Function and application of SiDRe1 for regulating drought tolerance of millet

Abstract

The invention discloses a function and application of SiDRe1 for regulating and controlling the drought resistance of millet, and aims at solving the technical problem of how to improve the drought resistance of the millet. Specifically disclosed are applications of a protein, a substance for knocking out or inhibiting or reducing or down-regulating expression of a coding gene of the protein, or a substance for knocking out or inhibiting or reducing or down-regulating activity or content of the protein in any one of the following: A1) an application in improving drought resistance of green bristlegrass plants; a2) application in preparation of products for improving drought resistance of green bristlegrass plants; a4) plant breeding and / or preparation of plant breeding products; the protein is any one of the following: B1) a protein with an amino acid sequence as shown in SEQ ID No.3; b2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues of the protein B1), has 80% or more of identity with the protein B1) and has the same function as the protein B1); and B3) a fusion protein obtained by connecting the N terminal or / and the C terminal of B1) or B2) with a protein tag. The drought resistance of the millet can be improved by inhibiting or reducing or down-regulating the expression of the GmPHD6 gene of the millet, and the GmPHD6 gene can be used for production.

Description

Technical Field

[0001] The present invention specifically relates to the function and application of SiDRe1 in regulating drought tolerance of millet. Background Art

[0002] Drought is a major factor affecting agricultural production. With the advancement of molecular biotechnology and the increasing maturity of plant genetic engineering, the introduction of key genes into target materials through genetic engineering methods and the cultivation of new drought-tolerant crop varieties are effective ways to alleviate drought stress in agricultural production.

[0003] Millet, my country's largest coarse grain, boasts drought resistance, stress tolerance, wide adaptability, rich nutrition, and a unique flavor. Millet is widely recognized as a drought-resistant crop and plays a crucial role in adjusting crop production structures and developing sustainable dryland agriculture. Analyzing millet's drought tolerance mechanisms and identifying drought-resistant genes are crucial for improving crop resilience.

[0004] bZIP transcription factors are involved in multiple biological processes such as plant growth and development, dormancy, aging, and stress resistance. The characteristics, classification, and biological functions of bZIP transcription factors in plants have been thoroughly studied (Lu Zixian et al., 2002). However, due to the late start of research on millet molecular biology, research on bZIP transcription factors in millet is still not abundant, and there are also fewer transcription factors of the bZIP family cloned from millet. How to provide a millet drought tolerance-related gene is a technical problem faced by those in this field. Summary of the Invention

[0005] The purpose achieved by the present application is to provide a method for improving the drought tolerance of millet, especially in the seedling stage.

[0006] In a first aspect, the present application discloses the use of a protein, a substance for knocking out or inhibiting or reducing or down-regulating the expression of a gene encoding the protein, or a substance for knocking out or inhibiting or reducing or down-regulating the activity or content of the protein in any of the following:

[0007] A1) Application in improving drought resistance of Setaria plants;

[0008] A2) application in the preparation of a product for improving drought resistance of Setaria plants;

[0009] A4) Use in plant breeding and / or in the preparation of plant breeding products;

[0010] The protein is any one of the following:

[0011] B1) the amino acid sequence is the protein shown in SEQ ID No. 3;

[0012] B2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein described in B1) and having at least 80% identity with the protein described in B1) and having the same function;

[0013] B3) A fusion protein obtained by linking the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

[0014] In the above, the Setaria plant may be a Setaria plant in the seedling stage.

[0015] In the above text, the seedling stage may be 3 days after emergence.

[0016] In the above, the breeding objectives include drought resistance. Drought resistance can be high drought resistance or low drought resistance.

[0017] In the above, the drought resistance index of the drought resistance may be the survival rate.

[0018] A substance that knocks out, inhibits, reduces or down-regulates the expression of the gene encoding the above protein in Setaria plants, or knocks out, inhibits, reduces or down-regulates the activity or content of the protein can improve drought resistance.

