SiERF109 gene for regulating drought resistance, salt tolerance and flavonoid accumulation of millet and application thereof

Abstract

This invention discloses a method for regulating the drought resistance, salt tolerance, and flavonoid accumulation of millet. SiERF109 This invention relates to genes and their applications, belonging to the field of plant breeding technology. Specifically, it describes the isolation and cloning of a gene from millet with the nucleotide sequence shown in SEQ ID NO. 1. SiERF109 Quantitative fluorescence expression analysis showed SiERF109 This gene is involved in the drought and salt stress response of millet. After overexpression of this gene in millet, overexpression showed significant effects in drought and salt stress experiments. SiERF109 Compared with wild-type lines, the gene-derived lines showed significantly higher survival rates and chlorophyll content, flavonoid content increased by 33.5%–39.5%, while reactive oxygen species accumulation was significantly reduced; this demonstrates that the gene provided by this invention... SiERF109 Genes and their encoded proteins can significantly improve a plant's drought resistance, salt tolerance, and flavonoid content.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for regulating drought resistance, salt tolerance, and flavonoid accumulation in millet. SiERF109 Genes and their applications. Background Technology

[0002] In recent years, the consumption demand for millet, as well as farmers' planting income and willingness to cultivate it, have all shown a significant upward trend. However, the unclear molecular mechanisms regulating drought and salt tolerance and the synthesis of health-promoting substances in millet, along with the scarcity of high-quality drought- and salt-tolerant germplasm available, are the main factors limiting its production. Therefore, identifying genes related to drought and salt tolerance and the synthesis of health-promoting substances in millet can provide new gene resources and bio-breeding technology support for molecular breeding of millet to improve stress resistance and quality, which is of great significance for production.

[0003] The AP2 / ERF family is one of the largest transcription factor families in the plant kingdom. The ERF subfamily contains a conserved AP2 / ERF domain, whose cis-acting elements can respond to a variety of responses.

[0004] Studies have shown that ERF transcription factors are closely related to plant growth and development, biotic and abiotic stress responses, and biosynthesis. In Arabidopsis, the AP2 / ERF transcription factor TINY regulates drought by activating drought-responsive genes and closing stomata. In eggplant, silencing the SmERF1 transcription factor significantly downregulates the expression levels of salt stress defense-related genes, reduces the synthesis of superoxide dismutase and catalase, and promotes the production of hydrogen peroxide (H2O2) and proline. Overexpression of soybean ERF7 in tobacco increases chlorophyll content and decreases malondialdehyde content, thereby improving the salt tolerance of tobacco. ERF transcription factors respond to drought and salt stress damage through multiple physiological and metabolic pathways within plants.

[0005] Therefore, ERF is essential for plant growth, development, and stress resistance. However, there are few reports on the simultaneous regulation of drought resistance, salt tolerance, and flavonoid content by members of the ERF family in millet. Summary of the Invention

[0006] To address the problem of insufficient genetic resources for stress resistance and high-quality breeding in millet in existing technologies, this invention provides a method for regulating drought resistance, salt tolerance, and flavonoid accumulation in millet. SiERF109 Genes and their applications provide new resources for molecular breeding of stress-resistant and high-quality millet.

[0007] To achieve the above objectives, on the one hand, the present invention provides a method for regulating the drought resistance, salt tolerance, and flavonoid accumulation of millet. SiERF109 Gene, SiERF109 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Preferred, SiERF109 The CDS nucleotide sequence of the gene is shown in SEQ ID NO.2; SiERF109 The amino acid sequence of the SiERF109 protein encoded by the CDS nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0009] On the other hand, the present invention provides the above-mentioned method for regulating the drought resistance, salt tolerance, and flavonoid accumulation of millet. SiERF109 Application of genes in the breeding of new millet varieties.

[0010] Preferably, by constructing millet SiERF109 Gene overexpression lines were achieved.

