CN120758525B

CN120758525B - SiERF109 gene for regulating drought resistance, salt tolerance and flavonoid accumulation of millet and application thereof

Info

Publication number: CN120758525B
Application number: CN202511262710.6A
Authority: CN
Inventors: 吴长艾; 张萌; 王康伟; 彭树伟
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2025-09-05
Filing date: 2025-09-05
Publication date: 2025-12-05
Anticipated expiration: 2045-09-05
Also published as: CN120758525A

Abstract

The invention discloses SiERF gene for regulating drought resistance, salt tolerance and flavonoid accumulation of millet and application thereof, belonging to the technical field of plant breeding. The invention separates and clones the gene SiERF with the nucleotide sequence shown as SEQ ID NO.1 from millet, and fluorescence quantitative expression analysis shows that SiERF109 participates in drought and salt stress response of millet. After the gene is over-expressed in millet, compared with a wild type strain, the strain of the over-expressed SiERF gene has the advantages that the survival rate and chlorophyll content are obviously increased, the flavonoid content is increased by 33.5% -39.5%, and the active oxygen accumulation is obviously reduced in drought and salt stress experiments, so that the SiERF gene and the protein encoded by the gene can obviously improve the drought resistance, salt tolerance and flavonoid content of plants.

Description

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

Technical Field

The invention relates to the field of biotechnology, in particular to SiERF gene for regulating drought resistance, salt tolerance and flavonoid accumulation of millet and application thereof.

Background

In recent years, the consumption demand of millet, the planting income and willingness of farmers all have obvious growing trend. However, the problems of unclear molecular mechanism of drought resistance and salt tolerance of millet and synthesis regulation of health care substances, few available drought resistance and salt tolerance high-quality germplasm and the like are main factors for limiting the production of the millet. Therefore, the identification of the genes related to drought resistance, salt tolerance and health care substance synthesis of the millet can provide new gene resources and biological breeding technical support for stress resistance and high-quality molecular breeding of the millet, and has important production significance.

The AP2/ERF family is one of the largest families of transcription factors in the plant kingdom, and the ERF subfamily structurally contains a conserved AP2/ERF domain on which cis-acting elements are capable of responding to a variety of responses.

Research shows that ERF transcription factor is closely related to plant growth, biological and abiotic stress response, biosynthesis and other processes. Arabidopsis AP2/ERF transcription factor TINY can regulate drought by activating drought response genes and closing stomata. In eggplants, the silenced SmERF1 transcription factor can obviously lower the expression level of the salt stress defense related genes, reduce the synthesis of superoxide dismutase and catalase and promote the generation of hydrogen peroxide (H ₂O₂) and proline. The over-expression of the soybean ERF7 in the tobacco can increase the chlorophyll content of the tobacco and reduce the malondialdehyde content, so that the salt tolerance of the tobacco is improved. ERF transcription factors can respond to drought and salt stress injury through a variety of physiological metabolic pathways in plants.

Thus, ERF is essential for plant growth and development and stress resistance. However, there are few reports of ERF family members in millet that regulate drought resistance, salt tolerance and flavonoid content simultaneously.

Disclosure of Invention

Aiming at the problems of stress resistance and insufficient high-quality breeding gene resources of the millet in the prior art, the invention provides SiERF gene for regulating drought resistance, salt tolerance and flavonoid accumulation of the millet and application thereof, and provides new resources for stress-resistant high-quality molecular breeding of the millet.

In order to achieve the aim, in one aspect, the invention provides SiERF gene for regulating drought resistance, salt tolerance and flavonoid accumulation of millet, and the nucleotide sequence of SiERF gene is shown as SEQ ID NO. 1.

Preferably, the CDS nucleotide sequence of SiERF109,109 gene is shown as SEQ ID NO.2, and the amino acid sequence of SiERF109,109 protein produced by coding the CDS nucleotide sequence of SiERF109,109 gene is shown as SEQ ID NO. 3.

On the other hand, the invention provides an application of SiERF gene for regulating drought resistance, salt tolerance and flavonoid accumulation of millet in breeding new variety of millet.

Preferably, this is achieved by constructing a millet SiERF109,109 gene over-expression line.

