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

Abstract

The invention discloses a SiERF109 gene for regulating drought resistance, salt tolerance and flavonoid accumulation of millet and application of the SiERF109 gene, and belongs to the technical field of plant breeding. According to the invention, a gene SiERF109 with a nucleotide sequence shown as SEQ ID NO.1 is separated and cloned from millet, and fluorescent quantitative expression analysis shows that SiERF109 participates in drought and salt stress response of millet. After the gene is over-expressed in foxtail millet, in drought and salt stress experiments, compared with a wild type strain, the survival rate and chlorophyll content of a strain over-expressed with the SiERF109 gene are remarkably increased, the flavonoid content is increased by 33.5%-39.5%, and active oxygen accumulation is remarkably reduced; it is shown that the SiERF109 gene and the protein coded by the SiERF109 gene can remarkably improve the drought resistance, the salt resistance and the flavonoid content of the plant.

Description

Technical Field

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

[0002] In recent years, millet consumption, farmers' returns, and willingness to cultivate millet have all shown a significant upward trend. However, the unclear molecular mechanisms regulating millet drought and salt tolerance, as well as the synthesis of health-promoting substances, and the limited availability of high-quality, drought- and salt-tolerant germplasm are major factors limiting its production. Therefore, identifying millet genes associated with drought and salt tolerance and the synthesis of health-promoting substances can provide new genetic resources and biobreeding technology support for molecular breeding of millet for stress resistance and high-quality production, 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 structurally contains a conserved AP2 / ERF domain, on which the cis-acting elements can respond to a variety of responses.

[0004] Research has shown that ERF transcription factors are closely associated with plant growth and development, responses to biotic and abiotic stresses, and biosynthesis. The Arabidopsis AP2 / ERF transcription factor TINY regulates drought stress by activating drought-responsive genes and closing stomata. In eggplant, silencing the SmERF1 transcription factor significantly downregulates the expression of genes involved in salt stress defense, 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 reduces malondialdehyde content, thereby improving salt tolerance. ERF transcription factors can respond to drought and salt stress through multiple physiological and metabolic pathways within the plant.

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

[0006] In view of the problem of insufficient genetic resources for millet stress resistance and high-quality breeding in the prior art, the present invention provides a method for regulating millet drought resistance, salt tolerance and flavonoid accumulation. SiERF109 Genes and their applications provide new resources for the molecular breeding of stress-resistant and high-quality millet.

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

[0008] Preferably, 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 a method for regulating millet drought resistance, salt tolerance and flavonoid accumulation. SiERF109 Application of genes in breeding new millet varieties.

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

[0011] Preferably, millet SiERF109 Methods for constructing gene overexpression lines include: S1. Construction SiERF109 Gene overexpression vector; S2, will SiERF109 The gene overexpression vector was transformed into Agrobacterium, which was then transformed into millet by the inflorescence infection method, and the overexpression strain was obtained by hygromycin selection.

[0012] Preferably, SiERF109 Gene overexpression vectors were cloned SiERF109 The gene was connected with the 35S::pCAMBIA1305.1 vector to construct.

[0013] Preferably, the upstream primer sequence for gene cloning is shown as SEQ ID NO.8, and the downstream primer sequence is shown as SEQ ID NO.9.

[0014] Therefore, the present invention provides a SiERF109 gene for regulating drought resistance, salt tolerance and flavonoid accumulation in millet and its application. SiERF109 The full-length cDNA of the gene was connected to the 1305.1 expression vector driven by 35S promoter and transformed into millet using the inoculum infection method. The results were verified by experiments: (1) Under drought stress conditions, overexpression SiERF109 The survival rate and chlorophyll content of millet lines with the gene were significantly higher than those of the wild type, thereby improving the drought stress tolerance of millet.

[0015] (2) Under salt stress conditions, overexpression SiERF109 The survival rate and chlorophyll content of millet lines with the gene were significantly higher than those of the wild type, thereby improving the salt stress tolerance of millet.

[0016] (3) Under salt stress conditions, overexpression SiERF109 The accumulation of H2O2 and superoxide anions in millet lines with the gene ( ) was significantly lower than that of the wild type.

[0017] (4) Under drought stress, overexpression SiERF109 The accumulation of H2O2 and significantly lower than the wild type.

