Application of ZmHB77 protein and its coding gene in regulating drought resistance of plants

Abstract

The application discloses application of ZmHB77 protein and a coding gene thereof in regulation of drought resistance of plants. The application discloses application of ZmHB77 protein (SEQ ID No. 1) or a related biological material thereof in all or part of the following: regulation of drought resistance of plants, regulation of seed root number of plants, regulation of lateral root density of plants, and the related biological material is a nucleic acid molecule capable of expressing the ZmHB77 protein or an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant bacterium or a transgenic cell line. After gene knockout of the ZmHB77 gene drought resistance protein coding gene of the application, the drought resistance of corn is significantly improved. The application provides a more precise, efficient and safe technical method for creating a corn overexpression plant and / or corn breeding, realizes precise improvement of the drought resistance of corn, can promote the commercial corn breeding process, overcomes the short board of traditional breeding, shortens the breeding cycle and does not affect other properties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to the application of the ZmHB77 protein and its encoding gene in regulating plant drought resistance. Background Technology

[0002] Corn (Zea mays L.) is one of the most important food, feed, and economic energy crops.

[0003] Drought is one of the most serious abiotic stresses globally. It hinders plant growth and development, causes stunted growth, and reduces crop yield and quality, posing a serious threat to agricultural production and is a global problem affecting agricultural production.

[0004] Therefore, breeding drought-resistant maize varieties is an effective way to reduce drought losses. Traditional maize breeding methods for improving drought resistance are time-consuming and inefficient. Utilizing transgenic and gene-editing technologies to directionally express genes with superior drought resistance traits in different species provides a more direct and effective method for targeted genetic improvement of these superior genes, offering a more direct and efficient approach to drought-resistant maize breeding. Summary of the Invention

[0005] This invention claims protection for the application of the ZmHB77 protein and its encoding gene in regulating plant drought resistance.

[0006] In a first aspect, the present invention claims protection for the use of ZmHB77 protein or related biological materials thereof in all or part of the following:

[0007] P1. Regulates plant drought resistance;

[0008] P2, regulates the number of seed roots in plants;

[0009] P3. Regulate the density of lateral roots in plants.

[0010] The expression cassette refers to DNA capable of expressing ZmHB77 in host cells. This DNA may include not only a promoter to initiate ZmHB77 gene transcription but also a terminator to terminate ZmHB77 gene transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the ubiqutin promoter (pUbi) for the ubiquitin gene; the constitutive promoter 35S for cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); the chemically induced promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); the tomato protease inhibitor II promoter (PIN2) or the LAP promoter (both induced by jasmonic acid methyl ester); the heat shock promoter (US Patent 5,187,267); the tetracycline-inducible promoter (US Patent 5,057,422); and seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 1)). 0099169.7), seed storage protein-specific promoters (e.g., promoters of beta-conglycin, napin, oleosin, and soybean beta-conglycin (Beachy et al. (1985) EMBO J.4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator (see, e.g., Odell et al. (I 985 Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res. 15:9627.

[0011] Construct a recombinant expression vector containing the ZmHB77 gene expression cassette. The plant expression vector used can be a binary Agrobacterium vector or a Gateway system vector, such as pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pGWB411, pGWB412, pGWB405, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When constructing recombinant expression vectors using ZmGW3, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0012] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants that encode enzymes or luminescent compounds that produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0013] In the above applications, the vector can be a plasmid, granule, bacteriophage, or viral vector.

[0014] In the above applications, the microorganisms can be yeast, bacteria, algae, or fungi. Among them, bacteria can be from the genera *Escherichia*, *Erwinia*, *Agrobacterium* (such as *Agrobacterium tumefaciens* EHA105), *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, *Bacillus*, etc.

[0015] The ZmHB77 protein can be any of the following:

[0016] (A1) A protein with the amino acid sequence SEQ ID No. 1;

[0017] (A2) A protein derived from maize with the same function, derived from the amino acid sequence shown in SEQ ID No. 1 by substitution and / or deletion and / or addition of one or more amino acid residues.

[0018] (A3) has 99%, 95%, 90%, 85% or more of the same amino acid sequence as any of (A1)-(A2) and is derived from a maize protein with the same function.

[0019] (A4) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of any of the proteins defined in (A1)-(A3).

[0020] 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.

[0021] 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.

[0022] In the aforementioned proteins, the homology of 95% or more can be at least 96%, 97%, or 98%. The homology of 90% or more can be at least 91%, 92%, 93%, or 94%. The homology of 85% or more can be at least 86%, 87%, 88%, or 89%. The homology of 80% or more can be at least 81%, 82%, 83%, or 84%.

[0023] In the plant, the expression level and / or activity of the ZmHB77 protein are reduced, the drought resistance of the plant is increased and / or the number of seed roots is reduced and / or the density of lateral roots is increased.

[0024] Secondly, the present invention claims the use of a substance capable of reducing the expression level and / or activity of ZmHB77 protein in plants in any of (a1)-(a4):

[0025] (a1) Improve plant drought resistance;

[0026] (a2) Reduce the number of seed roots in plants;

[0027] (a3) Increase the density of lateral roots in plants.