[0019] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0020] In order to facilitate the purification of the protein in B1), a tag as shown in Table 1 below may be connected to the amino terminus or carboxyl terminus of the protein shown in SEQ ID No. 1 in the sequence listing.

[0021] Table 1. Sequences of tags

[0022]

[0023]

[0024] In the above-mentioned proteins, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0025] In the above proteins, the above 80% or greater identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0026] In the above protein, sequence 3 (SEQ ID No. 3) consists of 403 amino acid residues and is named SiDRe1 protein or protein SiDRe1 or protein SiDRe1. Its encoding gene is SiDRe1 gene.

[0027] In certain embodiments, the protein is derived from foxtail millet.

[0028] In the above, the millet may be the millet variety Ci846.

[0029] In the above, the substance that knocks out or inhibits or reduces or downregulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene).

[0030] In the above, the "substance that expresses the gene encoding the protein or the substance that knocks out or inhibits or reduces or downregulates the activity or content of the protein" can achieve the purpose of inhibiting the expression level and / or activity of the protein through methods well known in the art such as RNA interference, homologous recombination, and gene site editing.

[0031] In certain embodiments, the substance that knocks out, inhibits, reduces, or downregulates the expression of the gene encoding the protein is any one of the following:

[0032] D1) a nucleic acid molecule that inhibits, reduces or downregulates the expression of the gene encoding the above protein;

[0033] D2) expressing the gene encoding the nucleic acid molecule described in D1);

[0034] D3) an expression cassette containing the gene described in D2);

[0035] D4) a recombinant vector containing the gene described in D2) or a recombinant vector containing the expression cassette described in D3);

[0036] D5) a recombinant microorganism containing the gene described in D2), or a recombinant microorganism containing the expression cassette described in D3), or a recombinant microorganism containing the recombinant vector described in D4);

[0037] D6) a transgenic plant cell line containing the gene described in D2), or a transgenic plant cell line containing the expression cassette described in D3), or a transgenic plant cell line containing the recombinant vector described in D4);

[0038] D7) transgenic plant tissue containing the gene described in D2), or transgenic plant tissue containing the expression cassette described in D3), or transgenic plant tissue containing the recombinant vector described in D4);

[0039] D8) A transgenic plant organ containing the gene described in D2), or a transgenic plant organ containing the expression cassette described in D3), or a transgenic plant organ containing the recombinant vector described in D4).

[0040] In the nucleic acid molecule D1), those skilled in the art can readily mutate the nucleotide sequence of the present invention that inhibits, reduces, or down-regulates the expression of the gene encoding the protein SiDRe1 using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that are 80% or more identical to the nucleotide sequence of the present invention that inhibits, reduces, or down-regulates the expression of the gene encoding the protein SiDRe1 and that have the function of inhibiting, reducing, or down-regulating the expression of the gene encoding the protein SiDRe1 are derived from and are equivalent to the nucleotide sequence of the present invention.

[0041] The aforementioned 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0042] As used herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search to calculate the identity of the amino acid sequence, the value (%) of identity can then be obtained.

[0043] In the above D4), the recombinant vector may be a plant gene editing vector. The plant gene editing vector may be pYLCRISPR / Cas9P ubi -H.

[0044] The microorganism described in D5) above may be Agrobacterium, and the Agrobacterium is EHA105.

[0045] In certain embodiments, the nucleic acid molecule D1) is a gRNA targeting the above-mentioned protein-coding gene, and the target sequence of the gRNA is SEQ ID No. 1.

[0046] In the above, the target sequence may be a DNA molecule represented by the sequence 5'-CCCCGGCACACGCTCACCG-3'.

[0047] In certain embodiments, the Setaria plant is foxtail millet.

[0048] In the above, the millet may be the millet variety Ci846.

[0049] In certain embodiments, a method for improving drought resistance of Setaria plants comprises improving drought resistance of Setaria plants by knocking out or inhibiting or reducing or down-regulating the expression of the gene encoding the above-mentioned protein in Setaria plants, and / or the activity and / or content of the above-mentioned protein.