[0018] (5) Under normal conditions, overexpression SiERF109 The accumulation of flavonoids in millet grains of the gene was significantly higher than that of the wild type.

[0019] Given that SiERF109 The conservation of the gene in plants and the phenotype exhibited in transgenic millet indicate that the gene has application value. SiERF109 The genes provide new genetic resources for millet drought and salt resistance and high-quality molecular breeding, and will play an important role in improving drought and salt-resistant and high-quality crop varieties, with broad application prospects.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0022] Figure 1 for SiERF109 Fluorescence quantitative RT-qPCR analysis results of expression levels in different tissues of foxtail millet; Figure 2 for SiERF109 Schematic diagram of gene ligation into 1305.1 expression vector; Figure 3 300mM NaCl treatment for different time SiERF109 Fluorescence quantitative RT-qPCR analysis results of gene expression in the aboveground and underground parts of millet; A represents the aboveground part, and B represents the underground part; Figure 4 20% PEG6000 treated for different time SiERF109 Fluorescence quantitative RT-qPCR analysis results of gene expression in the aboveground and underground parts of millet; A represents the aboveground part, and B represents the underground part; Figure 5 For overexpression SiERF109 Fluorescence quantitative RT-qPCR analysis results of the expression level of the target gene in the strain; Figure 6 For overexpression SiERF109and wild-type foxtail millet phenotypes under normal conditions, drought stress treatments, and rewatering after drought; Figure 7 For overexpression SiERF109 and chlorophyll contents of wild-type foxtail millet after drought stress treatment; Figure 8 For overexpression SiERF109 and H2O2 contents of wild-type foxtail millet after drought stress treatment; Figure 9 For overexpression SiERF109 and wild-type millet after drought stress treatment content; Figure 10 For overexpression SiERF109 and wild-type foxtail millet phenotypes under normal conditions and after salt stress treatment; Figure 11 For overexpression SiERF109 and chlorophyll contents of wild-type foxtail millet after salt stress treatment; Figure 12 For overexpression SiERF109 and H2O2 contents of wild-type foxtail millet after salt stress treatment; Figure 13 For overexpression SiERF109 and wild-type millet after salt stress treatment content; Figure 14 For overexpression SiERF109 NBT and DAB staining results of foxtail millet and wild type foxtail millet under normal conditions, drought stress and salt stress; Figure 15 For overexpression SiERF109 and wild-type foxtail millet under normal conditions; Figure 16 For overexpression SiERF109 The results of flavonoid content determination in seeds of millet and wild type millet under normal conditions. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0024] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0025] The wild-type millet used in the examples was the commercially available Ton Gu No. 1.

[0026] The gene was found by using the millet genome database website phytozome SiERF109 The amino acid sequence encoded by the gene was aligned on the ClustalW website, and it was found that SiERF109 protein was highly homologous in different species.

[0027] SiERF109 The full-length genomic sequence is 1307 bp, as shown in SEQ ID NO. 1, the CDS sequence is 834 bp, as shown in SEQ ID NO. 2, and the SiERF109 protein sequence produced by the coding is as shown in SEQ ID NO. 3.