[0011] Preferred millet SiERF109 Methods for constructing gene overexpression lines include:

[0012] S1, Construction SiERF109 Gene overexpression vectors;

[0013] S2, will SiERF109 Gene overexpression vectors were transformed into Agrobacterium, which was then used to transform millet seeds via the fluff infection method. Overexpression lines were obtained through hygromycin screening.

[0014] Preferred, SiERF109 Gene overexpression vectors are created through cloning. SiERF109 The gene was constructed by ligating it into the 35S::pCAMBIA1305.1 vector.

[0015] Preferably, the upstream primer sequence for gene cloning is shown in SEQ ID NO.8, and the downstream primer sequence is shown in SEQ ID NO.9.

[0016] Therefore, this invention relates to a SiERF109 gene that regulates drought resistance, salt tolerance, and flavonoid accumulation in millet, and its application. SiERF109 The full-length cDNA of the gene was ligated into the 1305.1 expression vector with a 35S promoter, and millet was transformed using the fluffy infection method. Experimental results confirmed that:

[0017] (1) Under drought stress, overexpression SiERF109 The survival rate and chlorophyll content of the millet lines with the gene were significantly higher than those of the wild type, thus improving the drought stress tolerance of millet.

[0018] (2) Under salt stress, overexpression SiERF109 The survival rate and chlorophyll content of the millet lines with the gene were significantly higher than those of the wild type, thus improving the salt stress tolerance of millet.

[0019] (3) Under salt stress, overexpression SiERF109 H2O2 and superoxide anions accumulated in millet strains of the gene (Significantly lower than wild type)

[0020] (4) Under drought stress, overexpression SiERF109 H2O2 and accumulated in millet strains of the gene Significantly lower than the wild type.

[0021] (5) Under normal conditions, overexpression SiERF109 The flavonoids accumulated in the millet grains of the genetically modified type were significantly higher than those of the wild type.

[0022] Given SiERF109 The conservation of the gene in plants and the phenotype exhibited in transgenic millet suggest that the gene has practical value. SiERF109 The gene provides new genetic resources for molecular breeding of drought-resistant, salt-tolerant, and high-quality millet varieties, and will play an important role in improving drought-resistant, salt-tolerant, and high-quality crop varieties, with broad application prospects.

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 for SiERF109 Results of quantitative real-time RT-qPCR analysis of expression levels in different tissues of millet;

[0026] Figure 2 for SiERF109 A schematic diagram of the gene being linked to the 1305.1 expression vector;

[0027] Figure 3 Treatment with 300mM NaCl for different times SiERF109 Results of quantitative real-time RT-qPCR analysis of gene expression levels in the aboveground and underground parts of millet; where A represents the aboveground part and B represents the underground part.

[0028] Figure 4 Treatment with 20% PEG6000 for different times SiERF109 Results of quantitative real-time RT-qPCR analysis of gene expression levels in the aboveground and underground parts of millet; where A represents the aboveground part and B represents the underground part.

[0029] Figure 5 For overexpression SiERF109Results of quantitative real-time RT-qPCR analysis of the expression levels of the target gene in the strain;

[0030] Figure 6 For overexpression SiERF109 Phenotypic characteristics of wild-type millet under normal conditions, drought stress treatment, and post-drought rehydration;

[0031] Figure 7 For overexpression SiERF109 Chlorophyll content of wild-type millet after drought stress treatment;

[0032] Figure 8 For overexpression SiERF109 H2O2 content of wild-type millet after drought stress treatment;

[0033] Figure 9 For overexpression SiERF109 Compared with wild-type millet after drought stress treatment content;

[0034] Figure 10 For overexpression SiERF109 Phenotypes of wild-type millet under normal conditions and salt stress treatment;

[0035] Figure 11 For overexpression SiERF109 Chlorophyll content of wild-type millet after salt stress treatment;

[0036] Figure 12 For overexpression SiERF109 H2O2 content of wild-type millet after salt stress treatment;

[0037] Figure 13 For overexpression SiERF109 Compared with wild-type millet after salt stress treatment content;

[0038] Figure 14 For overexpression SiERF109 NBT and DAB staining results of wild-type millet under normal conditions, drought stress, and salt stress;

[0039] Figure 15 For overexpression SiERF109 Results of flavonoid content determination in flag leaves of wild-type millet under normal conditions;

[0040] Figure 16 For overexpression SiERF109 Results of flavonoid content determination in seeds of wild-type millet under normal conditions. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] The wild-type millet used in the examples were all commercially available TunGu No. 1 millet.