[0050] In certain embodiments, a method for cultivating a highly drought-resistant Setaria plant comprises knocking out or inhibiting or reducing or downregulating the expression level of the gene encoding the above-mentioned protein in the target Setaria plant, and / or, the activity and / or content of the protein to obtain a highly drought-resistant Setaria plant, wherein the highly drought-resistant Setaria plant has higher drought resistance than the target Setaria plant.

[0051] In certain embodiments, the knocking out, inhibiting, reducing or down-regulating the expression of the gene encoding the above protein in the plant comprises introducing a gene knockout vector with SEQ ID No. 1 as the target sequence into the target plant.

[0052] On the other hand, the present application discloses a method for cultivating highly drought-resistant millet, wherein the genome of the target millet is subjected to any of the following operations:

[0053] 1) inserting an adenine deoxyribonucleotide residue between positions 656 and 657 of SEQ ID No. 1 in the target millet genome;

[0054] 2) Deletion mutation was performed between positions 627 and 662 of SEQ ID No. 1 in the target millet genome.

[0055] In certain embodiments, the Setaria plant is foxtail millet.

[0056] In the above, the millet may be the millet variety Ci846.

[0057] Beneficial effects

[0058] The present invention discloses the function and application of SiDRe1 in regulating millet drought tolerance, addressing the technical problem of improving millet drought resistance. Specifically disclosed are the applications of a protein, a substance for knocking out, inhibiting, reducing, or downregulating the expression of the gene encoding the protein, or a substance for knocking out, inhibiting, reducing, or downregulating the activity or content of the protein, in any of the following: A1) improving drought resistance in Setaria plants; A2) preparing products for improving drought resistance in Setaria plants; and A4) plant breeding and / or preparing plant breeding products. The protein is any of the following: B1) a protein having an amino acid sequence as shown in SEQ ID No. 3; B2) a protein obtained by substituting, deleting, and / or adding amino acid residues to the protein in B1) and having greater than 80% identity and the same function as the protein in B1); or B3) a fusion protein obtained by linking a protein tag to the N-terminus or / and C-terminus of B1) or B2). Inhibiting, reducing, or downregulating the expression of the millet GmPHD6 gene can improve millet drought resistance and can be used in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Figure 2: SiDRe1 target sgRNA expression cassette and pYLCRISPR / Cas9Pubi-H-SiDRe1 recombination vector framework.

[0060] Figure 2Figure 3 is a diagram showing the gene structure, target sequence and sequence alignment of the homozygous gene-edited strain of SiDRe1; Note: A: Gene structure of SiDRe1, with scissors indicating the target position; B: Schematic diagram of the SiDRe protein structure, in which editing results in a frameshift mutation and a loss of the bZIP domain; CD: Schematic diagram of sidre-i gene editing, sequencing peak diagram of the target position C, the black underline indicates the "NGG" PAM sequence, and the red arrow indicates the base insertion position; D: Sequence alignment of the coding chain between the wild type and the sidre-i target site, with "-" indicating a loss; EF: Schematic diagram of sidre-d34 gene editing, sequencing peak diagram of the target position E, the black underline indicates the "NGG" PAM sequence, and the red arrow indicates the base insertion position; F: Sequence alignment of the coding chain between the wild type and the sidre-d34 target site, with "-" indicating a loss.

[0061] Figure 3 Figure 1 shows that the SiDRe1 homozygous edited line is more sensitive to ABA than the wild-type Ci846. Notes: A-E: Germination rate statistics at different ABA concentrations; F: Germination phenotypes under different ABA concentrations. * indicates P < 0.05.