Preferably, the construction method of the millet SiERF109,109 gene overexpression line comprises the following steps:

s1, constructing SiERF109,109 gene over-expression vector;

S2, transforming the SiERF109,109 gene over-expression vector into agrobacterium, transforming millet by a flocculation infection method, and screening by hygromycin to obtain an over-expression strain.

Preferably, siERF109 gene overexpression vectors are constructed by cloning SiERF gene and ligating the vector of 35 S:pCAMBIA 1305.1.

Preferably, the sequence of the upstream primer of the gene clone is shown as SEQ ID NO.8, and the sequence of the downstream primer is shown as SEQ ID NO. 9.

Therefore, the SiERF gene for regulating and controlling the drought resistance, salt tolerance and flavonoid accumulation of the millet and the application thereof are disclosed, wherein the full-length cDNA of the SiERF gene is connected to a 1305.1 expression vector started by 35S, the millet is transformed by using a cotton-wool infection method, and the experiment shows that:

(1) Under drought stress conditions, the survival rate and chlorophyll content of the millet strain over-expressing SiERF gene are obviously higher than those of wild type, so that the drought stress tolerance of the millet is improved.

(2) Under the condition of salt stress, the survival rate and chlorophyll content of the millet strain over-expressing SiERF109,109 genes are obviously higher than those of wild type, thereby improving the salt stress tolerance of the millet.

(3) H ₂O₂ and superoxide anion accumulating in millet strain over-expressing SiERF gene under salt stress condition) Significantly lower than the wild type.

(4) H ₂O₂ and H ₂O₂ accumulated in millet strain overexpressing SiERF gene under drought stressSignificantly lower than the wild type.

(5) Under normal conditions, flavonoids accumulated in the grains of millet over-expressing SiERF gene were significantly higher than that of the wild type.

The SiERF109,109 gene can be considered to have application value in view of its conservation in plants and phenotype exhibited in transgenic millet. SiERF109 genes provide new gene resources for drought-resistant salt-tolerant and high-quality molecular breeding of millet, and play an important role in improving drought-resistant salt-tolerant high-quality varieties of crops, and have wide application prospects.

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments of the present invention will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of a fluorescent quantitative RT-qPCR analysis of SiERF109 expression levels in different tissues of millet;

FIG. 2 is a schematic representation of SiERF gene transfer to 1305.1 expression vector;

FIG. 3 shows the results of a fluorescent quantitative RT-qPCR analysis of the expression levels of SiERF genes in the aerial and subsurface portions of millet treated with 300mM NaCl for different times, wherein A is the aerial portion and B is the subsurface portion;

FIG. 4 shows the results of fluorescent quantitative RT-qPCR analysis of the expression levels of SiERF109,109 genes in the aerial part and the underground part of millet treated with 20% PEG6000 for different times, wherein A is the aerial part and B is the underground part;

FIG. 5 shows the result of a fluorescent quantitative RT-qPCR analysis of the expression level of a target gene in an over-expressed SiERF strain;

FIG. 6 is a phenotype of overexpression SiERF109,109 and rehydration of wild-type millet after normal conditions, drought stress treatment and drought;

FIG. 7 is chlorophyll content of over-expressed SiERF109 and wild-type millet after drought stress treatment;

FIG. 8 is H ₂O₂ content of over-expressed SiERF109 and wild-type millet after drought stress treatment;

FIG. 9 is a graph of over-expression SiERF109,109 and wild-type millet after drought stress treatment The content is as follows;

FIG. 10 is a phenotype of over-expressed SiERF109,109 and wild-type millet after normal conditions, salt stress treatment;

FIG. 11 is chlorophyll content of over-expressed SiERF109 and wild-type millet after salt stress treatment;

FIG. 12 is H ₂O₂ content of over-expressed SiERF109 and wild-type millet after salt stress treatment;

FIG. 13 is a graph showing overexpression SiERF109,109 and wild-type millet after salt stress treatment The content is as follows;

FIG. 14 shows NBT and DAB staining results of over-expressed SiERF and wild-type millet under normal conditions, drought stress and salt stress;

FIG. 15 shows flavonoid content measurements in flag leaves under normal conditions for over-expressed SiERF109,109 and wild-type millet;

FIG. 16 shows the results of flavonoid content measurements in seeds under normal conditions for over-expressed SiERF and wild-type millet.