[0044] Using the millet genome database website phytozome to find genes SiERF109 The amino acid sequence encoded by this gene was compared on the ClustalW website, and it was found that the SiERF109 protein is highly homologous in different species.

[0045] SiERF109 The full-length genome sequence is 1307 bp, as shown in SEQ ID NO.1. The CDS sequence is 834 bp, as shown in SEQ ID NO.2. The sequence of the SiERF109 protein encoded by the CDS is shown in SEQ ID NO.3.

[0046]

[0047] SEQ ID NO.2: atgaccaaccgcatcttctccgccatggcagcgaaccaagcgtacatgatccgattcgacggccacctcgacgacccctccccgagctccgccggcgcggagccgccggaggtgtcgcagcagcagccgccgccgccgttcgcagggagggtgatctcccccgagcaggagcaccaggtgatcgtcgccgccctgctccacgtcgtctccgggtacaccacgccgccgccggagatcttccctgccgcggcggcgggcgcggcatgccgggtatgcgggatggagcggtgcctcggctgcgagttcttcgggggggagggcgccgaggtgatcgcgctggatggcggcgcggcggagaacaacaatgcggccgtggcggcgggagggcagaggaggcggaggaagaagaagaacaagtaccgcggcgtgcggcagcggccgtggggcaagtgggcggcggagatccgcgacccgcgccgcgcggtgcgcaagtggctcgggacgttcgacaccgccgaggaggcggccaaggcctacgaccgcgccgccatcgagttccgtggcccgcgcgccaagctcaacttcccgtttcccgagcagctcgcccacgacgaggccagcaacggcgacgccagcgccgccgccaggtcgtcggacaacacgcagtcgccgtcgctctgcagcggggatgccgaggagcgggggcagccggcggagtggccgccgcggggcgggcaggaaacaggggagcagctctgggaaggactgcaggacctgatgaagctggacgagggcgagctctggttcccgccaacttcgagcgcttggaattga.

[0048] SEQ ID NO.3: MTNRIFSAMAANQAYMIRFDGHLDDPSPSSAGAEPPEVSQQQPPPPFAGRVISPEQEHQVIVAALLHVVSGYTTPPPEIFPAAAAGAACRVCGMERCLGCEFFGGEGAEVIALDGGAAEN NNAAVAAGGQRRRRKKKNKYRGVRQRPWGKWAAEIRDPRRAVRKWLGTFDTAEEAAKAYDRAAIEFRGPRAKLNFPFPEQLAHDEASNGDASAAARSSDNTQSPSLCSGDAEERGQPAEWPPRGGQETGEQLWEGLQDLMKLDEGELWFPPTSSAWN.

[0049] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0050] Example 1

[0051] Under normal growth conditions, for SiERF109 Gene expression analysis was performed on tissues.

[0052] Roots, stems, leaves, ears, and mature seeds of *Tun Gu No. 1* were collected under normal growth conditions, flash-frozen in liquid nitrogen, and ground into powder in liquid nitrogen. RNA was extracted using the Biotech Universal Plant RNA Extraction Kit. Using the RNA extracted from each part as a template, reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit. Real-time quantitative RT-PCR analysis was employed. SiERF109 Gene expression levels. (Based on millet) SiACTIN Genes used as internal reference genes SiACTIN The primers for gene amplification are Actin_F and Actin_R; the primers for SiERF109 gene amplification are... SiERF109 _F and SiERF109 _R.