[0062] Figure 4 Figure 2 shows that the SiDRe1 homozygous edited line has stronger drought resistance than the wild type Ci846; A: Drought treatment test of sidre1-i and sidre-d34 seedlings; B: Survival rate statistics of sidre1-i and sidre-d34. DETAILED DESCRIPTION

[0063] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0064] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0065] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0066] Example 1 Obtaining Millet with SiDRe1 Gene Knockout

[0067] Design of SiDRe1 gene knockout targets

[0068] According to the SiDRe1 gene sequence, the target was designed using the online target design website CRISPR-P 2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The appropriate target was selected according to the target location (before the bZIP domain) and the specificity of the target. The final SiDRe1 gene knockout target was as follows: 5'-CCCCGGCACACGCTCACCG-3'( Figure 1 ).

[0069] The gene sequence of the SiDRe1 gene is shown in SEQ ID No. The CDS sequence thereof is shown in SEQ ID No. 2. The protein encoded by the SiDRe1 gene is named SiDRe1 protein, and its sequence is shown in SEQ ID No. 3.

[0070] SEQ ID No. 1 is as follows:

[0071]

[0072] SEQ ID No. 2 is specifically mentioned as follows:

[0073]

[0074] SEQ ID No.3 is as follows:

[0075] MSDDSDGQRQCRRGVTVDVELDAAMALADMAGVGPAATPGGGNHPRSSHQAAAAAPPTAEGEDDLASTRLSLELGKVGIQSASPCSSSSSAGHPHPQPSAQHQPHPVPEAAATGYGPRPRHTLTEAEKEAKRLRRVLANRESARQTILRRQAIRDELARKVVDLSSQNENMKKEKDMVMQEYLSLKEANKQLKEQVARTTK KAPVTTAVTAAVPMHVAAVQQQAAEPMAAAAASPPATPPQPGFLYTAAPPVPYVWGSWPPGAGYEHHHHHGSPPALCLPPCAWYYPVVADPRGSPSAAYAGTQQQQQQAAFQPEPAGSGGGSPAGGATAEEDTDDDPCSLTLGLDIAADKRSAPVSIDARGGAGPSDRDKAATAAEARKRRKELTKLKHMHAAGRPGAEQW

[0076] Obtain the target fragment containing the target sequence for infusion connection

[0077] First-round PCR: Two primer pairs (UF / gR-R and Cris8.3 gRT#+ / Cris8.3 OsU6aT#-) were used for amplification using 2-5 ng of plasmid pYLsgRNA-OsU6a (AddGene: Plasmid #66194) as template. UF / gR-R primers were used at 0.2 μM each, while Cris8.3 gRT#+ / Cris8.3 OsU6aT#- primers were used at 0.1 μM each. PCR amplification was performed using the high-fidelity enzyme KOD FX. The reaction conditions were pre-denaturation at 94°C for 2 min, followed by 25-28 cycles of 94°C for 10 s, 58°C for 15 s, and 68°C for 20 s, and a final extension at 68°C for 10 min. First-round PCR products were obtained and detected by agarose gel electrophoresis.

[0078] Second-round PCR: Dilute the first-round PCR product 10-fold. 1 μl of this diluted product was used as the template for the second-round PCR. Primers U-GAL and Pgs-GAR were used, and amplification was performed using KOD FX. The reaction conditions were 94°C for 2 minutes of initial denaturation, followed by 28-30 cycles of 94°C for 10 seconds, 58°C for 15 seconds, and 68°C for 20 seconds, followed by a final extension at 68°C for 10 minutes. After amplification, electrophoresis revealed a target band approximately 700 bp in size. The target fragment was recovered and purified from the gel to obtain recovered fragment 1, which contained the target sequence and was used for infusion ligation.

[0079] Primer sequences used in this section:

[0080] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3';

[0081] gR-R: 5'-CGGAGGAAAATTCCATCCAC-3';

[0082] U-GAL: 5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGG-3';

[0083] Pgs-GAR: 5'-TAGCTCGAGAGGCGCGCCAATGATACCGACGCGTATCCATCCACTCCAAGTCTTG-3';

[0084] Cris8.3 gRT#+: 5'-CCCCGGCACACGCTCACCGGTTTTAGAGCTAGAAAT-3';

[0085] Cris8.3 OsU6aT#-: 5'-CGGTGAGCGTGTGCCGGGGCGGCAGCCAAGCCAGCA-3'.