Detailed Description

The technical scheme of the invention is further described below through the attached drawings and the embodiments.

In order to make the objects, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application will be clearly and completely described below through the accompanying drawings and examples. The following detailed description is of embodiments, and is intended to provide further details of the application. Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The wild type millet used in the examples is commercially available ton millet number one.

The gene SiERF109,109 was found using the millet genome database website phytozome, and the amino acid sequences encoded by this gene were aligned on the ClustalW website, finding that SiERF protein was highly homologous in different species.

SiERF109,109 genome sequence is 1307bp in total length, the sequence is shown as SEQ ID NO.1, CDS sequence is 834bp, the sequence is shown as SEQ ID NO.2, and the coded SiERF109,109 protein sequence is shown as SEQ ID NO. 3.

SEQ ID NO.1:ccgctcagttcaaagcagcgcctcgccatcagacaagcacacgagctcagaggaagaacacacacatgaccaaccgcatcttctccgccatggcagcgaaccaagcgtacatgatccgattcgacggccacctcgacgacccctccccgagctccgccggcgcggagccgccggaggtgtcgcagcagcagccgccgccgccgttcgcagggagggtgatctcccccgagcaggagcaccaggtgatcgtcgccgccctgctccacgtcgtctccgggtacaccacgccgccgccggagatcttccctgccgcggcggcgggcgcggcatgccgggtatgcgggatggagcggtgcctcggctgcgagttcttcgggggggagggcgccgaggtgatcgcgctggatggcggcgcggcggagaacaacaatgcggccgtggcggcgggagggcagaggaggcggaggaagaagaagaacaagtaccgcggcgtgcggcagcggccgtggggcaagtgggcggcggagatccgcgacccgcgccgcgcggtgcgcaagtggctcgggacgttcgacaccgccgaggaggcggccaaggcctacgaccgcgccgccatcgagttccgtggcccgcgcgccaagctcaacttcccgtttcccgagcagctcgcccacgacgaggccagcaacggcgacgccagcgccgccgccaggtcgtcggacaacacgcagtcgccgtcgctctgcagcggggatgccgaggagcgggggcagccggcggagtggccgccgcggggcgggcaggaaacaggggagcagctctgggaaggactgcaggacctgatgaagctggacgagggcgagctctggttcccgccaacttcgagcgcttggaattgaatcgtctgtgattagatcccagccgttgaagtagtttgaaaaatgaacatccctagcctttaacaatatgcttcttttggctctctctgtttttttttcctcagctctgttgtaacgtatttgatcatagcagagttttgtacattatcagtagagtcctaccaactgtattcaccgggcaattttgggagtgtaccttttctacttgaaaagcaaggaacacgagctgactactgacttgttaagggtacatataaaagtactgagatcactccctgcctatttgtgattactatcaatttgaaccaatttctacgcacgggatttggatgaaacatttttggactgtgctatttcgtgcctcaatatgatccacgtgctaatctcaatcattggttggcaaattaa.

SEQ ID NO.2: atgaccaaccgcatcttctccgccatggcagcgaaccaagcgtacatgatccgattcgacggccacctcgacgacccctccccgagctccgccggcgcggagccgccggaggtgtcgcagcagcagccgccgccgccgttcgcagggagggtgatctcccccgagcaggagcaccaggtgatcgtcgccgccctgctccacgtcgtctccgggtacaccacgccgccgccggagatcttccctgccgcggcggcgggcgcggcatgccgggtatgcgggatggagcggtgcctcggctgcgagttcttcgggggggagggcgccgaggtgatcgcgctggatggcggcgcggcggagaacaacaatgcggccgtggcggcgggagggcagaggaggcggaggaagaagaagaacaagtaccgcggcgtgcggcagcggccgtggggcaagtgggcggcggagatccgcgacccgcgccgcgcggtgcgcaagtggctcgggacgttcgacaccgccgaggaggcggccaaggcctacgaccgcgccgccatcgagttccgtggcccgcgcgccaagctcaacttcccgtttcccgagcagctcgcccacgacgaggccagcaacggcgacgccagcgccgccgccaggtcgtcggacaacacgcagtcgccgtcgctctgcagcggggatgccgaggagcgggggcagccggcggagtggccgccgcggggcgggcaggaaacaggggagcagctctgggaaggactgcaggacctgatgaagctggacgagggcgagctctggttcccgccaacttcgagcgcttggaattga.