[0053] The real-time quantitative RT-PCR reaction system is shown in Table 1 below:

[0054] Table 1 Real-time quantitative RT-PCR reaction system

[0055] ;

[0056] The amplification procedure is as follows:

[0057] ① 95.0℃, 60s;

[0058] ②95.0℃, 10s;

[0059] ③60.0℃, 10s;

[0060] ④ 72.0℃, 15s;

[0061] ⑤Plate Read;

[0062] ⑥ Incubate at 65℃ for 20 seconds;

[0063] ⑦ The melting curve from 65℃ to 95℃ was read every 0.5℃ and held for 1 second;

[0064] ⑧ End.

[0065] The Actin_F sequence, as shown in SEQ ID NO.4, is: 5′-TTGCTGACAGGATGAATGGC-3′;

[0066] The Actin_R sequence, as shown in SEQ ID NO.5, is: 5′-CACATCTGCTGGAATGTGCT-3′;

[0067] SiERF109 The _F sequence, as shown in SEQ ID NO.6, is: 5′-GAAACAGGGGAGCAGCTCTG-3′;

[0068] SiERF109 The _R sequence is shown in SEQ ID NO.7 as: 5′-ATTCCAAGCGCTCGAAGTTG-3′.

[0069] The results are as follows Figure 1 As shown, it can be seen SiERF109 It is mainly expressed in roots during the seedling stage, with lower expression levels in stems, leaves, spikes, and seeds.

[0070] Example 2

[0071] Millet SiERF109 Gene sequence analysis, cloning, and vector construction:

[0072] according to SiERF109 Primers were designed based on the CDS sequence of the gene for cloning. The cloning method is as follows:

[0073] (1) RNA extraction: Total RNA was extracted from millet using the Biotech Universal Plant Total RNA Extraction Kit.

[0074] (2) Synthesis of first strand of reverse transcribed cDNA: The extracted RNA was dissolved and the RNA concentration was measured. Then, reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit.

[0075] Take 5 μg of total RNA, add 10 μL of 2× reaction buffer, 1 μL of primer oligo dT (0.5 μg / μL), 1 μL of reverse transcriptase, 1 μL of genomic degenerating enzyme, and add water to 20 μL. Incubate at 42℃ for 30 minutes, and then inactivate the enzyme at 85℃ for 5 minutes.

[0076] (3) SiERF109 Gene cloning:

[0077] The upstream primer sequence is shown in SEQ ID NO.8, which is: 5′-ATGACCAACCGCATCTTCT-3′;

[0078] The downstream primer sequence is shown in SEQ ID NO.9: 5′-CGCATTGTTGTTCTCCGC-3′.

[0079] Amplification was performed using Novizan high-fidelity enzyme (Vazyme#P505). The reaction system consisted of: 12.5 μL of 2× reaction buffer, 0.5 μL of deoxyribonucleic acid (dNTP), 1 μL of upstream primer, 1 μL of downstream primer, 0.5 μL of high-fidelity enzyme, 1 μL of cDNA template, and water to a final volume of 25 μL.

[0080] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 15 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 1 minute, for a total of 35 cycles; 72℃ extension for 5 minutes; and incubation at 16℃.

[0081] After the reaction was completed, agarose gel electrophoresis was performed. Once the target band was detected, the gel was cut and recovered. The gel recovery method was performed according to the Rapid Agarose Gel DNA Recovery Kit (Cat#DP1722).

[0082] (4) Take 3.5 μL of the gel recovery product and ligate it with the pCAMBIA 1305.1 expression vector started at 35 s, as follows: Figure 2 As shown, the operation steps were performed according to the EasyFusion Assembly Master Mix instructions. The ligation product was transformed into E. coli DH5α strain using the heat shock method and grown overnight on LB agar plates containing kanamycin to obtain positive clones. Single white colonies were picked for colony PCR, using the same reaction system as above, and positive colonies were selected and incubated overnight in LB liquid medium.