[0028] The ZmHB77 protein may be any of the proteins shown in (A1)-(A4) above.

[0029] In a specific embodiment of the present invention, the substance capable of reducing the expression level and / or activity of ZmHB77 protein in plants is specifically a CRISPR / Cas9 system (used to knock out nucleic acid molecules in recipient plants capable of expressing the ZmHB77 protein), and the target sequence targeted by the CRISPR / Cas9 system is positions 437-456 of SEQ ID No. 4, positions 437-456 of SEQ ID No. 5, and / or positions 437-456 of SEQ ID No. 6.

[0030] Thirdly, the present invention claims protection for a method of cultivating plants with increased drought resistance and / or fewer seed roots and / or increased lateral root density.

[0031] The method for cultivating plants with increased drought resistance and / or fewer seed roots and / or increased lateral root density claimed in this invention may include the step of reducing the expression level and / or activity of the ZmHB77 protein in the recipient plant.

[0032] The ZmHB77 protein may be any of the proteins shown in (A1)-(A4) above.

[0033] The method can be achieved through hybridization or through genetic modification.

[0034] Fourthly, the present invention claims protection for a method of cultivating transgenic plants with improved drought resistance and / or fewer seed roots and / or increased lateral root density.

[0035] The method for cultivating transgenic plants with improved drought resistance and / or fewer seed roots and / or increased lateral root density, as claimed in this invention, may include the following steps: inhibiting the expression of nucleic acid molecules capable of expressing ZmHB77 protein in a recipient plant to obtain a transgenic plant; the transgenic plant having improved drought resistance and / or fewer seed roots and / or increased lateral root density compared to the recipient plant.

[0036] The ZmHB77 protein may be any of the proteins shown in (A1)-(A4) above.

[0037] In a specific embodiment of the present invention, the inhibition of expression of nucleic acid molecules capable of expressing the ZmHB77 protein in the recipient plant is achieved using CRISPR / Cas9 technology. More specifically, in the method, the nucleic acid molecules capable of expressing the ZmHB77 protein in the recipient plant are knocked out using CRISPR / Cas9 technology, with the target sequence being positions 437-456 of SEQ ID No. 4, positions 437-456 of SEQ ID No. 5, and / or positions 437-456 of SEQ ID No. 6.

[0038] Furthermore, in the method described, knocking out the gene encoding the aforementioned protein in maize can be achieved by performing any of the following mutations on the maize DNA molecule whose coding sequence is shown in SEQ ID No. 2:

[0039] (1) The DNA molecule shown in SEQ ID No.2 is mutated to ZmHB77 / -59bp. ZmHB77 / -59bp is a DNA molecule obtained by deleting nucleotides at positions 40-58, 228-250, and 342-358 of the DNA molecule shown in SEQ ID No.2, while keeping the other nucleotide sequences unchanged (this mutation leads to a frameshift mutation, and translation cannot proceed normally).

[0040] (2) The DNA molecule shown in SEQ ID No.2 is mutated to ZmHB77 / -34bp. ZmHB77 / -34bp is a DNA molecule obtained by deleting nucleotides at positions 42-58 and 228-244 of the DNA molecule shown in SEQ ID No.2 while keeping the other nucleotide sequences unchanged (this mutation leads to a frameshift mutation, and translation cannot proceed normally).

[0041] In the above method, the transgenic plant is understood to include not only first- and second-generation transgenic plants, but also their progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The transgenic plant includes seeds, callus tissue, complete plants, and cells.

[0042] In all of the above aspects, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA.

[0043] Furthermore, the nucleic acid molecule capable of expressing the ZmHB77 protein can be any of the following:

[0044] (B1) The DNA molecule shown in SEQ ID No. 2 (CDS sequence) or SEQ ID No. 3 (genome sequence);

[0045] (B2) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined in (B1) and encodes the ZmHB77 protein;

[0046] A DNA molecule that has 99%, 95%, 90%, 85% or more homology to the DNA sequence defined in (B3) and (B1)-(B2) and encodes the ZmHB77 protein.

[0047] The stringent conditions for the above nucleic acid molecules can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA. Hybridization can be performed in a mixed solution of EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS; alternatively, hybridization can be performed at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 65°C in 0.1×SSC and 0.1% SDS; alternatively, hybridization can be performed in a solution of 6×SSC and 0.5% SDS at 65°C, followed by washing once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS.

[0048] In the aforementioned nucleic acid molecules, homology refers to the similarity of nucleotide sequences. The similarity of nucleotide sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI website. 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, 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 for the similarity of a pair of nucleotide sequences, the homology value (%) can be obtained.

[0049] In the aforementioned nucleic acid molecules, the homology of 95% or more can be at least 96%, 97%, or 98%. The homology of 90% or more can be at least 91%, 92%, 93%, or 94%. The homology of 85% or more can be at least 86%, 87%, 88%, or 89%. The homology of 80% or more can be at least 81%, 82%, 83%, or 84%.