SEQ ID NO.3: MTNRIFSAMAANQAYMIRFDGHLDDPSPSSAGAEPPEVSQQQPPPPFAGRVISPEQEHQVIVAALLHVVSGYTTPPPEIFPAAAAGAACRVCGMERCLGCEFFGGEGAEVIALDGGAAEN NNAAVAAGGQRRRRKKKNKYRGVRQRPWGKWAAEIRDPRRAVRKWLGTFDTAEEAAKAYDRAAIEFRGPRAKLNFPFPEQLAHDEASNGDASAAARSSDNTQSPSLCSGDAEERGQPAEWPPRGGQETGEQLWEGLQDLMKLDEGELWFPPTSSAWN.

The instrumentation and reagent materials used in the examples are all commercially available.

Example 1

Under normal growth conditions, tissue expression analysis was performed on SiERF109,109 genes.

Collecting ton-valley No. one root, stem, leaf, spike and mature seed under normal growth condition, quick freezing in liquid nitrogen, grinding into powder in liquid nitrogen, and extracting RNA by using a Baitaike universal plant RNA extraction kit. RNA extracted from each part is used as a template, and reverse transcription is carried out by using TRANSSCRIPT ONE-Step gDNA Removal AND CDNA SYNTHESIS Supermix reverse transcription kit. The expression level of SiERF109,109 genes was analyzed by real-time fluorescent quantitative RT-PCR. The millet SiACTIN gene is used as an internal reference gene, the SiACTIN gene amplification primers are actin_F and actin_R, and the SiERF109 gene amplification primers are SiERF109_F and SiERF109 _109_R.

The real-time fluorescent quantitative RT-PCR reaction system is shown in the following table 1:

TABLE 1 real-time fluorescent quantitative RT-PCR reaction system

;

The amplification procedure was as follows:

①95.0°C,60s;

②95.0°C,10s;

③60.0°C,10s;

④72.0°C,15s;

⑤Plate Read;

⑥ Incubation at 65 ℃ for 20 seconds;

⑦ A melting curve from 65 ℃ to 95 ℃ is read once every 0.5 ℃ and kept for 1 second;

⑧ And (5) ending.

The sequence of the Actin_F is shown as SEQ ID NO.4, which is 5'-TTGCTGACAGGATGAATGGC-3';

the sequence of the Actin_R is shown as SEQ ID NO.5, which is 5'-CACATCTGCTGGAATGTGCT-3';

SiERF _F sequence is shown as SEQ ID NO.6, 5'-GAAACAGGGGAGCAGCTCTG-3';

The SiERF _R sequence is shown as SEQ ID NO.7, 5'-ATTCCAAGCGCTCGAAGTTG-3'.

As a result, as shown in FIG. 1, siERF109 was found to be expressed mainly in roots during seedling stage, and the expression level was low in stems, leaves, ears and seeds.

Example 2

Sequence analysis, cloning and vector construction of millet SiERF109,109 genes:

primers were designed based on the CDS sequence of SiERF109,109 genes and cloned as follows:

(1) RNA extraction total RNA from millet using the total plant RNA extraction kit of the biotak.

(2) Reverse transcription first strand cDNA Synthesis the RNA concentration was determined after dissolving the extracted RNA, and then reverse transcription was performed using TRANSSCRIPT ONE-Step gDNA Removal AND CDNA SYNTHESIS Supermix reverse transcription kit.

Mu.g of total RNA was taken, 10. Mu.L of 2 Xreaction buffer, 1. Mu.L of primer oligo dT (0.5. Mu.g/. Mu.L), 1. Mu.L of reverse transcriptase, 1. Mu.L of genome-deleted enzyme, water was added to 20. Mu.L, and incubated at 42℃for 30 minutes and 85℃for 5 minutes.

(3) Cloning SiERF109,109 Gene:

The sequence of the upstream primer is shown as SEQ ID NO.8, 5'-ATGACCAACCGCATCTTCT-3';

The sequence of the downstream primer is shown as SEQ ID NO.9 and is 5'-CGCATTGTTGTTCTCCGC-3'.

