.

[0051] Example 1 Verification of the involvement of the Ntwrky99 gene in tobacco leaf aging

[0052] The following are the specific steps of the cloning method and application of the tobacco negative regulatory senescence gene Ntwrky99:

[0053] 1. Using the coding region sequence of tobacco Ntwrky99 gene as a reference, design specific primers Ntwrky99-F and Ntwrky99-R. The primer sequences are as follows:

[0054] Ntwrky99-F:GGATCCATGGAGTCTCCGTTGCCG-3';

[0055] Ntwrky99-R:GGATCCAGAATTGTACCCTTCAAAGTTCCA-3'.

[0056] 2. Extract the mRNA from the seedling leaves of the tobacco cultivar "K326", reverse transcribe it into cDNA, and use it as a template for PCR amplification. Use the primers in step 1 to perform PCR amplification to obtain the amplified fragment of the Ntwrky99 gene.

[0057] The PCR amplification system is:

[0058]

[0059] The PCR reaction conditions were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 57°C for 10 s, and 72°C for 50 s, for 32 cycles; and post-extension at 72°C for 10 min.

[0060] 3. Analysis of the expression pattern of Ntwrky99 in tobacco leaves at different stages

[0061] The tobacco variety "K326" and tobacco leaves at the mature and senescent stages were used as materials, and the changes in the expression of the Ntwrky99 gene were analyzed using fluorescent quantitative PCR technology.

[0062] Among them, the primer sequences of fluorescent quantitative PCR are:

[0063] RT-Nt-NtWRKY99-F:5'-AACTACTCTTCAATCCAAAACGTT-3'

[0064] RT-Nt-NtWRKY99-R:5'-CCGTCGGAGGCTTCAAATACT-3'

[0065] The results are as follows Figure 1 As shown, the Ntwrky99 gene expression in the early stage of tobacco leaf maturity was used as the control ( Figure 1, where the vertical axis is the relative expression level and the horizontal axis is the leaf development period), it was found that the expression of Ntwrky99 gene was significantly downregulated in the late mature stage of tobacco leaves, decreasing by about 3 times. It was preliminarily verified that Ntwrky99 gene was involved in the senescence process of tobacco leaves, and Ntwrky99 gene negatively regulated leaf senescence.

[0066] Example 2 Construction of Ntwrky99 overexpression vector and transgenic plants

[0067] In this example, tobacco leaf cDNA was used as the test material, and the Ntwrky99 gene fragment was obtained by PCR amplification; after the expression vector was constructed by enzyme cutting and ligation, it was transferred to Arabidopsis thaliana by Agrobacterium infection, and the transgenic Arabidopsis thaliana plants with Ntwrky99 gene were obtained, and it was confirmed that the gene can significantly delay aging, thereby proving that the Ntwrky99 gene is a gene that negatively regulates plant aging. Therefore, the functional identification of the tobacco Ntwrky99 gene analyzed the diverse functions of tobacco WRKY transcription factors, enriched the breeding gene resources of plants in aging, and has important theoretical significance and application value. Specifically, it includes the following steps:

[0068] (1) Construct an overexpression vector of Ntwrky99.

[0069] Extract the mRNA from the seedling leaves of tobacco cultivar "K326" and reverse transcribe it into cDNA as a template for PCR amplification. Use the specific primers Ntwrky99-F and Ntwrky99-R in Example 1 to perform PCR amplification to obtain the amplified fragment of the Ntwrky99 gene.

[0070] The PCR amplification system is:

[0071]

[0072] The PCR reaction conditions were as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 57°C for 10 s, and 72°C for 50 s, for 32 cycles; and post-extension at 72°C for 10 min.