[0083] (5) Extraction of plasmid DNA: Plasmid DNA was extracted using a high-purity plasmid mini extraction kit (CW0500A).

[0084] (6) Sequencing: The extracted plasmid DNA was sent to the company for sequencing.

[0085] Example 3

[0086] Under salt and drought stress conditions, SiERF109 Gene expression analysis:

[0087] (1) Wild-type millet was used as material and grown for 1 week. It was then subjected to stress treatment with 300mM NaCl and 20% PEG6000 culture medium, respectively. Fresh material was collected after each treatment lasted for 0, 3, 6, 9 and 12 hours and was flash-frozen in liquid nitrogen.

[0088] (2) Total RNA was extracted from millet material using a universal plant RNA extraction kit and synthesized using a reverse transcription kit. SiERF109 The first-strand cDNA of the gene was processed using the same steps as in Example 2.

[0089] (3) Analysis using quantitative real-time RT-PCR SiERF109 Gene expression. The quantitative RT-PCR experiment used the SYBR Premix Ex Taq II kit to prepare the PCR system, and real-time quantitative PCR detection was performed on a CFX96 Touch quantitative PCR instrument.

[0090] The results are as follows Figure 3 , Figure 4 As shown, in the aboveground parts, salt stress treatment and PEG6000 treatment induced... SiERF109 Gene expression was significantly upregulated in millet; in the underground parts, salt stress for 3, 6, and 9 hours and PEG6000 treatment for 3 hours induced [the gene's expression]. SiERF109 Gene expression was significantly upregulated in millet.

[0091] In summary SiERF109 Genes are expressed in millet roots and seeds. SiERF109 The gene is a salt and drought stress response gene and may be involved in the millet's response to high salt and drought stress.

[0092] Example 4

[0093] Millet SiERF109 Genetic transformation and screening of homozygous transgenic lines:

[0094] (1) Take 2 μL of the vector plasmid constructed in Example 2 and transform it into Agrobacterium EHA105 to culture a single colony.

[0095] (2) Pick a single colony from the culture medium and incubate it in 100 μL LB (with kanamycin added) liquid medium for 4 h.

[0096] (3) Transfer 100 μL of bacterial culture to 3 mL LB (with kanamycin added) liquid medium and incubate overnight for 7 h.

[0097] (4) Take 500 μL of bacterial culture and culture it overnight in 50 mL of liquid pre-culture medium (pH 7.2) for 14 h.

[0098] (5) Section OD 600 =1.0; collect bacterial cells at 2400g for 5min.

[0099] (6) Resuspend the bacterial cells in 40 mL of infection solution.

[0100] The composition of the inoculum is as follows: 2.1g K2HPO4, 0.9g KH2PO4, 0.2g (NH4)2SO4, 0.112g citric acid, 0.66mL glycerol, 0.0493g MgSO4·7H2O, 3g ascorbic acid, 0.4264g MES, 7.2g glucose, 200mL distilled water, pH 5.8.

[0101] (7) Treat the ears of grain (before flowering) with 1 mL of permeate for 20 min before infection.

[0102] The permeate composition is as follows: MgSO4·7H2O (0.493g), MES (0.3184g), 200mL distilled water, pH 5.8.

[0103] (8) Infect the ears of grain with pre-induced Agrobacterium for 20 min.

[0104] (9) After the infected ears of grain are covered with a light-transmitting plastic bag for 24 hours, the bag is removed and the grain continues to grow until the seeds mature.

[0105] (10) After the seeds were matured and infected, they were screened on MS solid medium containing 90 mg / L hygromycin, and the strains with more green leaves were selected as the transgenic material of 35S::SiERF109.

[0106] (11) Total RNA was further extracted from the leaves of transgenic seedlings, reverse transcribed into cDNA, and detected by qPCR. Six stable overexpression lines were obtained, and the results are as follows: Figure 5 As shown.