[0050] Fifthly, the present invention claims protection for the application of the method described in the third or fourth aspect above in plant breeding.

[0051] In all of the above aspects, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0052] Furthermore, the monocotyledonous plant is a member of the Poaceae family.

[0053] Furthermore, the grasses mentioned are plants of the genus *Zea*, such as maize.

[0054] This invention successfully obtained transgenic maize plants with the ZmHB77 protein-coding gene knocked out using a CRISPR-Cas9 knockout experiment. Phenotypic identification and comparison of homozygous plants obtained by self-pollination of transgenic positive plants with wild-type plants showed that knocking out the ZmHB77 gene can improve the drought resistance of plants by increasing lateral root density and reducing the number of seed roots, thus proving that the ZmHB77 gene has an important biological function in regulating plant drought resistance.

[0055] Compared with the prior art, the present invention has the following advantages:

[0056] (1) After knocking out the gene encoding the drought-resistant protein of the ZmHB77 gene in this invention, the drought resistance of maize is significantly improved.

[0057] (2) This invention provides a more precise, efficient and safe technical method for drought-resistant maize breeding, realizes precise improvement of maize drought resistance, can promote the commercial maize breeding process, overcome the shortcomings of traditional breeding, shorten the breeding cycle without affecting other traits. Attached Figure Description

[0058] Figure 1 These are the results of genome-wide association analysis (GWAS) based on seed root number and the phenotypic detection results of mutant plants in this invention. a) shows the results of GWAS based on seed root number. b) shows the genotype of mutant plants obtained from the knockout lines. c and d show the seed root phenotype and seed root number statistics of the mutant plants, respectively. e and f show the lateral root density phenotype and statistics of the mutant plants, respectively. g and h show the survival rate phenotype and statistics of the mutant plants, respectively.

[0059] Figure 2 This diagram illustrates the construction of the recombinant vector CPB-sgRNA and shows the vector map of the recombinant vector. Detailed Implementation

[0060] 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.

[0061] 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.

[0062] The maize inbred line B104 is derived from the US National Plant Germplasm System (https: / / npgsweb.ars-grin.gov / gringlobal / search) and is referred to below as wild-type maize.

[0063] The maize inbred line B73 was derived from the National Germplasm Resource Bank.

[0064] The vector pCAMBIA3301 was purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., catalog number ZK868.

[0065] The CPB vector was provided by Professor Xie Chuanxiao's research group at the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and is disclosed in the literature "Zhao, Y., Zhang, C., Liu, W. et al. An alternative strategy for targeted gene replacement inplants using a dual-sgRNA / Cas9 design. Sci Rep 6, 23890 (2016)." The public can obtain the above-mentioned biological material from the applicant. The obtained biological material is only used for repeating the experiments of this invention and cannot be used for other purposes.

[0066] The pLB vector was purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number VT205.

[0067] Fast-T1 Escherichia coli competent cells were purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number C505-03.

[0068] EHA105 Agrobacterium competent cells were purchased from Beijing Bomed Gene Technology Co., Ltd., catalog number BC303.

[0069] The following examples use Excel software to process the data. The experimental results are expressed as mean ± standard deviation. P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0070] Example 1: Determination and acquisition of the target gene ZmHB77

[0071] I. Determination of the target gene ZmHB77

[0072] 1. Identification of ZmHB77, a gene related to the number of maize seed roots.

[0073] Based on the phenotypic data of seed root number from 1604 maize germplasm resources and the genotypic data obtained from resequencing, a genome-wide association analysis was conducted, locating an association signal controlling seed root number on chromosome 9, such as... Figure 1 As shown in Figure a. Based on bioinformatics analysis, a candidate gene Zm00001d045398 was discovered on this associated signal. This gene encodes a transcription factor and was named ZmHB77.

[0074] The genomic sequence of the ZmHB77 gene is a DNA molecule as shown in SEQ ID No. 3, and its CDS sequence is a DNA molecule as shown in SEQ ID No. 2. It encodes a protein with an amino acid sequence as shown in SEQ ID No. 1. The encoded protein is named ZmHB77 protein or protein ZmHB77.

[0075] II. Amplification and recovery sequencing of the ZmHB77 genome and its coding genes

[0076] Select plump seeds from the maize inbred line B73 and plant three seedlings in each pot filled with nutrient soil. Place the pots in a light incubator (28℃, light). When the seedlings reach the six-leaf stage, collect leaves. Use some leaves for genomic DNA extraction and the remaining leaves for RNA extraction. Store the extracted leaves at -80℃.

[0077] 1. Extraction of genomic DNA from maize materials

[0078] (1) Quickly place the leaves into a sterilized mortar and grind them thoroughly with liquid nitrogen, adding liquid nitrogen continuously during the process. After grinding, add the leaves to a 2.0ml centrifuge tube, filling the tube to 1 / 3 full.