[0073] Then the amplified fragment of the Ntwrky99 gene was connected to the subcloning vector pCE2 TA / Blunt-Zero, and the plasmid was extracted after sequencing. Then the purified amplified product was connected to the pCHF3 vector digested with BamH I by homologous recombination. The primers with adapters for amplification of the Ntwrky99 gene used in the homologous recombination process have the following primer sequences:

[0074] pCHF3-NtWRKY99-F:

[0075] 5'-GAGAACACGGGGGGACGGATCCATGGAGTCTCCGTTGCCG-3'

[0076] pCHF3-NtWRKY99-R:

[0077] 5'-GGATCCCCGGGTACCGGATCCTCAAGAATTGTACCCTTCAAAGTTCC-3'

[0078] After the obtained pCHF3 vector was sequenced correctly, the pCHF3 vector was transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation to obtain Arabidopsis thaliana plants overexpressing Ntwrky99.

[0079] Agrobacterium-mediated transformation is an efficient plant transgenic technology that uses Agrobacterium to introduce foreign genes into plant cells to improve genetic characteristics. This method has many advantages, including high transformation frequency, the ability to introduce large fragments of DNA, low copy number, compliance with Mendelian inheritance, and a wide host range. Agrobacterium is a Gram-negative bacterium whose T-DNA on the Ti plasmid can be transferred to plant cells through conjugation. Scientists use molecular cloning technology to replace the tumorigenic gene in T-DNA with the target gene, making Agrobacterium an effective gene transfer tool. When Agrobacterium infects a plant, the T-DNA is cut out and integrated into the plant genome, a process achieved through the DNA repair mechanism of plant cells.

[0080] Agrobacterium GV3101 was transformed by freeze-thaw method, and Arabidopsis thaliana was genetically transformed by inflorescence infiltration method to obtain transgenic Ntwrky99 Arabidopsis thaliana plants. Two overexpression strains were obtained by the above method and named "OX-NtWRKY99-2" and "OX-NtWRKY99-6" respectively.

[0081] Test example

[0082] Two overexpression strains were created in Arabidopsis thaliana by the steps of Example 2, and were named "OX-NtWRKY99-2" and "OX-NtWRKY99-6" respectively. The relative expression levels of the genes were detected by RT-PCR and compared with those of the non-overexpressed Arabidopsis thaliana plant Col-0:

[0083] 1) Extract RNA from the overexpression strain and convert it into cDNA for qRT-PCR to detect the expression level. The qRT-PCR primer sequences are as follows:

[0084] RT-At-NtWRKY99-F:5'-AACTACTCTTCAATCCAAAACGTT-3'

[0085] RT-At-NtWRKY99-R:5'-CCGTCGGAGGCTTCAAATACT-3'

[0086] The results are as follows Figure 2 As shown in c, the relative expression level of NtWRKY99 gene in the two overexpression strains is as high as more than 200 times, proving that these two strains have great reliability for phenotypic identification.

[0087] 2) Identification and analysis of the aging phenotype of transgenic Ntwrky99 Arabidopsis plants.

[0088] The above two expression lines "OX-NtWRKY99-2" and "OX-NtWRKY99-6" were cultivated to obtain the T3 generation homozygous lines of "OX-NtWRKY99-2" (abbreviated as OX-2) and "OX-NtWRKY99-6" (abbreviated as OX-6), respectively. The T3 generation homozygous lines were sown in 1 / 2MS medium, vernalized in a refrigerator at 4°C for two days, and cultured under light for 6 days. The seedlings with relatively consistent growth were selected and planted in mixed nutrient soil (nutrient soil: vermiculite = 3:1, v / v). T3 generation transgenic plants and Col-0 were planted side by side in a 32-hole plug tray, with one plant planted in each hole. The planting conditions of all lines involved in phenotypic identification were kept consistent and planted twice. When the leaves showed obvious mature and yellowing phenotypes, the wild-type plants and transgenic plants under the same planting conditions were photographed and the phenotypes were recorded. Twelve rosette leaves of Arabidopsis were taken and arranged in the order of growth for photography and observation. The photosynthetic rate, chlorophyll content, ion leakage and relative expression of SAG12 in the detached leaves of the overexpression lines OX-NtWRKY99-6 and Col-0 with obvious differences in leaf yellowing were measured. All indicators were measured with three biological and technical replicates.

