Function and application of sirdre1 in drought tolerance of foxtail millet

Abstract

The application discloses SiDRe1 regulation of foxtail millet drought resistance function and application, solves the technical problem of how to improve the drought resistance of foxtail millet, specifically discloses the application of protein, knocking out or inhibiting or reducing or down-regulating the expression of the coding gene of the protein or knocking out or inhibiting or reducing or down-regulating the activity or content of the protein in any one of the following: A1) application in improving the drought resistance of poaceae plants; A2) application in preparing products for improving the drought resistance of poaceae plants; A4) application in plant breeding and / or preparing plant breeding products; the protein is any one of the following: B1) the protein with the amino acid sequence shown in SEQ ID No. 3; B2) the protein obtained by substitution, deletion and / or addition of amino acid residues of the protein in B1) and having more than 80% identity with the protein shown in B1) and having the same function; B3) the fusion protein obtained by connecting the N terminal or / and C terminal of B1) or B2) with a protein tag. Inhibiting or reducing or down-regulating the expression of foxtail millet GmPHD6 gene can improve the drought resistance of foxtail millet, and can be used for production.

Description

Technical Field

[0001] This invention specifically relates to the function and application of SiDRe1 in regulating the drought resistance of millet. Background Technology

[0002] Drought is a significant factor affecting agricultural production. With the advancement of molecular biotechnology and the increasing maturity of plant transgenic technology, introducing key genes into target materials using genetic engineering methods to cultivate drought-resistant crop varieties is one of the effective ways to alleviate drought stress in agricultural production.

[0003] Millet is my country's largest coarse grain crop, characterized by its drought resistance, stress tolerance, wide adaptability, rich nutrition, and unique flavor. Millet is widely recognized as a drought-resistant crop and plays a crucial role in adjusting the agricultural structure and developing sustainable dryland agriculture. Analyzing the drought-resistant mechanisms of millet and identifying its drought-resistant genes is of great significance for improving crop stress resistance.

[0004] bZIP transcription factors participate in multiple biological processes in plants, including growth, development, dormancy, senescence, and stress resistance. The characteristics, classification, and biological functions of bZIP transcription factors in plants have been extensively studied (Lu et al., 2002). However, due to the relatively late start of molecular biology research on millet, research on bZIP transcription factors in millet is still limited, and few bZIP family transcription factors have been cloned from millet. How to provide a gene related to drought resistance in millet is a technical problem faced by researchers in this field. Summary of the Invention

[0005] The purpose of this application is to provide a method for improving the drought resistance of millet, especially during the seedling stage.

[0006] In a first aspect, this application discloses the use of proteins, substances that knock out, inhibit, reduce, or downregulate the expression of genes encoding said proteins, or substances that knock out, inhibit, reduce, or downregulate the activity or content of said proteins in any of the following:

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

[0008] A2) Application in the preparation of products that enhance the drought resistance of plants in the genus Setaria;

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

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

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

[0012] B2) A protein having more than 80% identity and the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1).

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

[0014] The *Setaria* species mentioned above can refer to seedling *Setaria* species.

[0015] The seedling stage mentioned above can be 3 days after emergence.

[0016] The breeding objectives mentioned above include drought resistance. Drought resistance can be high or low.

[0017] In the above text, the drought resistance index can be the survival rate.

[0018] Knocking out, inhibiting, reducing, or downregulating the expression of genes encoding the aforementioned proteins in *Setaria* plants, or knocking out, inhibiting, reducing, or downregulating the activity or content of the proteins, can improve drought resistance.

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

[0020] To facilitate the purification of proteins in B1), tags as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein represented by SEQ ID No. 1 in the sequence listing.

[0021] Table 1. Sequence of Labels

[0022]

[0023]

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

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

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

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

[0028] The millet mentioned above may be the millet variety Ci846.

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

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

[0031] In some 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) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the genes encoding the above proteins;

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

[0034] D3) contains 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) Recombinant microorganisms containing the gene described in D2), or recombinant microorganisms containing the expression cassette described in D3), or recombinant microorganisms 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 described in D1), those skilled in the art can easily mutate the nucleotide sequence that inhibits, reduces, or downregulates the expression of the SiDRe1 protein-encoding gene of the present invention using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides that have 80% or more of the same nucleotide sequence as the one isolated in the present invention that inhibits, reduces, or downregulates the expression of the SiDRe1 protein-encoding gene, and that have the function of inhibiting, reducing, or downregulating the expression of the SiDRe1 protein-encoding gene, are all derived from and equivalent to the nucleotide sequence of the present invention.

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

[0042] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, 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 amino acid sequences, then the identity value (%) can be obtained.

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

[0044] The microorganism mentioned in D5 above can be Agrobacterium. The Agrobacterium is EHA105.