[0086] Construction of SiDRe1 knockout vector

[0087] The pYLCRISPR / Cas9Pubi-H vector (AddGene: Plasmid #66187) was digested with BsaI-HF (NEB #R3535) at 37°C for 4 h. After digestion, the linearized vector fragment was cut out and purified (Nanjing Novozymes Biotech Co., Ltd. #DC301-01) to obtain linearized vector 1.

[0088] The recovered fragment 1 obtained above was ligated with the linearized vector 1 using In-fusion ligase (Nanjing Novozymes Biotech Co., Ltd. #C112-01), transformed into E. coli competent cells DH5α, and plated on solid LB medium containing kanamycin. After overnight culture, single clones were picked and positive single clones were identified by PCR and sequenced using primers CasMHV3F (5'-GTGGTGATAAGCGTCCTG-3') and CasMHV3R (5'-AAGGCGATTAAGTTGGGT-3') (Shanghai Sangon Biotech Co., Ltd.). The recombinant vector with correct sequencing was named SiDRe1 gene knockout vector. SiDRe1 gene knockout vector

[0089] SEQ ID No. 4 (5′-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGGATTTTT -3′) was inserted between sequence A (5′-ACCGGTAAGGCGCGCCGTAGTGCTCGA-3′) and sequence B (5′-CGGTATCATTGGCGCGCCTCTC GAGCTA-3′) of the pYLCRISPR / Cas9Pubi-H vector, while keeping the other nucleotide sequences of the pYLCRISPR / Cas9Pubi-H vector unchanged. The resulting recombinant expression vector was named SiDRe1 gene knockout vector.

[0090] Acquisition and identification of SiDRe1 knockout millet

[0091] The correct SiDRe1 gene knockout vector ( Figure 1) was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium; millet Ci846 callus tissue was transformed using Agrobacterium-mediated genetic transformation (millet Ci846 and its transformation method are recorded in the following literature: Cheng, Z., Sun, Y., Yang, S., Zhi, H., Yin, T., Ma, X., Zhang, H., Diao, X., Guo, Y., Li, X., Wu, C., & Sui, Y. (2021). Establishing in planta haploid inducer line by edited SiMTLin foxtailmillet (Setaria italica). Plant biotechnology journal, 19(6), 1089–1091. https: / / doi.org / 10.1111 / pbi.13584, millet Ci846 is named Ci846 or Ci846 wild type in this literature) to obtain T0 generation transgenic plants.

[0092] Positive transgenic plants were identified using primers CasMHV3F (5'-GTGGTGATAAGCGTCCTG-3') and CasMHV3R (5'-AAGGCGATTAAGTTGGGT-3'). Genomic sequences near the target site in T0-generation transgenic plants were sequenced using primers CR8.3SeqF (5'-GCCTTCGGCACAGCATC-3') and CR8.3SeqR (5'-TTCACGACCGTCAGTTGGA-3') (Sangon Biotech (Shanghai) Co., Ltd.) to confirm the editing pattern of the target site in the transgenic plants. T0-generation seeds were harvested and sown in a greenhouse. The editing pattern of T1-generation transgenic plants was identified using the above primers. Continuous self-pollination ultimately yielded plants homozygous for a single-base A insertion and a 34-bp deletion (GTACGGCCCCCGGCCCCGGCACACGCTCACCGAG), designated sidre1-i and sidre1-d34, respectively.