[0089] Leaf photosynthetic rate determination: The photosynthetic rate (Fv / Fm) of Arabidopsis leaves was measured using an OS-5p+ chlorophyll fluorescence meter. The specific operating steps are as follows: mark and number the leaf positions to be measured, and place them in the dark for 2 hours to obtain more accurate photosynthetic rate values; insert the measuring probe into the leaf clamp, clamp the leaf to be measured with the leaf clamp, and adjust the parameters so that the Ft range is 150-250 for measurement; perform three biological replicates and three technical replicates for each leaf position.

[0090] Determination of leaf chlorophyll content: When Arabidopsis leaves show obvious differences in senescence, the 4th, 5th, 6th, 7th, and 8th rosette leaves of the overexpression plants and wild-type Arabidopsis are taken for chlorophyll content determination. The specific method is as follows: After weighing the leaf position to be measured, place it in a 15mL centrifuge, add a certain amount of anhydrous ethanol to ensure that the leaves are completely immersed in anhydrous ethanol, wrap the centrifuge tube with tin foil to protect it from light, and collect the solution after placing it at room temperature for 24 hours. Using anhydrous ethanol as a blank control, use the Infinite M200 PRO microplate reader to measure the absorbance of each group of solutions at 665nm and 649nm, and record the data. Three biological replicates and three technical replicates were performed for each group; the chlorophyll content of Arabidopsis leaves was calculated according to the following formula:

[0091] Total chlorophyll content C (mg / g) = (C a +C b )×V / W

[0092] C a =13.95*A665-6.88*A649

[0093] C b =24.96*A649-7.32*A665

[0094] Wherein, V = 10 mL, W is the fresh weight of leaves.

[0095] Leaf ion leakage assay: Thermo Fisher Scientific A122 conductivity meter was used to measure the ion leakage rate of the leaves of the expression plants and wild-type Arabidopsis. The experimental steps are as follows: the leaf positions of the overexpression plants with obvious leaf senescence phenotype and the Col-0 wild-type plants were selected and numbered; the leaves were placed in a 15mL centrifuge tube, and a certain amount of ddH2O was added to immerse the leaves. The leaves were slowly shaken on a MULAB orbital shaker at room temperature for 30 minutes, and the conductivity R1 of the aqueous solution was measured with a conductivity meter; the centrifuge tube containing the leaves was heated and boiled for 10 minutes, and then the conductivity R2 of the aqueous solution was measured again after it was quickly cooled to room temperature on ice; the percentage of the conductivity measured twice before and after was the ion leakage rate of the leaves, and the calculation formula was: R1 / R2*100%.

[0096] Senescence marker gene expression determination: SAG12 gene was used as senescence marker gene, and OX-NtWRKY99-6 and Col-0 Arabidopsis plants were selected to determine the relative expression of SAG12 gene. SAG12 is a gene related to plant leaf senescence and belongs to senescence-specific promoter. The expression of SAG12 gene in Arabidopsis is highly tissue-specific and time-specific, and is mainly expressed in senescent leaves, so it is called senescence-specific gene. SAG12 gene plays an important regulatory role in the senescence of plant leaves. Its expression is regulated by a variety of environmental factors, especially when the leaves are senescent, its expression is upregulated. The protein encoded by SAG12 gene may be involved in metabolic processes related to leaf senescence, such as chlorophyll degradation and cell wall degradation. Therefore, SAG12 gene was used as senescence marker gene in this experiment.

[0097] Fluorescence quantitative PCR was used to determine the relative expression of the SAG12 gene, wherein the fluorescent quantitative PCR primer sequences used were:

[0098] RT-SAG12-F: 5'-ATGTTGTTGGGCGTTTTCAGC-3'

[0099] RT-SAG12-R: 5'-GCCGCCAGTCGCTTTTATAT-3'.