[0079] (2) Preheat the prepared CTAB extract in a 65°C water bath, then add 800 μl of preheated CTAB buffer to the centrifuge tube from the previous step and shake vigorously to mix thoroughly.

[0080] (3) Place the mixed extract in a 65°C constant temperature water bath for 30 minutes, shaking it every 10 minutes during the process to ensure a full reaction.

[0081] (4) Take out the centrifuge tube that has undergone sufficient reaction, add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1), mix slowly for 15 min, and let stand for 10 min.

[0082] (5) Place the centrifuge tubes into the centrifuge and centrifuge (12000 rpm, 15-20 min);

[0083] (6) Carefully aspirate the supernatant into another clean 2.0 ml centrifuge tube, add an equal volume of pre-cooled (-20℃) isopropanol, gently mix and place the centrifuge tube in a -20℃ refrigerator for 30 min until a large amount of white precipitate is formed.

[0084] (7) Use a sterile pipette tip to remove the white precipitate and place it in another clean centrifuge tube. Add 500 μl of 75% ethanol and rinse 2-3 times.

[0085] (8) Centrifuge, discard 75% ethanol, remove excess ethanol with pipette tip and dry at room temperature, then dissolve DNA with 1×TE.

[0086] (9) Add RNase for purification (final concentration of 10 μg / μl), and keep in a constant temperature of 37℃ for 1 h;

[0087] (10) Add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) for extraction once, and centrifuge at high speed (12000 rpm) for 10 min.

[0088] (11) Carefully aspirate the supernatant, and extract it once again with an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1). Centrifuge at high speed (12000 rpm) for 10 min.

[0089] (12) Precipitate DNA with pre-cooled anhydrous ethanol and centrifuge (12000 rpm) for 10 min;

[0090] (13) Discard the ethanol, air dry thoroughly to avoid inhibiting the downstream PCR reaction, add an appropriate amount of TE to dissolve, and store in a -20℃ refrigerator for later use.

[0091] 2. Extraction and purification of total RNA from maize materials

[0092] The main method used is a phenol-free, chloroform-free rapid RNA extraction technique to extract total RNA from plants.

[0093] For specific methods, please refer to the instructions of the Gene-better Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (with gDNA filter). All experimental instruments used below have been sterilized at high temperature to remove RNase.

[0094] (1) Pour liquid nitrogen into a mortar that has been treated at 180℃ without RNase, then take out the frozen leaves from the -76℃ ultra-low temperature freezer, add liquid nitrogen and grind them thoroughly, continuously replenishing liquid nitrogen during the process until they are fully ground.

[0095] (2) Before adding the sample to the centrifuge tube, immerse the 1.5 ml RNase-free centrifuge tube completely in liquid nitrogen for quick freezing. Then quickly add the ground sample. Do not add too much sample, generally add to 1 / 3 (about 50 mg). Add 500 μl of lysis buffer and 50 μl of PLANTaid to the centrifuge tube and vortex for 20 s to allow it to fully lyse.

[0096] (3) Centrifuge the lysate at 13,000 rpm for 10 minutes to precipitate the fragments that cannot be lysed and PLANTaid bound with polysaccharides and polyphenols.

[0097] (4) Transfer the supernatant of the lysate to a new centrifuge tube. Add anhydrous ethanol equal to the volume of the supernatant, and immediately mix by pipetting. Do not centrifuge.

[0098] (5) Add the mixture (less than 720 μl each time, which can be added in two batches) into a genomic DNA filter, centrifuge at 13000 rpm for 2 min, and discard the waste liquid.

[0099] (6) Place the genomic DNA filter in a clean 2ml centrifuge tube, add 500μl of lysis buffer RLT PLUS to the genomic DNA filter, centrifuge at 13000rpm for 30s, collect the filtrate, estimate the filtration volume more accurately with a pipette, add 0.5 times the volume of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Immediately mix by pipetting and do not centrifuge.

[0100] (7) Immediately add the mixture to an adsorption column RA, centrifuge at 13000 rpm for 2 min, and discard the waste liquid.

[0101] (8) Add 700 μl of protein removal solution RW1, let stand at room temperature for 1 min, centrifuge at 13000 rpm for 30 s, and discard the waste liquid.

[0102] (9) Add 500 μl of wash buffer RW, centrifuge at 13000 rpm for 30 s and discard the waste liquid. Add 500 μl of wash buffer RW and repeat once. Put the adsorption column RA back into the empty collection tube, centrifuge at 13000 rpm for 2 min to remove as much wash buffer as possible to avoid residual ethanol in the wash buffer inhibiting the downstream reaction.

[0103] (10) Take out the RA adsorption column, put it into an RNase-free centrifuge tube, add 30-50ul RNase-free water in the middle of the adsorption membrane, let it stand at room temperature for 1min, and centrifuge at 13000rpm for 1min.

[0104] (11) The quality of the extracted RNA was identified by 1% agarose gel electrophoresis (about 18 min), and the concentration of the extracted RNA was determined by spectrophotometer. An appropriate amount of RNA was used for purification and reverse mixing, and the remaining RNA was stored at -76℃ for long-term storage.