[0045] In some embodiments, the nucleic acid molecule described in D1) is a gRNA that targets the protein-coding gene described above, and the target sequence of the gRNA is SEQ ID No. 1.

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

[0047] In some embodiments, the foxtail grass is millet.

[0048] The millet mentioned above may be the millet variety Ci846.

[0049] In some embodiments, a method for improving the drought resistance of *Setaria* plants includes improving the drought resistance of *Setaria* plants by knocking out or inhibiting or reducing or downregulating the expression of genes encoding the aforementioned proteins in *Setaria* plants, and / or the activity and / or content of the aforementioned proteins.

[0050] In some embodiments, a method for cultivating highly drought-resistant Setaria species includes knocking out, inhibiting, reducing, or downregulating the expression level of the gene encoding the aforementioned protein in the target Setaria species, and / or, the activity and / or content of the protein to obtain highly drought-resistant Setaria species, the highly drought-resistant Setaria species having higher drought resistance than the target Setaria species.

[0051] In some embodiments, knocking out, inhibiting, reducing, or downregulating the expression of the gene encoding the protein described above in the plant includes introducing a gene knockout vector with SEQ ID No. 1 as the target sequence into the target plant.

[0052] On the other hand, this 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) Insert an adenine deoxyribonucleotide residue between positions 656 and 657 of SEQ ID No. 1 in the target millet genome;

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

[0055] In some embodiments, the foxtail grass plant is millet.

[0056] The millet mentioned above may be the millet variety Ci846.

[0057] Beneficial effects

[0058] This invention discloses the function and application of SiDRe1 in regulating drought resistance in millet. The technical problem it addresses is how to improve the drought resistance of millet. Specifically, it discloses the application of the protein, substances that knock out, inhibit, reduce, or downregulate the expression of the gene encoding the protein, or substances that knock out, inhibit, reduce, or downregulate the activity or content of the protein in any of the following: A1) application in improving the drought resistance of *Setaria* species; A2) application in preparing products that improve the drought resistance of *Setaria* species; A4) application in plant breeding and / or preparation of plant breeding products; the protein is any of the following: B1) a protein with the amino acid sequence shown in SEQ ID No. 3; B2) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in B1) that has more than 80% identity with and the same function as the protein shown in B1); B3) a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2). Inhibiting or reducing or downregulating the expression of the millet GmPHD6 gene can improve the drought resistance of millet and can be used in production. Attached Figure Description

[0059] Figure 1 Here is a diagram of the SiDRe1 target sgRNA expression cassette and the pYLCRISPR / Cas9Pubi-H-SiDRe1 recombinant vector framework.

[0060] Figure 2The diagram shows the SiDRe1 gene structure, target sequence, and sequence alignment of homozygous gene-edited lines. Note: A: SiDRe1 gene structure, with scissors indicating the target location; B: SiDRe protein structure diagram, showing frameshift mutations resulting in the deletion of the bZIP domain after editing; CD: Schematic diagram of sidre-i gene editing, with sequencing peaks at the target location (C), black underlined "NGG" PAM sequence, and red arrows indicating base insertion positions; alignment of wild-type and sidre-i target coding strand sequences (D), with "-" indicating deletion; EF: Schematic diagram of sidre-d34 gene editing, with sequencing peaks at the target location (E), black underlined "NGG" PAM sequence, and red arrows indicating base insertion positions; alignment of wild-type and sidre-d34 target coding strand sequences (F), with "-" indicating deletion.

[0061] Figure 3 The SiDRe1 homozygous edited line showed stronger ABA sensitivity than the wild-type Ci846. Note: A-E: Germination rate statistics at different ABA concentrations; F: Germination phenotype under different ABA concentrations. * indicates P < 0.05.

[0062] Figure 4 The drought resistance of the SiDRe1 homozygous edited line is stronger than that of the wild type Ci846 (Figure A: Drought treatment test of sidre1-i and sidre-d34 seedlings; Figure B: Survival statistics of sidre1-i and sidre-d34). Detailed Implementation

[0063] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

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

[0065] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0066] Example 1: Obtaining millet with SiDRe1 gene knocked out

[0067] Design SiDRe1 gene knockout target

[0068] Based on the SiDRe1 gene sequence, target sites were designed using the online target design website CRISPR-P 2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Suitable target sites were selected based on their location (before the bZIP domain) and targeting specificity. The final SiDRe1 gene knockout target was: 5'-CCCCGGCACACGCTCACCG-3' ( Figure 1 ).