[0028] SEQ ID NO. 2: atgaccaaccgcatcttctccgccatggcagcgaaccaagcgtacatgatccgattcgacggccacctcgacgacccctccccgagctccgccggcgcggagccgccggaggtgtcgcagcagcagccgccgccgccgttcgcagggagggtgatctcccccgagcaggagcaccaggtgatcgtcgccgccctgctccacgtcgtctccgggtacaccacgccgccgccggagatcttccctgccgcggcggcgggcgcggcatgccgggtatgcgggatggagcggtgcctcggctgcgagttcttcgggggggagggcgccgaggtgatcgcgctggatggcggcgcggcggagaacaacaatgcggccgtggcggcgggagggcagaggaggcggaggaagaagaagaacaagtaccgcggcgtgcggcagcggccgtggggcaagtgggcggcggagatccgcgacccgcgccgcgcggtgcgcaagtggctcgggacgttcgacaccgccgaggaggcggccaaggcctacgaccgcgccgccatcgagttccgtggcccgcgcgccaagctcaacttcccgtttcccgagcagctcgcccacgacgaggccagcaacggcgacgccagcgccgccgccaggtcgtcggacaacacgcagtcgccgtcgctctgcagcggggatgccgaggagcgggggcagccggcggagtggccgccgcggggcgggcaggaaacaggggagcagctctgggaaggactgcaggacctgatgaagctggacgagggcgagctctggttcccgccaacttcgagcgcttggaattga. SEQ ID NO. 3: MTNRIFSAMAANQAYMIRFDGHLDDPSPSSAGAEPPEVSQQQPPPPFAGRVISPEQEHQVIVAALLHVVSGYTTPPPEIFPAAAAGAACRVCGMERCLGCEFFGGEGAEVIALDGGAAENNNAAVAAGGQRRRRKKKNKYRGVRQRPWGKWAAEIRDPRRAVRKWLGTFDTAEEAAKAYDRAAIEFRGPRAKLNFPFPEQLAHDEASNGDASAAARSSDNTQSPSLCSGDAEERGQPAEWPPRGGQETGEQLWEGLQDLMKLDEGELWFPPTSSAWN. The instrument equipment and reagent materials used in the examples were obtained through commercial channels.

[0029] Example 1 Under normal growth conditions, the expression of the genes in different tissues was analyzed. SiERF109 The expression of the genes in different tissues was analyzed.

[0030] The roots, stems, leaves, spikes and mature seeds of Tonggu No. 1 under normal growth conditions were collected, quickly frozen in liquid nitrogen, ground into powder in liquid nitrogen, and RNA was extracted using a general plant RNA extraction kit. The extracted RNA from each part was used as a template for reverse transcription using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit. The expression of the genes was analyzed by real-time fluorescent quantitative RT-PCR. SiERF109 The expression of the genes was analyzed by real-time fluorescent quantitative RT-PCR. SiACTIN The expression of the genes was analyzed by real-time fluorescent quantitative RT-PCR. SiACTIN The expression of the genes was analyzed by real-time fluorescent quantitative RT-PCR. SiERF109 The expression of the genes was analyzed by real-time fluorescent quantitative RT-PCR. SiERF109 The expression of the genes was analyzed by real-time fluorescent quantitative RT-PCR.

[0031] The real-time fluorescent quantitative RT-PCR reaction system is shown in Table 1 below: Table 1 Real-time fluorescent quantitative RT-PCR reaction system ;

[0032] The amplification program is as follows: ① 95.0℃, 60s; ② 95.0℃, 10s; ③ 60.0℃, 10s; ④ 72.0℃, 15s; ⑤ Plate Read; ⑥ Incubate at 65°C for 20 seconds; ⑦ Melting curve from 65℃ to 95℃, read once every 0.5℃ and hold for 1 second; ⑧End.

[0033] The sequence of Actin_F is shown in SEQ ID NO. 4: 5′-TTGCTGACAGGATGAATGGC-3′; The sequence of Actin_R is shown in SEQ ID NO. 5: 5′-CACATCTGCTGGAATGTGCT-3′; SiERF109 The _F sequence is shown in SEQ ID NO. 6, which is: 5′-GAAACAGGGGAGCAGCTCTG-3′; SiERF109 The _R sequence is shown in SEQ ID NO.7, which is: 5′-ATTCCAAGCGCTCGAAGTTG-3′.

[0034] The results are as follows Figure 1 As shown, it can be seen SiERF109 During the seedling stage, it is mainly expressed in roots, with lower expression levels in stems, leaves, ears and seeds.

[0035] Example 2 millet SiERF109 Gene sequence analysis, cloning and vector construction: according to SiERF109 Primers were designed based on the CDS sequence of the gene and cloned. The cloning method was as follows: (1) RNA extraction: The total RNA of foxtail millet was extracted using the Biotek Universal Plant Total RNA Extraction Kit.

[0036] (2) Synthesis of the first-strand cDNA by reverse transcription: The extracted RNA was dissolved and the RNA concentration was determined. Then, the reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit.

[0037] 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 de novo genomic enzyme, add water to 20 μL, incubate at 42°C for 30 minutes, and inactivate the enzyme at 85°C for 5 minutes.