The amplification was performed using Northenzan high-fidelity enzyme (Vazyme #P505) in a reaction system of 12.5. Mu.L of 2 Xreaction buffer, 0.5. Mu.L of deoxyribonucleic acid (dNTP), 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 0.5. Mu.L of high-fidelity enzyme, 1. Mu.L of cDNA template, and water to 25. Mu.L.

The PCR reaction conditions were 95℃for 5 minutes, 95℃for 15 seconds, 60℃for 15 seconds, 72℃for 1 minute, 35 cycles total, 72℃for 5 minutes, and 16℃for incubation.

After the reaction, agarose gel electrophoresis was performed, and after the target band was detected, the gel was cut and recovered, and the gel recovery method was performed according to the rapid agarose gel DNA recovery kit (Cat#DP1722).

(4) The 3.5. Mu.L of the gel recovery product was ligated with the 35S-driven pCAMBIA 1305.1 expression vector as shown in FIG. 2, and the procedure was followed according to EasyFusion Assembly Master Mix instructions. The ligation product was transformed into E.coli DH 5. Alpha. Strain by heat shock method, and grown overnight on LB plate containing kanamycin to give positive clones. White single colonies were picked for colony PCR, the reaction system was the same as above, and positive colonies were selected and placed in LB liquid medium overnight.

(5) Plasmid DNA extraction plasmid DNA was extracted using the high purity plasmid miniextraction kit (CW 0500A).

(6) Sequencing the extracted plasmid DNA was sent to the company for sequencing.

Example 3

Analysis of SiERF109,109 gene expression under salt and drought stress treatment conditions:

(1) Wild millet is used as a material and grows for 1 week, and 300mM NaCl and 20% PEG6000 culture solution are respectively adopted for stress treatment. Fresh material was collected for each treatment time of 0, 3, 6, 9 and 12 hours and stored in liquid nitrogen by flash freezing.

(2) Total RNA of millet material was extracted using a universal plant RNA extraction kit, and first strand cDNA of SiERF gene was synthesized using a reverse transcription kit, and the procedure was as in example 2.

(3) The expression of SiERF109,109 genes was analyzed by fluorescent quantitative RT-PCR. Wherein, the fluorescent quantitative RT-PCR experiment is to configure a PCR system according to SYBRPremix Ex Taq II kit, and to carry out real-time quantitative fluorescent PCR detection on CFX96 Touch fluorescent quantitative PCR instrument.

As shown in FIG. 3 and FIG. 4, the salt stress treatment and the PEG6000 treatment induced a significant up-regulation of SiERF gene expression in millet in the aerial part, and the salt stress treatment for 3, 6 and 9 hours and the PEG6000 treatment for 3 hours induced a significant up-regulation of SiERF gene expression in millet in the underground part.

Taken together, siERF genes expressed in Gu Zigen and seeds, siERF genes are salt stress and drought stress response genes, likely involved in millet responses to high salt and drought stress.

Example 4

Genetic transformation of millet SiERF109,109 and selection of homozygous transgenic lines:

(1) 2. Mu.L of the vector plasmid constructed in example 2 was transformed into Agrobacterium EHA105 and single colonies were cultured.

(2) Single colonies were picked from the medium and cultured in 100. Mu.L LB (kanamycin) liquid medium for 4h.

(3) Mu.L of the bacterial liquid was transferred to 3mL of LB (kanamycin) liquid medium and cultured overnight for 7 hours.

(4) Mu.L of the bacterial liquid was cultured in 50mL of liquid preculture solution (pH 7.2) overnight for 14 hours.

(5) Section OD ₆₀₀ = 1.0, 2400g, 5 min.

(6) The cells were resuspended with 40mL of the counterstain.

The dip ingredients were ：2.1g K₂HPO₄、0.9g KH₂PO₄、0.2g (NH₄)₂SO₄、0.112g citric acid, 0.66mL glycerol, 0.0493g MgSO ₄·7H₂ O, 3g ascorbic acid, 0.4264g MES, 7.2g glucose, 200mL distilled water, pH5.8.

(7) The ears (prior to anthesis) were pre-treated with 1mL of permeate for 20min before infestation.

The permeate was composed of MgSO ₄·7H₂ O (0.493 g), MES (0.3184 g), 200mL distilled water, pH 5.8.