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

[0070] The specific details of SEQ ID No. 1 are as follows;

[0071]

[0072] SEQ ID No. 2 is detailed below:

[0073]

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

[0075] MSDDDSDGQRQCRRGVTVDVELDAAMALADMAGVGPAATPGGGNHPRSSHQAAAAAPPTAEGEDDLASTRLSLELGKVGIQSASPCSSSSSAGHPHPQPSAQHQPHPVPEAAATGYGPRPRHTLTEAEKEAKRLRRVLANRESARQTILRRQAIRDELARKVVDLSSQNENMKKEKDMVMQEYLSLKEANKQLKEQVARTTK KAPVTTAVTAAVPMHVAAVQQQAAEPMAAAAASPPATPPQPGFLYTAAPPVPYVWGSWPPGAGYEHHHHHGSPPALCLPPCAWYYPVVADPRGSPSAAYAGTQQQQQQAAFQPEPAGSGGGSPAGGATAEEDTDDDPCSLTLGLDIAADKRSAPVSIDARGGAGPSDRDKAATAAEARKRRKELTKLKHMHAAGRPGAEQW

[0076] Obtain the target fragment containing the target sequence for Infusion ligation.

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

[0078] Second round PCR: The first round PCR amplification product was diluted 10-fold. 1 μl of this diluted product was used as the template for the second round PCR. Using U-GAL and Pgs-GAR primers, KOD FX enzyme was used for amplification. The reaction conditions were: 94℃ for 2 min pre-denaturation, followed by 28-30 cycles of 94℃ for 10 s, 58℃ for 15 s, and 68℃ for 20 s, with a final extension at 68℃ for 10 min. Electrophoresis showed the target band size to be approximately 700 bp. The target fragment was recovered and purified using gel extraction to obtain fragment 1, which is the target fragment containing the target sequence for infusion ligation (Seq).

[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℃ for 4 hours. After digestion, the results were detected by agarose gel electrophoresis. The linearized vector fragment was extracted and purified (Nanjing Novizan Biotechnology Co., Ltd. #DC301-01) to obtain linearized vector 1.

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

[0089] Insert SEQ ID No. 4 (5′-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGCAGCAAAGGATTTTT-3′) between sequence A (5′-ACCGGTAAGGCGCGCCGTAGTGCTCGA-3′) and sequence B (5′-CGGTATCATTGGCGCGCCTCTCGAGCTA-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 is named the SiDRe1 gene knockout vector.

[0090] Obtaining and identifying SiDRe1 knockout millet

[0091] Construct the correct SiDRe1 gene knockout vector ( Figure 1Agrobacterium EHA105 was introduced to obtain recombinant Agrobacterium; Agrobacterium-mediated genetic transformation was used to transform millet Ci846 callus (Millet Ci846 and its transformation method are described 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, where millet Ci846 is referred to as Ci846 or Ci846 wild type in this literature), to obtain T0 generation transgenic plants.

[0092] Positive transgenic Zhihu plants were identified using primers CasMHV3F (5'-GTGGTGATAAGCGTCCTG-3') and CasMHV3R (5'-AAGGCGATTAAGTTGGGT-3'). The genome sequence near the target site in T0 generation transgenic positive plants was sequenced using primers CR8.3SeqF (5'-GCCTTCGGCACAGCATC-3') and CR8.3SeqR (5'-TTCACGACCGTCAGTTGGA-3') to determine the editing mode of the transgenic plant target site. T0 generation seeds were harvested and sown in a greenhouse. The editing mode of T1 generation transgenic plants was identified using the same primers. Continuous self-pollination yielded homozygous edited plants with a single A insertion and homozygous edited plants with a 34bp deletion (GTACGGCCCCCGGCCCCGGCACACGCTCACCGAG), named sidre1-i and sidre1-d34, respectively.

[0093] Compared to wild-type millet Ci846, the SiDRe1 gene in both sets of 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 insertion of an adenine deoxyribonucleotide residue between positions 370 and 371 of the SiDRe1 gene coding region (SEQ ID No. 2). This caused a frameshift and premature termination of protein translation, leading to loss of function of the SiDRe1 protein (loss of the bZIP domain and nuclear localization signal). A schematic diagram of the sequence alignment between wild-type millet Ci846 and the gene-edited homozygous line sidre1-i is shown below. Figure 2 As shown in B and C of section 2.

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

[0096] A 34bp (5'-GTACGGCCCCCGGCCCCGGCACACGCTCACCGAG-3') deletion exists between positions 627 and 662 of the SiDRe1 gene (SEQ ID No. 1), resulting in a 34bp (5'-GTACGGCCCCCGGCCCCGGCACACGCTCACCGAG-3') deletion between positions 341 and 376 of the SiDRe1 gene coding region (SEQ ID No. 2). This causes a frameshift and premature termination of protein translation, leading to loss of SiDRe1 protein function (loss of the bZIP domain and nuclear localization signal). A schematic diagram of sequence alignment between wild-type millet Ci846 and the gene-edited homozygous line sidre1-d34 is shown below. Figure 2 As shown in D in section 2 and E in section 2.