[0038] (3) SiERF109 Gene cloning: The upstream primer sequence is shown in SEQ ID NO. 8, which is: 5′-ATGACCAACCGCATCTTCT-3′; The downstream primer sequence is shown in SEQ ID NO.9, which is: 5′-CGCATTGTTGTTCTCCGC-3′.

[0039] Vazyme high-fidelity enzyme (Vazyme#P505) was used for amplification. The reaction system was: 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 was added to 25 μL.

[0040] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 1 minute, for a total of 35 cycles; post-extension at 72°C for 5 minutes; and insulation at 16°C.

[0041] After the reaction, agarose gel electrophoresis was performed. After the target band was detected, the gel was cut and the gel was recovered. The gel recovery method was performed according to the Quick Agarose Gel DNA Recovery Kit (Cat# DP1722).

[0042] (4) Take 3.5 μL of the gel recovery product and connect it with the 35S-activated pCAMBIA 1305.1 expression vector, as shown in the following example: Figure 2 As shown, the steps were performed according to the EasyFusion Assembly Master Mix instructions. The ligation product was transformed into E. coli DH5α using the heat shock method and grown overnight on LB plates containing kanamycin to obtain positive clones. Single white colonies were selected for colony PCR using the same reaction system as above, with positive colonies selected and incubated overnight in LB liquid medium.

[0043] (5) Extraction of plasmid DNA: Use a high-purity plasmid mini-extraction kit (CW0500A) to extract plasmid DNA.

[0044] (6) Sequence determination: The extracted plasmid DNA is sent to the company for sequence determination.

[0045] Example 3 Under salt and drought stress conditions, SiERF109 Gene expression analysis: (1) Wild-type foxtail millet was grown for one week and subjected to stress treatments in 300 mM NaCl and 20% PEG6000 medium. Fresh samples were collected at 0, 3, 6, 9, and 12 h after each treatment and quickly frozen in liquid nitrogen.

[0046] (2) The total RNA of millet material was extracted using a universal plant RNA extraction kit and synthesized using a reverse transcription kit. SiERF109 The first-strand cDNA of the gene was prepared using the same steps as in Example 2.

[0047] (3) Fluorescence quantitative RT-PCR analysis SiERF109 The fluorescence quantitative RT-PCR experiment was performed using the SYBR Premix Ex Taq Ⅱ kit to configure the PCR system, and the real-time quantitative fluorescence PCR detection was performed on a CFX96 Touch fluorescence quantitative PCR instrument.

[0048] The results are as follows Figure 3 、 Figure 4 As shown, in the aboveground part, salt stress and PEG6000 treatment induced SiERF109 The gene expression was significantly up-regulated in millet; in the underground part, salt stress for 3, 6 and 9 hours and PEG6000 treatment for 3 hours induced SiERF109 The gene expression was significantly upregulated in foxtail millet.

[0049] In summary SiERF109 The gene is expressed in millet roots and seeds. SiERF109 The gene is a salt stress and drought stress response gene and may be involved in the response of millet to high salt and drought stress.

[0050] Example 4 millet SiERF109 Genetic transformation and screening of homozygous transgenic lines: (1) Take 2 μL of the vector plasmid constructed in Example 2 and transform it into Agrobacterium EHA105, and culture a single colony.

[0051] (2) Pick a single colony from the culture medium and culture it in 100 μL LB (with kanamycin) liquid medium for 4 h.

[0052] (3) Transfer 100 μL of bacterial solution into 3 mL of LB (with kanamycin) liquid culture medium and culture overnight for 7 h.

[0053] (4) Take 500 μL of bacterial solution and culture it in 50 mL of liquid pre-culture medium (pH 7.2) overnight for 14 h.

[0054] (5) Section OD 600 =1.0; 2400g, 5min to collect the bacteria.

[0055] (6) Resuspend the bacteria in 40 mL of infection solution.

[0056] The composition of the infection solution 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.

[0057] (7) Before infection, pre-treat the ears of grain (before flowering) with 1 mL of penetrant for 20 minutes.

[0058] The permeate composition was as follows: MgSO4·7H2O (0.493 g), MES (0.3184 g), 200 mL distilled water, pH 5.8.

[0059] (8) Infect the ears of grain with pre-induced Agrobacterium for 20 minutes.