[0105] 3. Reverse transcription to synthesize first-strand cDNA

[0106] The main reference was to Transgen's One-Step gDNA Removal and Cdna Synthesis SuperMix instruction manual.

[0107] The 20 μl reverse transcription reaction mixture (placed in a 0.2 ml RNase-free centrifuge tube) is as follows:

[0108]

[0109] Incubate at 50°C for 30 min, heat at 85°C for 5 s to inactivate the enzyme, and quickly transfer to ice to obtain cDNA.

[0110] Dilute the cDNA stock solution 5 times, perform PCR amplification with the internal reference gene GADPH, test the reverse inversion effect, and store at -20℃ for later use.

[0111] 4. Amplification of candidate genomic DNA

[0112] Download the candidate gene sequence corresponding to the reference genome B73 from the Maize GDB database (https: / / www.maizegdb.org / ). Use Primer5 software to design primers in the 5'UTR and 3'UTR regions of the genome and amplify the full-length candidate genome DNA using the genomic DNA of the maize inbred line B73 seedlings obtained in step 1 above as a template.

[0113] The amplification system (20 μl) is as follows: DNA template 1 μl; upstream primer 1 μl; downstream primer 1 μl; MCLAB SuperMix 10 μl; ddH2O 7 μl.

[0114] The primer sequences are as follows:

[0115] Upstream primer: 5'-TATTTATAACACCACCACTTCCC-3';

[0116] Downstream primer: 5'-CGAGATCCTCTCCTAATCGTC-3'.

[0117] The PCR amplification program was as follows: 98℃ for 2 min; 98℃ for 10 s, 58℃ for 15 s, 72℃ for 15 s, 35 cycles; 72℃ for 5 min.

[0118] 5. Amplification of full-length cDNA of candidate genes

[0119] Primers were designed based on the amplified genomic DNA sequence. The cDNA of maize inbred line B73 seedlings obtained in step 2 above was used as the template for gene amplification. Primers were designed to amplify the full-length cDNA sequence of the gene. The amplification system was as follows: 1 μl cDNA template; 1 μl upstream primer; 1 μl downstream primer; 10 μl MCLAB SuperMix; 7 μl ddH2O.

[0120] The primer sequences are as follows:

[0121] Upstream primer: 5'-ATGATGCCCCAGGCCAGCGCT-3';

[0122] Downstream primer: 5'-TCAGAAGAGTTCTCTGGTGACG-3'.

[0123] The PCR amplification program was as follows: 98℃ for 2 min; 98℃ for 10 s, 58℃ for 15 s, 72℃ for 15 s, 35 cycles; 72℃ for 5 min.

[0124] 6. Recovery and sequencing of candidate genomic DNA and cDNA fragments

[0125] Step 1: Recovery of candidate genomic DNA and cDNA fragments

[0126] Fragment recovery primarily referenced the instruction manual for the FastPure Gel DNAExtraction Mini Kit from Beijing Novizan Biotechnology Co., Ltd., as follows:

[0127] (1) Add 1g of agarose to 100ml TAE solution, heat and boil in microwave oven, then add 10ul of 10000×GelStain dye, pour into a plate with a comb, prepare 1% agarose gel, perform agarose gel electrophoresis (125v) on the PCR product with loading buffer added for 25min, cut the target band under long-wave ultraviolet light and put it into a 1.5ml centrifuge tube;

[0128] (2) Add 400 μl of Buffer GDP to the centrifuge tube for sol-gel preparation;

[0129] (3) Place the centrifuge tube in a 55°C water bath and incubate. Invert the tube every 5 minutes to mix until the gel is completely melted and the solution is pale yellow. Let it stand at room temperature until the temperature drops to room temperature before proceeding to the next step of the reaction.

[0130] (4) Transfer the solution in the centrifuge tube from the previous step into the adsorption column, put the centrifuge tube into the centrifuge, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and then put the adsorption column back into the empty collection tube.

[0131] (5) Add 300 μL of Buffer GDP to the adsorption column, let stand for 1 min, add the centrifuge tube to the centrifuge, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0132] (6) Place the adsorption column in the collection tube, add 700 μl of Buffer GW to the adsorption column, centrifuge at 12000 rpm for 30 s; discard the filtrate. Wash twice.

[0133] (7) Place the adsorption column back into the empty collection tube and centrifuge at 12,000 rpm for 2 min;

[0134] (8) Take out the adsorption column, put it into a clean 1.5ml sterile centrifuge tube, let it stand at room temperature for 5min, add 25ul of elution buffer to the middle part of the adsorption membrane, let it stand at room temperature for 5min, and centrifuge at 12000rpm for 2min.

[0135] (9) Repeat step (8).

[0136] Step 2: Sequencing of candidate genomic DNA and cDNA fragments

[0137] Referring to the instructions for Tiangen Biotech's pLB zero-background rapid cloning kit, the operating steps are as follows:

[0138] (1) The DNA fragment recovered above was ligated into the pLB vector. The ligation system (10 μl) is as follows:

[0139]

[0140] Gently tap the centrifuge tube to mix the reaction solution.