(8) The ears were infected with pre-induced agrobacteria for 20min.

(9) And (5) taking down the infected cereal ears after covering the light-transmitting plastic bags for 24 hours, and continuing to grow until the seeds are mature.

(10) Mature infected seeds are screened on MS solid medium containing 90mg/L hygromycin, and a strain with more green leaves is selected as a transgenic material of 35S SiERF < 109 >.

(11) Further extracting total RNA of transgenic seedling leaves, reversely transcribing into cDNA, and obtaining 6 stable over-expression lines by qPCR detection, wherein the result is shown in figure 5.

Example 5

Drought resistance identification of the SiERF109,109 transgenic lines:

Mixing vermiculite and nutrient soil uniformly according to the proportion of 1:1, weighing the same weight, then placing the mixture in small basins, fully wetting the soil through water absorption of small holes at the bottom, dibbling the transgenic seeds and the seeds of wild millet which are swelled overnight in the small basins, covering 9 grains of each small basin with a layer of soil after the seeds are planted, lightly compacting, normally growing in a greenhouse for 30 days, selecting millet seedlings with consistent growth for drought stress treatment, and changing the positions of the small basins frequently, thereby reducing the influence of the positions on the growth of the millet seedlings under stress. Until the plants grew for 7d in stress, watering was resumed for 3-5 days, and chlorophyll, H ₂O₂, and O ₂ ^•− contents were determined at 10 days of drought treatment.

The phenotype of the over-expressed SiERF109,109 gene millet and wild-type millet in normal conditions, drought stress treatment and rehydration after drought is shown in figure 6, compared with the wild-type millet, the over-expressed SiERF gene millet after drought treatment maintains a better growth state, a small amount of leaves wilt, and most of leaves are green.

Overexpression SiERF Gene millet and wild-type millet chlorophyll content, H ₂O₂ content and after drought stress treatmentThe content is shown in figures 7-9, respectively, and the result shows that the chlorophyll content of the millet over-expressed SiERF gene is significantly higher than that of wild type, H ₂O₂ and HThe content is obviously lower than that of a wild type, which indicates that the drought resistance of the millet over-expressed with SiERF gene is obviously higher than that of a wild plant.

Example 6

Salt tolerance identification of the SiERF gene transgenic millet strain:

Mixing vermiculite and nutrient soil uniformly according to the proportion of 1:1, weighing the same weight, then placing the mixture in small basins, fully wetting the soil through water absorption of small holes at the bottom, dibbling the transgenic seeds and the seeds of wild millet which are swelled overnight in the small basins, covering 9 grains of each small basin with a layer of soil after the seeds are planted, lightly compacting, normally growing in a greenhouse for 14 days, selecting millet seedlings with consistent growth for salt stress treatment, and changing the positions of the small basins frequently, thereby reducing the influence of the positions on the growth of the millet seedlings under stress. After 7d of plant growth in 300mM NaCl, the chlorophyll content of the plants was determined.

The phenotype of the over-expressed SiERF < 109 > and wild millet after normal condition and salt stress treatment is shown in figure 10, and the result shows that the millet with the over-expressed SiERF < 109 > gene has better growth vigor after 300mM NaCl treatment.

Overexpression SiERF Gene millet and wild-type millet chlorophyll content, H ₂O₂ content and after salt stress treatmentThe content is shown in figures 11-13, respectively, and the result shows that the chlorophyll content of the millet with over-expressed SiERF109,109 gene is obviously higher than that of a wild plant, the H ₂O₂ content andThe content is significantly lower than that of the wild type. The salt tolerance of the millet over-expressed SiERF gene is obviously higher than that of a wild plant.

Example 7

And (3) detecting the active oxygen content of the transgenic SiERF109,109 gene millet strain:

Mixing vermiculite and nutrient soil uniformly according to the proportion of 1:1, weighing the same weight, then placing the mixture in small basins, fully wetting the soil through water absorption of small holes at the bottom, dibbling the transgenic seeds and the seeds of wild millet which are swelled overnight in the small basins, covering 9 grains of each small basin with a layer of soil after the seeds are planted, lightly compacting, normally growing in a greenhouse for 14 days, selecting millet seedlings with consistent growth for salt stress treatment, and changing the positions of the small basins frequently, thereby reducing the influence of the positions on the growth of the millet seedlings under stress. After plants were grown for 3d in 300mM NaCl, transgenic millet and wild millet leaves were placed in centrifuge tubes containing Diaminobenzidine (DAB) and azulene tetrazolium (NBT) dye solutions, left overnight in dark at room temperature, the dye solutions were decanted, the fixative solution was prepared as lactic acid: glycerol: ethanol=1:1:4, the fixative solution was decanted, added, boiled for 30min, and photographs were retained.