[0060] (9) After the infection, the ears of grain are covered with a transparent plastic bag and then removed after 24 hours, and allowed to continue growing until the seeds mature.

[0061] (10) The mature infected seeds were screened on MS solid medium containing 90 mg / L hygromycin, and the strains with more green leaves were selected as the transgenic materials of 35S::SiERF109.

[0062] (11) Total RNA from the leaves of transgenic seedlings was further extracted and reverse transcribed into cDNA. After qPCR detection, 6 stable overexpression lines were obtained. The results are as follows: Figure 5 shown.

[0063] Example 5 change SiERF109 Identification of drought resistance of genetic lines: Vermiculite and nutrient soil were mixed in a 1:1 ratio, and the same weight was weighed and placed in a small pot. Water was absorbed through the small holes at the bottom to completely moisten the soil. Transgenic and wild-type millet seeds that had been swollen overnight were planted in the small pots, with 9 seeds in each small pot. After planting, a layer of soil was covered and lightly compacted. The seeds were grown normally in the greenhouse for 30 days. Millet seedlings with consistent growth were selected for drought stress treatment, and the position of the small pots needed to be changed frequently to reduce the impact of the position on the growth of millet seedlings under stress. Chlorophyll, H2O2, and O2 were measured after the plants grew for 7 days under stress and watering was resumed for 3-5 days. Chlorophyll, H2O2, and O2 were measured after 10 days of drought treatment. •− content.

[0064] Overexpression SiERF109 Phenotypes of genetically modified millet and wild-type millet under normal conditions, drought stress treatment and rewatering after drought Figure 6 As shown, compared with the wild type, the overexpression SiERF109The genetically modified millet maintained a good growth state, with a small number of leaves wilting and most of the leaves remaining green.

[0065] Overexpression SiERF109 Chlorophyll content, H2O2 content and The contents are as follows Figure 7-9 The results showed that overexpression SiERF109 The chlorophyll content of genetically modified millet was significantly higher than that of wild type, and H2O2 and The content was significantly lower than that of wild type, indicating overexpression SiERF109 The drought resistance of genetically modified millet is significantly higher than that of wild plants.

[0066] Example 6 change SiERF109 Identification of Salt Tolerance of Genetic Millet Lines: Vermiculite and nutrient soil were mixed in a 1:1 ratio, weighed, and placed in small pots. Water was then drawn through the small holes at the bottom to completely moisten the soil. Transgenic and wild-type millet seeds, which had imbibed overnight, were then sown in the pots, with nine seeds per pot. After planting, the seeds were covered with a layer of soil and gently compacted. The plants were grown normally in a greenhouse for 14 days. Millet seedlings with consistent growth were then selected for salt stress treatment. The position of the pots was frequently rotated to minimize the impact of position on the stress-induced growth of the millet seedlings. Chlorophyll content was measured after the plants had been grown in 300 mM NaCl for 7 days.

[0067] Overexpression SiERF109 Phenotypes of wild-type millet under normal conditions and after salt stress treatment are as follows Figure 10 As shown, the results showed that 300mM NaCl treatment overexpressed SiERF109 The genetically modified millet is growing well.

[0068] Overexpression SiERF109 Chlorophyll content, H2O2 content and The contents are as follows Figure 11-13 The results showed that overexpression SiERF109 The chlorophyll content of genetically modified millet was significantly higher than that of wild-type plants, and the H2O2 content and The content is significantly lower than that of wild type. SiERF109 The salt tolerance of genetically modified millet is significantly higher than that of wild plants.

[0069] Example 7 change SiERF109 Detection of active oxygen content in genetically modified millet strains: Vermiculite and nutrient soil were mixed in a 1:1 ratio, weighed, and placed in small pots. Water was then absorbed through the small holes at the bottom to completely moisten the soil. Transgenic and wild-type millet seeds, which had imbibed overnight, were then sown in the pots, with nine seeds per pot. After planting, the seeds were covered with a layer of soil and gently compacted. The pots were grown normally in the greenhouse for 14 days. Millet seedlings with consistent growth were selected for salt stress treatment. The pots were rotated frequently to minimize the effects of position on the stress-induced growth of the millet seedlings. After the plants had grown in 300 mM NaCl for 3 days, transgenic and wild-type millet leaves were placed in centrifuge tubes containing diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stains. The leaves were placed in the dark at room temperature overnight. The stain was then discarded, and a fixative solution of lactic acid:glycerol:ethanol (1:1:4) was prepared. The stain was discarded, the fixative solution was added, and the leaves were boiled for 30 minutes. The leaves were then photographed.