[0141] (2) Place the mixed reaction solution in a 22°C constant temperature metal bath and react for 15 min. After the reaction is complete, place the centrifuge tube on ice and carry out subsequent conversion experiments.

[0142] (3) Prepare LB agarose plates containing ampicillin at a final concentration of 100 μg / ml. Place the plates at 37°C and preheat for 20 min.

[0143] (4) Add 10 μL of the ligation product to 100 μL of TOP10 competent cells (the competent cells were taken out of the -80℃ freezer and placed on ice. The ligation product was added when the cells were just thawed). Mix gently by hand and incubate on ice for 30 min.

[0144] (5) Then place the centrifuge tubes in a 42°C water bath for 90 seconds, and immediately place them in an ice bath for 5 minutes.

[0145] (6) Add 500 μl of LB (antibiotic-free) medium to the centrifuge tube and incubate at 150 rpm and 37°C for 60 min to allow the bacteria to recover.

[0146] (7) Mix the bacterial culture in the centrifuge tube, add 100 μl to LB solid agar medium containing ampicillin, and gently spread the bacterial culture with a sterile bent glass rod. After the surface of the plate is dry, invert the plate and incubate at 37°C for 12 h.

[0147] (8) Take out the plate, pick the milky white positive single clones onto 600 μl of LBA liquid medium (with antibiotics added), and culture at 37°C with shaking for 8 h. Perform bacterial PCR using pLB vector universal primers. The clones identified as positive by agarose gel electrophoresis are sequenced for verification. The plasmids are extracted for subsequent experiments.

[0148] The universal primer sequences for the pLB vector are as follows:

[0149] Forward primer: 5'-CGACTCACTATAGGGAGAGCGGC-3'

[0150] Reverse primer: 5'-AAGAACATCGATTTTCCATGGCAG-3'

[0151] The results are as follows:

[0152] The nucleotide sequence of the genomic DNA of the ZmHB77 gene is SEQ ID No. 3.

[0153] The open reading frame in the cDNA nucleotide sequence of ZmHB77 is SEQ ID No. 2, which encodes the ZmHB77 protein shown in SEQ ID No. 1.

[0154] Example 2: Functional Verification of ZmHB77 Gene Transgenic Maize

[0155] I. Construction of the ZmHB77 CRISPR / Cas9 transgenic maize line

[0156] 1. Constructing a ZmHB77 knockout vector

[0157] (1) Linearization of CPB vector

[0158] The CPB vector was linearized by digesting it with HindIII restriction enzyme at 37°C. The enzyme digestion system is as follows:

[0159] 1 μg of plasmid

[0160] HindⅢ 1μl

[0161] Buffer 10μl

[0162] Bath in a 37°C water bath for 3 hours, then cut and recycle the rubber.

[0163] (2) Screening of ZmHB77 gene target genes

[0164] Generate a target list using the online target prediction website (http: / / crispor.tefor.net / ), and select the following targets:

[0165] T1: 5′-TGAGGGTGAGGCCCAGGCTG AGG-3′;

[0166] T2: 5′-CGACACCACCACCGCCAAGA GGG-3′;

[0167] T3: 5′-GCTCAGGCTCTCCAAGGACC AGG-3′.

[0168] (3) Construction of sgRNA expression cassette

[0169] sgRNA expression cassette template, such as Figure 2 As shown, the target sequences T1, T2, and T3 were replaced with the nnnnnnnnnnnnnnnnnnn (where n represents any base A, T, C, or G) positions in the template of the sgRNA expression cassette (as shown in SEQ ID No. 7) to obtain the sgRNA1, sgRNA2, and sgRNA3 sequences. These were then sent to a biotechnology company for synthesis. Positions 1-17 and 543-568 of SEQ ID No. 7 are the upstream and downstream homologous arms, positions 42-436 are the U6 promoter sequence, and positions 457-539 are the sgRNA backbone sequence.

[0170] The expression cassette of sgRNA1 is a DNA molecule with the nucleotide sequence shown in SEQ ID No. 4, the expression cassette of sgRNA2 is a DNA molecule with the nucleotide sequence shown in SEQ ID No. 5, and the expression cassette of sgRNA3 is a DNA molecule with the nucleotide sequence shown in SEQ ID No. 6.

[0171] (4) The linear fragment of the CPB vector obtained in step (1) and the sgRNA expression cassette fragment obtained in step (3) were ligated using homologous recombination. The ligation principle was in-fusion, and the reagent used was the Seamless Assembly Cloning Kit from Clone Smarter Technologies. The ligation system is as follows:

[0172]

[0173]

[0174] Gently mix and react at 50°C for 15 minutes to obtain recombinant vectors CPB-sgRNA1, CPB-sgRNA2, and CPB-sgRNA3, respectively.