[0097] Example 2: The SiDRe1 homozygous edited line showed stronger drought resistance in the seedling stage than the wild type.

[0098] Seeds of two homozygous edited lines of SiDRe1 (sidre1-i and sidre1-d34) and the wild type (wild-type millet Ci846 or millet Ci846 or Ci846) were sown separately in square flowerpots (10cm×10cm top diameter, 10cm depth). Three days after germination, seedlings were thinned and water was withheld (no additional watering). Photos were taken and survival rates were recorded one week after germination, seven days after drought treatment, before rewatering (12 days after drought treatment), and two days after rewatering. The survival rates of both homozygous edited lines of SiDRe1 were significantly higher than those of the wild type. Figure 4 The specific steps are as follows:

[0099] Seeds of wild-type millet Ci846(WT), sidre1-i, and sidre1-d34 were sown separately in square flowerpots [(top diameter 10cm × 10cm, depth 10cm). The cultivation substrate in the flowerpots was prepared by mixing vermiculite and nutrient soil in a volume ratio of 3:1. After transplanting, watering was carried out with 200g of cultivation substrate to 100ml of water]. The flowerpots were labeled with the planted species and placed randomly. They were kept at 25℃-30℃ under 10 hours of light / 14 hours of darkness for 3 days. Cultivate under 0%-40% humidity conditions. Three days after emergence, keep 25 seedlings per pot and start water control (no additional watering). After 12 days of water control, rehydrate (immerse the flowerpots in water for 5 minutes and then remove them). Place them in 25℃-30℃, 10 hours of light / 14 hours of darkness, and 30%-40% humidity conditions for rehydration culture. Calculate the survival rate 2 days after rehydration. Phenotypic records are 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 are as follows Figure 4 China A ( Figure 4 In Figure A, WT represents wild-type millet Ci846(WT), sidre1-i represents the self-pollinated progeny of sidre1-i, and sidre1-d34 represents the self-pollinated progeny of sidre1-d34. The values ​​before water control are 3 days after emergence, 7 days after water control is 7 days after water control treatment, 12 days after water control treatment is 12 days after water control treatment, and 2 days after rehydration are 2 days after rehydration. As shown in the figure, after 7 days of water control treatment, 12 days of water control treatment, and 2 days after rehydration treatment, 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 wild-type millet Ci846(WT).

[0102] Survival results as follows Figure 4 B ( Figure 4 In the data, Ci846 represents wild-type millet, sidre1-i represents the self-crossed offspring of sidre1-i, and sidre1-d34 represents the self-crossed offspring of sidre1-d34. The vertical axis represents the survival rate. The survival rates of all of them are significantly higher than those of the wild type.

[0103] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Substances that knock out, inhibit, reduce, or downregulate the expression of protein-coding genes, or substances that knock out, inhibit, reduce, or downregulate protein levels, in any of the following applications: A1) Application in improving the drought resistance of millet; A2) Application in the preparation of products that enhance the drought resistance of millet; A3) Application in millet breeding and / or preparation of millet breeding products, wherein the breeding objective is to improve drought resistance; The protein is any one of the following: B1) The amino acid sequence is that of the protein shown in SEQ ID No. 3; B2) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1); The substance is any one of the following: D1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein; D2) expresses the gene encoding the nucleic acid molecule described in D1); D3) contains 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) Recombinant microorganisms containing the gene described in D2), or recombinant microorganisms containing the expression cassette described in D3), or recombinant microorganisms 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).

2. The application according to claim 1, characterized in that, D1) The nucleic acid molecule is a gRNA that targets the protein-coding gene of claim 1, and the target sequence of the gRNA is SEQ ID No.

1.

3. A method for improving the drought resistance of millet, characterized in that, The method includes improving the drought resistance of millet by knocking out or inhibiting or reducing or downregulating the expression of the gene encoding the protein of claim 1 and / or the content of the protein of claim 1 in millet.

4. A method for cultivating highly drought-resistant millet, comprising knocking out or inhibiting or reducing or downregulating the expression level of the gene encoding the protein of claim 1 and / or the content of the protein in the target millet, to obtain highly drought-resistant millet, wherein the drought resistance of the highly drought-resistant millet is higher than that of the target millet.

5. The method according to claim 3 or 4, characterized in that, The knockout, inhibition, reduction, or downregulation of the expression of the gene encoding the protein of claim 1 in millet comprises introducing a gene knockout vector with SEQ ID No. 1 as the target sequence into the target millet.

6. A method for breeding highly drought-resistant millet, wherein the genome of the target millet is subjected to any of the following operations: 1) Insert an adenine deoxyribonucleotide residue between positions 656 and 657 of SEQ ID No. 1 in the target millet genome; 2) A deletion mutation was performed between positions 627 and 662 of SEQ ID No. 1 in the target millet genome.

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