Mixing vermiculite and nutrient soil uniformly according to the proportion of 1:1, weighing the same weight, then placing the mixture in small basins, fully wetting the soil through water absorption of small holes at the bottom, dibbling the transgenic seeds and the seeds of wild millet which are swelled overnight in the small basins, covering 9 grains of each small basin with a layer of soil after the seeds are planted, lightly compacting, normally growing in a greenhouse for 30 days, selecting millet seedlings with consistent growth for drought stress treatment, and changing the positions of the small basins frequently, thereby reducing the influence of the positions on the growth of the millet seedlings under stress. Until the plants grew for 5d in stress, the transgenic lines and wild millet H ₂O₂ content were determined using the H ₂O₂ kit. Is measured by referring to the modern plant physiology experimental guideline of the plant physiology institute of China academy of sciences and Shanghai city plant physiology instituteThe content is as follows.

The NBT and DAB staining results of the over-expressed SiERF109,109 gene millet and wild-type millet under normal conditions, drought stress and salt stress are shown in figure 14, and the result shows that the DAB and NBT staining of the SiERF109,109 gene millet leaf after drought and salt stress treatment is significantly shallower than that of the wild-type, which indicates that the over-expression of SiERF109,109 gene can reduce H ₂O₂ and H under drought and salt stress of the milletAccumulation and reduction of active oxygen damage.

Example 8

Flavonoid content detection of transgenic SiERF109,109 gene millet leaf and grain:

Mixing vermiculite and nutrient soil uniformly according to the proportion of 1:1, weighing the same weight, then placing the mixture in small basins, fully wetting the soil through small holes at the bottom, dibbling the transgenic and wild millet seeds which are swelled overnight into the small basins, covering 9 grains in each small basin, covering a layer of soil after the seed is planted, lightly compacting, normally culturing in a greenhouse, taking the flag leaves and mature seeds in the heading period, and detecting the content of flavonoid substances in the transgenic flag leaves and seeds and the wild flag leaves and seeds by using a flavonoid test box (M0118A).

The results of the flavonoid content measurement of the over-expressed SiERF and wild millet under normal conditions are shown in fig. 15 and 16, and the results show that the flavonoid content of the over-expressed SiERF gene millet flag leaf and seed is significantly higher than that of the wild millet, and the over-expressed SiERF gene millet accumulates more flavonoid active substances in the flag leaf and seed.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted by the same, and the modified or substituted technical solution may not deviate from the spirit and scope of the technical solution of the present invention.

Claims

The application of SiERF109 gene in preparing products for regulating drought resistance, salt tolerance and flavonoid accumulation of millet is characterized in that the nucleotide sequence of SiERF gene is shown as SEQ ID NO. 1;

the CDS sequence of SiERF109,109 gene is shown as SEQ ID NO.2, and the amino acid sequence of SiERF109,109 protein produced by coding the CDS nucleotide sequence of SiERF109,109 gene is shown as SEQ ID NO. 3;

Overexpression of SiERF gene can improve drought stress tolerance, salt stress tolerance and flavonoid accumulation in Gu Zizi grain of the cereal line.
2. The method according to claim 1, wherein the construction method of the millet SiERF109,109 gene overexpression line comprises the following steps:

s1, constructing SiERF109,109 gene over-expression vector;

S2, transforming the SiERF109,109 gene over-expression vector into agrobacterium, transforming millet by a flocculation infection method, and screening by hygromycin to obtain an over-expression strain.
3. The method of claim 2, wherein the SiERF109 gene over-expression vector is constructed by cloning SiERF gene and ligating 35 S:pCAMBIA 1305.1 vector.
4. The method of claim 3, wherein the upstream primer sequence of the SiERF109 gene is shown in SEQ ID NO.8 and the downstream primer sequence is shown in SEQ ID NO. 9.