[0093] Compared with the wild-type foxtail millet Ci846, the SiDRe1 gene in both chromosomes of sidre1-ide has the following mutations:

[0094] An adenine deoxyribonucleotide residue was inserted between positions 656 and 657 (on two exons) of the SiDRe1 gene (SEQ ID No. 1), resulting in an adenine deoxyribonucleotide residue inserted between positions 370 and 371 of the SiDRe1 gene coding region (SEQ ID No. 2), causing a frameshift and premature termination of protein translation, resulting in loss of SiDRe1 protein function (loss of the bZIP domain and nuclear localization signal). The sequence alignment of wild-type foxtail millet Ci846 and the gene-edited homozygous strain sidre1-i is shown in the figure below. Figure 2 Shown in B and 2C.

[0095] Compared with the wild-type foxtail millet Ci846, the SiDRe1 gene in both chromosomes of sidre1-d34 has the following mutations:

[0096] There is a 34bp (5'-GTACGGCCCCCGGCCCCGGCACACGCTCACCGAG-3') deletion between positions 627 and 662 of the SiDRe1 gene (SEQ ID No. 1), which results in a 34bp (5'-GTACGGCCCCCGGCCCCGGCACACGCTCACCGAG-3') deletion between positions 341 and 376 of the SiDRe1 gene coding region (SEQ ID No. 2), causing a frameshift and premature termination of protein translation, resulting in loss of SiDRe1 protein function (loss of the bZIP domain and nuclear localization signal). The sequence alignment diagram of the wild-type foxtail millet Ci846 and the gene-edited homozygous strain sidre1-d34 is shown in Figure 2. Figure 2 As shown in D and 2E.

[0097] Example 2: SiDRe1 homozygous edited lines have stronger drought tolerance at the seedling stage than the wild type

[0098] Seeds of two homozygous editing lines of SiDRe1 (sidre1-i and sidre1-d34) and wild type (wild type foxtail millet Ci846 or foxtail millet Ci846 or Ci846) were sown in square flower pots (upper diameter 10 cm × 10 cm, depth 10 cm). Three days after emergence, the seedlings were transplanted and water control began (no additional watering). Pictures were taken and the survival rate was calculated one week after emergence, seven days after drought treatment, before rewatering (drought treatment for 12 days), and two days after rewatering. The survival rate of the two homozygous editing lines of SiDRe1 was significantly higher than that of the wild type ( Figure 4 ), the specific operations are as follows:

[0099] Seeds of wild-type millet Ci846 (WT), sidre1-i, and sidre1-d34 were sown in square pots (10 cm × 10 cm in diameter, 10 cm in depth, with a volume ratio of vermiculite to nutrient soil of 3:1. After transplanting, 200 g of the culture medium was irrigated with 100 ml of water). The pots were marked with the species and randomly placed. The pots were kept at 25°C-30°C, 10 h light / 14 h dark, and 3 The culture was carried out under 0%-40% humidity conditions. 3 days after emergence, 25 seedlings were left in each tray and water control was started (no additional watering). After 12 days of water control, the seedlings were rehydrated (the flower pots were immersed in water for 5 minutes and then taken out). The seedlings were placed at 25°C-30°C, 10 hours of light / 14 hours of darkness, and 30%-40% humidity for rehydration and culture. The survival rate was calculated 2 days after rehydration. The phenotypic records were also made 3 days after emergence, 7 days after water control, 12 days after water control, and 2 days after rehydration.

[0100] Survival rate = number of surviving seedlings / total number of seedlings.

[0101] Phenotypic results such as Figure 4 Middle A( Figure 4 In middle A, WT is the wild-type millet Ci846 (WT), sidre1-i is the self-pollinated progeny of sidre1-i, sidre1-d34 is the self-pollinated progeny of sidre1-d34, before water control is 3 days after seedling emergence, water control 7 is 7 days after water control treatment, water control 12 days is 12 days after water control treatment, and rewatering 2 days is 2 days after rewatering. As shown in FIG. 7 days after water control treatment, 12 days after water control treatment, and 2 days after rewatering, the seedling growth phenotypes of the self-pollinated progeny of sidre1-i and the self-pollinated progeny of sidre1-d34 were significantly better than those of the wild-type millet Ci846 (WT).