[0070] Vermiculite and nutrient soil were mixed in a ratio of 1:1, and the same weight was weighed and placed in a small pot. Water was absorbed through the small holes at the bottom to completely moisten the soil. The transgenic and wild-type millet seeds that had been swollen overnight were planted in the small pots, with 9 seeds in each small pot. After planting, a layer of soil was covered and lightly compacted. The seeds were grown normally in the greenhouse for 30 days. Millet seedlings with consistent growth were selected for drought stress treatment, and the position of the small pots needed to be changed frequently to reduce the impact of the position on the growth of millet seedlings under stress. When the plants grew for 5 days under stress, the H2O2 content of the transgenic lines and wild-type millet was determined using an H2O2 kit. Refer to the "Modern Plant Physiology Experimental Guide" compiled by the Institute of Plant Physiology, Chinese Academy of Sciences and the Shanghai Society of Plant Physiology for determination. content.

[0071] Overexpression SiERF109 The results of NBT and DAB staining of genetically modified millet and wild-type millet under normal conditions, drought stress and salt stress are shown in the figure. Figure 14 The results showed that after drought and salt stress treatment, the SiERF109 The DAB and NBT staining of the leaves of genetically modified millet were significantly lighter than those of the wild type, indicating that SiERF109 Gene overexpression can reduce H2O2 and Accumulation, reducing reactive oxygen species damage.

[0072] Example 8 change SiERF109 Flavonoid content detection in leaves and seeds of genetically modified millet: Vermiculite and nutrient soil were mixed in a 1:1 ratio, and equal weights were weighed and placed in small pots. Water was absorbed through the small holes at the bottom to completely moisten the soil. Transgenic and wild-type millet seeds that had been imbibed overnight were planted in the small pots, with 9 seeds in each pot. After planting, a layer of soil was covered and lightly compacted. The seeds were cultured normally in a greenhouse. Flag leaves and mature seeds at the heading stage were collected, and the flavonoid content of the transgenic flag leaves and seeds and the wild-type flag leaves and seeds was tested using a flavonoid test kit (M0118A).

[0073] Overexpression SiERF109 The results of flavonoid content determination in flag leaves and grains of millet and wild type millet under normal conditions are as follows Figure 15 and Figure 16 As shown, the results showed that overexpression SiERF109 The flavonoid content in flag leaves and grains of genetically modified millet was significantly higher than that of wild type, indicating that overexpression SiERF109 Genetic millet accumulates more flavonoid active substances in its flag leaves and grains.

[0074] 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 the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for regulating drought resistance, salt tolerance and flavonoid accumulation in millet SiERF109 A gene characterized by: SiERF109 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A method for regulating millet drought resistance, salt tolerance and flavonoid accumulation according to claim 1 SiERF109 A gene characterized by: SiERF109 The CDS 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.

3. The method for regulating millet drought resistance, salt tolerance and flavonoid accumulation as claimed in claim 1 or 2 SiERF109 Application of genes in breeding new millet varieties.

4. The use according to claim 3, characterized in that: By building a grain SiERF109 Gene overexpression lines were achieved.

5. The use according to claim 4, characterized in that millet SiERF109 Methods for constructing gene overexpression lines include: S1. Construction SiERF109 Gene overexpression vector; S2, will SiERF109 The gene overexpression vector was transformed into Agrobacterium, which was then transformed into millet by the inflorescence infection method, and the overexpression strain was obtained by hygromycin selection.

6. The use according to claim 5, characterized in that: SiERF109 Gene overexpression vectors were cloned SiERF109 The gene was connected to the 35S::pCAMBIA 1305.1 vector for construction.

7. The use according to claim 6, characterized in that: clone SiERF109 The upstream primer sequence of the gene is shown in SEQ ID NO.8, and the downstream primer sequence is shown in SEQ ID NO.9.

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