[0107] Example 5

[0108] change SiERF109 Identification of drought resistance in genetically modified strains:

[0109] Mix vermiculite and potting soil in a 1:1 ratio, weigh out equal amounts, and place them in small pots. Allow the soil to fully moisten through the small holes at the bottom. Sow nine seeds of transgenic and wild-type millet, which have absorbed water overnight, into each pot. Cover with a layer of soil and gently compact. Allow to grow normally in a greenhouse for 30 days. Select millet seedlings with uniform growth and subject them to drought stress treatment. The pots should be rotated frequently to minimize the impact of location on seedling growth under stress. Continue this treatment until the plants have grown for 7 days under stress, followed by 3-5 days of watering to allow them to recover. Measure chlorophyll, H2O2, and O2 levels after 10 days of drought treatment. •− content.

[0110] overexpression SiERF109 Phenotypic characteristics of genetically modified and wild-type millet under normal conditions, drought stress treatment, and post-drought rehydration: Figure 6 As shown, compared with the wild type, overexpression after drought treatment SiERF109 Genetically modified millet maintains good growth, with a small number of leaves wilting and most leaves remaining green.

[0111] overexpression SiERF109 Chlorophyll content, H2O2 content, and [other parameters] of genetically modified and wild-type millet after drought stress treatment. The contents are as follows Figure 7-9 As shown, the results indicate that overexpression SiERF109 Genetically modified millet has significantly higher chlorophyll content and H2O2 content than wild-type millet. The content was significantly lower than that of the wild type, indicating overexpression. SiERF109 Genetically modified millet exhibits significantly higher drought resistance than wild plants.

[0112] Example 6

[0113] change SiERF109 Salt tolerance identification of genetically modified millet lines:

[0114] Vermiculite and potting soil were mixed in a 1:1 ratio, and equal weights were placed in small pots. Water was drawn through the small holes at the bottom to fully moisten the soil. Nine seeds of transgenic and wild-type millet, which had absorbed water overnight, were sown in each pot. After sowing, a layer of soil was covered and gently compacted. The plants were allowed to grow normally in a greenhouse for 14 days. Millet seedlings with uniform growth were then subjected to salt stress treatment. The pots were frequently moved to minimize the impact of location on the seedlings' growth under stress. The chlorophyll content of the plants was measured after 7 days of growth in 300 mM NaCl.

[0115] overexpression SiERF109 Phenotypic characteristics of wild-type millet under normal conditions and salt stress treatment, as follows: Figure 10 As shown, the results indicate that treatment with 300 mM NaCl overexpression SiERF109The genetically modified millet is growing well.

[0116] overexpression SiERF109 Chlorophyll content, H2O2 content, and [other parameters] in genetically modified and wild-type millet after salt stress treatment. The contents are as follows Figures 11-13 As shown, the results indicate that overexpression SiERF109 Genetically modified millet plants have significantly higher chlorophyll content and H2O2 content than wild-type plants. The content was significantly lower than that of the wild type. This indicates overexpression. SiERF109 Genetically modified millet exhibits significantly higher salt tolerance than wild plants.

[0117] Example 7

[0118] change SiERF109 Detection of reactive oxygen species (ROS) content in genetically modified millet strains:

[0119] Mix vermiculite and potting soil in a 1:1 ratio, weigh out equal amounts, and place them in small pots. Water is drawn through the small holes at the bottom to fully moisten the soil. Sow 9 seeds of transgenic and wild-type millet, which have absorbed water overnight, into each pot. Cover with a layer of soil and gently compact. Allow to grow normally in a greenhouse for 14 days. Select millet seedlings with uniform growth for salt stress treatment, and frequently change the position of the pots to reduce the impact of location on the seedling growth under stress. After the plants have grown in 300mM NaCl for 3 days, take leaves from transgenic and wild-type millet and place them in centrifuge tubes containing diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining solutions. Incubate in the dark at room temperature overnight. Discard the staining solution. Prepare a fixative solution with a lactic acid:glycerol:ethanol ratio of 1:1:4. Discard the staining solution, add the fixative solution, boil for 30 minutes, and photograph the sample.