[0102] The survival results were as follows Figure 4 Middle B( Figure 4 As shown in Figure B, Ci846 is the wild-type millet Ci846, sidre1-i is the self-pollinated progeny of sidre1-i, and sidre1-d34 is the self-pollinated progeny of sidre1-d34. The vertical axis represents the survival rate. The survival rates are significantly higher than those of the wild type.

[0103] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of a protein, a substance for knocking out, inhibiting, reducing or down-regulating the expression of a gene encoding the protein, or a substance for knocking out, inhibiting, reducing or down-regulating the activity or content of the protein in any of the following: A1) Application in improving drought resistance of Setaria plants; A2) application in the preparation of a product for improving drought resistance of Setaria plants; A4) Use in plant breeding and / or in the preparation of plant breeding products; The protein is any of the following: B1) the amino acid sequence is the protein shown in SEQ ID No. 3; B2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein described in B1) and having at least 80% identity with the protein described in B1) and having the same function; B3) A fusion protein obtained by linking the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

2. The use according to claim 1, characterized in that The protein is derived from millet.

3. The use according to claim 1 or 2, characterized in that The substance that knocks out, inhibits, reduces or downregulates the expression of the gene encoding the protein is any one of the following: D1) a nucleic acid molecule that inhibits, reduces or downregulates the expression of a gene encoding a protein according to claim 1 or 2; D2) expressing the gene encoding the nucleic acid molecule described in D1); D3) an expression cassette containing the gene described in D2); D4) a recombinant vector containing the gene described in D2) or a recombinant vector containing the expression cassette described in D3); D5) a recombinant microorganism containing the gene described in D2), or a recombinant microorganism containing the expression cassette described in D3), or a recombinant microorganism containing the recombinant vector described in D4); D6) a transgenic plant cell line containing the gene described in D2), or a transgenic plant cell line containing the expression cassette described in D3), or a transgenic plant cell line containing the recombinant vector described in D4); D7) transgenic plant tissue containing the gene described in D2), or transgenic plant tissue containing the expression cassette described in D3), or transgenic plant tissue containing the recombinant vector described in D4); D8) A transgenic plant organ containing the gene described in D2), or a transgenic plant organ containing the expression cassette described in D3), or a transgenic plant organ containing the recombinant vector described in D4).

4. The use according to claim 3, characterized in that D1) The nucleic acid molecule is a gRNA targeting the protein-encoding gene of claim 1, and the target sequence of the gRNA is SEQ ID No.

1.

5. The method according to any one of claims 1 to 4, wherein: The Setaria plant is millet.

6. A method for improving drought resistance of Setaria plants, characterized in that: The method comprises improving the drought resistance of Setaria plants by knocking out or inhibiting or reducing or down-regulating the expression of the gene encoding the protein of claim 1 or 2 in Setaria plants, and / or the activity and / or content of the protein of claim 1 or 2.

7. A method for cultivating highly drought-resistant Setaria plants, comprising knocking out or inhibiting or reducing or downregulating the expression level of the gene encoding the protein described in claim 1 or 2 in the target Setaria plant, and / or the activity and / or content of the protein to obtain a highly drought-resistant Setaria plant, wherein the highly drought-resistant Setaria plant has higher drought resistance than the target Setaria plant.

8. The method according to claim 6 or 7, wherein: The knocking out, inhibiting, reducing or down-regulating the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing a gene knockout vector with SEQ ID No. 1 as the target sequence into the target plant.

9. A method for cultivating highly drought-resistant millet, comprising subjecting the genome of the target millet to any of the following operations: 1) inserting an adenine deoxyribonucleotide residue between positions 656 and 657 of SEQ ID No. 1 in the target millet genome; 2) Deletion mutation was performed between positions 627 and 662 of SEQ ID No. 1 in the target millet genome.

10. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9, characterized in that: The Setaria plant is millet.

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