[0175] (5) Transformation

[0176] Thaw Fast-T1 competent cells on ice, add 10 μl of the recombinant vector obtained in step (4) to each 50 μl competent cell, gently tap the centrifuge tube to mix, place on ice for 30 minutes, then heat shock in a 42°C water bath for 30 seconds, then immediately transfer to ice to cool for 2 minutes, add 450 μl of room temperature LB medium, then culture at 37°C and 250 rpm for 1 hour, then take 100 μl of cells and spread them evenly on LB plates containing kanamycin resistance, and culture overnight in a 37°C incubator.

[0177] (6) Screening for positive clones

[0178] The bacterial cells obtained from step (5) were screened for positive clones, and plasmids were extracted from the bacterial solutions with correct sequencing results for later use.

[0179] Sequencing results show that:

[0180] The structure of the recombinant vector CPB-sgRNA1 is: U6-2pro::target T1::sgRNA.

[0181] The structure of the recombinant vector CPB-sgRNA2 is: U6-2pro::target T2::sgRNA.

[0182] The structure of the recombinant vector CPB-sgRNA3 is: U6-2pro::target T3::sgRNA.

[0183] 2. Obtaining EHA105 / CPB-sgRNA1, EHA105 / CPB-sgRNA2, and EHA105 / CPB-sgRNA3: Recombinant vectors CPB-sgRNA1, CPB-sgRNA2, and CPB-sgRNA3 were introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which were named EHA105 / CPB-sgRNA1, EHA105 / CPB-sgRNA2, and EHA105 / CPB-sgRNA3.

[0184] A schematic diagram of the construction of the recombinant vector CPB-sgRNA and a vector map of the recombinant vector are shown below. Figure 2 As shown.

[0185] 3. Obtaining T0 generation ZmHB77 gene knockout maize

[0186] A transformation method using Agrobacterium-mediated transformation of maize embryos was employed. The EHA105 / CPB-sgRNA1, EHA105 / CPB-sgRNA2, and EHA105 / CPB-sgRNA3 prepared in step 2 were mixed and simultaneously transformed into B104 to obtain T0 generation transgenic maize. Basta was applied to the leaves of the T0 generation transgenic maize; plants whose leaves grew normally (resistant seedlings) were identified as T0 generation transgenic plants. The transgenic T0 plants were self-pollinated, and after maturity, they were harvested to obtain T1 generation transgenic seeds, including knockout lines KO#1 and KO#3 mutant plants. Further generations were then performed to obtain T3 generation homozygous knockout lines KO#1 and KO#3.

[0187] 4. Genotype and phenotype of the ZmHB77 knockout strain

[0188] Seeds of maize inbred line B104 (WT) and T3 generation transgenic seeds of knockout lines were planted in two rows for each material, with a row length of 3 meters, a plant spacing of 0.25 meters, a row spacing of 0.6 meters, and three replicates.

[0189] Leaves were taken from transgenic plants for DNA extraction (see previous text for DNA extraction methods). DNA levels were detected in ZmHB77 gene knockout mutant-positive plants. Genomic DNA was extracted from the knockout mutant plants and used as a template, with F2 and R2 as primers for amplification. Wild-type plant genomic DNA and ddH2O served as negative controls.

[0190] The reaction system is as follows:

[0191]

[0192] The nucleotide sequences of F2 and R2 are as follows:

[0193] F2: 5′-TATTTATAACACCACCACTTCCC-3′;

[0194] R2: 5′-GATGCTAGCTTACGGGGTTGAG-3′.

[0195] The amplification reaction program was as follows: first round: denaturation at 95℃ for 5 min; second round: denaturation at 95℃ for 10 sec, annealing at 56℃ for 15 sec, extension at 72℃ for 15 sec, 35 cycles; third round: extension at 72℃ for 5 min.

[0196] After the procedure, the samples were analyzed by 2.0% agarose gel electrophoresis.

[0197] Compared to wild-type materials, the mutant bands in the transgenic plants were smaller, indicating that the transgenic plants had small fragment deletions. Further sequencing of the target bands revealed two main mutant genotypes in the positive transgenic plants: KO#1 and KO#3. Figure 1 (b)

[0198] KO#1, ZmHB77 / -59bp, is a deletion of 59 nucleotides between positions 143-161, 331-353, and 445-461 in the SEQ ID No. 1 genome sequence (corresponding to the deletion of nucleotides at positions 40-58, 228-250, and 342-358 in SEQ ID No. 2 on the CDS), while keeping the other nucleotide sequences unchanged. This mutation results in a frameshift mutation, preventing normal translation.

[0199] KO#3, ZmHB77 / 34bp, is a deletion of 34 nucleotides between positions 145-161 and 331-347 in the genomic sequence of SEQ ID No.1 (corresponding to the deletion of nucleotides 42-58 and 228-244 in SEQ ID No.2 on the CDS). The other nucleotide sequences remain unchanged. This mutation results in a frameshift mutation, which prevents normal translation.

[0200] SEQ ID No. 8 is the coding region sequence of the ZmHB77 gene of KO#1, and SEQ ID No. 9 is the coding region sequence of the ZmHB77 gene of KO#3.