[0120] Vermiculite and potting soil were mixed in a 1:1 ratio, and equal weights were placed in small pots. Water was drawn through the bottom holes to fully moisten the soil. Nine seeds of transgenic and wild-type millet, which had absorbed water overnight, were sown in each pot. After sowing, a layer of soil was covered and gently compacted. The plants were allowed to grow normally in a greenhouse for 30 days. Millet seedlings with uniform growth were then subjected to drought stress treatment. The pots were frequently moved to minimize the impact of location on seedling growth under stress. The H2O2 content of the transgenic and wild-type millet was measured using an H2O2 kit after 5 days of stress treatment. The determination was based on the method described in *Experimental Guide to Modern Plant Physiology*, compiled by the Institute of Plant Physiology, Chinese Academy of Sciences and the Shanghai Plant Physiology Society. content.

[0121] overexpression SiERF109 The NBT and DAB staining results of genetically modified millet and wild-type millet under normal conditions, drought stress, and salt stress are as follows: Figure 14As shown, the results indicate that overexpression was observed after drought and salt stress treatment. SiERF109 The DAB and NBT staining of the leaves of the genetically modified millet was significantly lighter than that of the wild type, indicating that... SiERF109 Gene overexpression can reduce H2O2 and [other factors] in millet under drought and salt stress. Accumulation reduces reactive oxygen species damage.

[0122] Example 8

[0123] change SiERF109 Detection of flavonoid content in genetically modified millet leaves and grains:

[0124] Mix vermiculite and potting soil in a 1:1 ratio, weigh out equal amounts, and place them in small pots. Use the small holes at the bottom to absorb water and fully moisten the soil. Sow 9 seeds of transgenic and wild-type millet that have been absorbing water overnight in each small pot. After sowing, cover with a layer of soil and gently compact. Cultivate normally in a greenhouse. Take flag leaves and mature seeds at the heading stage, and use a flavonoid test kit (M0118A) to detect the flavonoid content in the transgenic flag leaves and seeds as well as the wild-type flag leaves and seeds.

[0125] overexpression SiERF109 The results of the determination of flavonoid content in flag leaves and grains of wild-type millet under normal conditions are as follows: Figure 15 and Figure 16 As shown, the results indicate that overexpression SiERF109 The flavonoid content in the flag leaves and grains of the overexpressing gene was significantly higher than that in the wild type, indicating that overexpression... SiERF109 Genetically modified millet accumulates a large amount of flavonoid active substances in its flag leaves and grains.

[0126] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. SiERF 109 The application of the gene in preparing a product for regulating drought resistance, salt tolerance and flavonoid accumulation of foxtail millet, characterized in that: SiERF 109 The nucleotide sequence of the gene is shown as SEQ ID NO. 1; SiERF 109 The CDS sequence of the gene is shown as SEQ ID NO. 2; SiERF 109 The amino acid sequence of the SiERF109 protein produced by the CDS nucleotide sequence of the gene is shown as SEQ ID NO. 3; Overexpression SiERF 109 The gene can improve drought stress tolerance, salt stress tolerance of foxtail millet strains and flavonoid accumulation in foxtail millet grains.

2. Use according to claim 1, characterized in that: millet SiERF 109 The method for constructing a gene overexpression strain includes: S1, construct SiERF 109 gene overexpression vector; S2, will SiERF 109 The gene overexpression vector was transformed into Agrobacterium, and millet was transformed by flower petal infection. The overexpression strain was obtained by hygromycin screening.

3. Use according to claim 2, characterized in that: SiERF 109 Gene overexpression vectors were constructed by cloning SiERF 109 Genes were ligated into 35S::pCAMBIA 1305.1 vectors.

4. Use according to claim 3, characterized in that: Cloning SiERF 109 SiERF 109 The upstream primer sequence of the gene is shown as SEQ ID NO. 8, and the downstream primer sequence is shown as SEQ ID NO. 9.

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