[0100] The results are as follows Figure 2As shown in the figure, after 40 days, the leaves of Col-0 showed obvious senescence phenotype, while the leaves of the overexpression lines "OX-NtWRKY99-2" and "OX-NtWRKY99-6" did not show or just showed senescence, and the yellowing degree of the 4th to 10th leaves was significantly lower than that of the wild type. The chlorophyll content of the leaves was detected, and it was found that the chlorophyll content of the 4th leaf of the overexpression line "OX-NtWRKY99-6" was significantly higher than that of the wild type. The photosynthetic rate measurement found that the photosynthetic rates of the 4th, 5th and 6th leaves of the overexpression line "OX-NtWRKY99-6" were significantly higher than those of the same leaves of Col-0. The ion leakage rate, as a sign of leaf senescence, is inversely proportional to the degree of senescence. The ion leakage rate of the 4th leaf of the overexpression line was significantly lower than that of the wild type, and the ion leakage rate of the 5th to 8th leaves was slightly lower than that of the wild type. This shows that the senescence degree of the 4th leaf of the overexpression plant is significantly lower than that of the wild type control. qRT-PCR was used to detect the differential expression level of the leaf senescence marker gene SAG12. The results showed that the expression level of the SAG12 gene in the overexpression strain "OX-NtWRKY99-6" was significantly lower than that in Col-0, indicating that the leaf development age of Col-0 was higher than that of the overexpression strain. Based on the above physiological index measurement results, it was shown that overexpression of the NtWRKY99 gene in Arabidopsis delayed the yellowing of leaves and delayed the maturity and yellowing of leaves compared with the wild type, which preliminarily indicated that NtWRKY99 was a negative regulatory factor regulating the maturity and yellowing of leaves, delaying the yellowing of leaves.

[0101] Obviously, the above embodiments are only examples for clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A transcription factor Ntwrky99 for regulating plant senescence, characterized in that: The amino acid sequence of the transcription factor Ntwrky99 for regulating plant senescence is shown in SEQ ID No.

1.

2. A nucleic acid encoding the transcription factor Ntwrky99 that regulates plant senescence as described in claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID No.

2.

4. A carrier, characterized in that The vector comprises the nucleic acid according to claim 2 or 3.

5. A primer for amplifying the nucleic acid of claim 2 or 3, characterized in that: include: Ntwrky99-upstream primer, the nucleotide sequence is shown in SEQ ID No.3; Ntwrky99-downstream primer, the nucleotide sequence is shown in SEQ ID No.

4.

6. A kit, characterized in that: It comprises at least one of the transcription factor Ntwrky99 for regulating plant senescence as described in claim 1, the nucleic acid as described in claim 2 or 3, the vector as described in claim 4, and the primer as described in claim 5.

7. Use of the transcription factor Ntwrky99 for regulating plant senescence according to claim 1, the nucleic acid according to claim 2 or 3, the vector according to claim 4, the primer according to claim 5, and the kit according to claim 6 in regulating plant senescence.

8. A method for regulating plant senescence, characterized in that: The steps include: The transcription factor Ntwrky99 for regulating plant senescence as claimed in claim 1 is overexpressed in plants to delay plant senescence.

9. The method for regulating plant senescence according to claim 8, characterized in that: The steps include: Step 1) amplifying the nucleic acid encoding the transcription factor Ntwrky99 for regulating plant senescence to obtain an amplified fragment of the Ntwrky99 gene; Step 2) constructing an Ntwrky99 overexpression vector using the amplified fragment of the Ntwrky99 gene; Step 3) The Ntwrky99 overexpression vector is introduced into plants to overexpress the transcription factor Ntwrky99 for regulating plant senescence in the plants.

10. The method for regulating plant senescence according to claim 9, characterized in that: The Ntwrky99 overexpression vector in the step 2) is a pCHF3 vector comprising a 35S promoter and a NOS terminator; Preferably, in the step 3), the Ntwrky99 overexpression vector is transferred into the plant by Agrobacterium-mediated method; Preferably, the plant is Arabidopsis thaliana.

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