[0201] The experiment also included an empty control line into which the CPB vector was introduced into the maize inbred line B104.

[0202] II. Knocking out the ZmHB77 gene leads to enhanced drought resistance in maize.

[0203] 1. Phenotypic identification of root systems in the ZmHB77 knockout line

[0204] Germination experiments were conducted using seeds of maize inbred line B104 (WT) and T3 generation transgenic seeds of knockout lines KO#1 and KO#3 obtained in step one. An empty vector control was also included in step one. Plump and uniform seeds were selected, soaked in 6% sodium hypochlorite solution for 10 minutes, rinsed three times with distilled water, and 10 seeds were rolled on each germination paper. The experiments were repeated three times, and the average value was taken. Seeds were first cultured in the dark at 28℃ until germination, then cultured at 28℃ / 25℃ for 16 hours of light / 8 hours of darkness for 9 days to identify the number of seed roots and lateral root phenotypes. Epson Scan was used to scan the 10cm root segment at the base of the primary root. Figure 1 In the cases of c and e), the number of lateral roots was determined by manually counting the roots by magnifying the root segment images using computer drawing software.

[0205] The results of knocking out ZmHB77 lines KO#1 and KO#3, as well as wild-type maize (B104), are as follows: Figure 1 As shown in figures d and f, under normal moisture conditions, the number of seed roots in the ZmHB77 knockout lines KO#1 and KO#3 was significantly lower than that in the wild-type maize B104, while the lateral root density was significantly higher. These results indicate that the knockout of the ZmHB77 gene significantly affects the number of seed roots and the density of lateral roots in maize. The empty vector control lines were essentially the same as the wild-type maize (B104), with no statistically significant difference.

[0206] 2. Survival rate assessment of the ZmHB77 knockout strain under drought stress

[0207] Survival rates of knockout lines and wild-type B104 were assessed under soil drought stress. Knockout lines KO#1, KO#3, and B104 were sown in small black pots, 5 plants per pot. After 7 days of growth, seedlings with consistent growth were selected, ensuring 4 seedlings per pot. These were allowed to grow normally to the three-leaf stage, after which no further watering was applied. The drought treatment lasted 15 days, followed by 4 days of rewatering. Survival rates of wild-type maize B104 (WT) and knockout lines KO#1 and KO#3 were then recorded. The experiment was repeated three times, and the average results were used. An empty control line obtained in step one was also included in the experiment.

[0208] The results are as follows Figure 1 As shown in g and h, no significant difference was found between the knockout lines and WT under normal water conditions. After 15 days of drought stress, WT and the two knockout lines resumed watering. The knockout lines KO#1 and KO#3 showed better recovery ability and significantly higher survival rate than WT. The empty control lines were basically the same as WT, with no statistical difference.

[0209] 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. The application of substances that can reduce the expression level and / or activity of ZmHB77 protein in plants in any of (a1)-(a3): (a1) Improve plant drought resistance; (a2) Reduce the number of seed roots in plants; (a3) Increase the density of lateral roots in plants; The ZmHB77 protein is any one of the following: (A1) A protein with the amino acid sequence SEQ ID No. 1; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1); The substance that can reduce the expression level and / or activity of ZmHB77 protein in plants is the CRISPR / Cas9 system, which is used to knock out nucleic acid molecules in recipient plants that can express the ZmHB77 protein; The plant in question is corn.

2. The application according to claim 1, characterized in that: The nucleic acid molecule capable of expressing the ZmHB77 protein is the DNA molecule shown in SEQ ID No. 2 or SEQ ID No.

3.

3. A method for cultivating plants with increased drought resistance and / or fewer seed roots and / or increased lateral root density, comprising the step of reducing the expression level and / or activity of ZmHB77 protein in the recipient plant; The ZmHB77 protein is any one of the following: (A1) A protein with the amino acid sequence SEQ ID No. 1; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1); The reduction in the expression level and / or activity of ZmHB77 protein in the recipient plant is achieved by knocking out the nucleic acid molecules in the recipient plant that can express the ZmHB77 protein using CRISPR / Cas9 technology; The plant in question is corn.

4. A method for cultivating transgenic plants with improved drought resistance and / or fewer seed roots and / or increased lateral root density, comprising the following steps: inhibiting the expression of nucleic acid molecules capable of expressing ZmHB77 protein in a recipient plant to obtain transgenic plants; the transgenic plants having improved drought resistance and / or fewer seed roots and / or increased lateral root density compared to the recipient plants; The ZmHB77 protein is any one of the following: (A1) A protein with the amino acid sequence SEQ ID No. 1; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1); The inhibition of the expression of nucleic acid molecules capable of expressing the ZmHB77 protein in the recipient plant is achieved by knocking out the nucleic acid molecules capable of expressing the ZmHB77 protein in the recipient plant using CRISPR / Cas9 technology; The plant in question is corn.

5. The method according to claim 4, characterized in that: The target sequences targeted by the CRISPR / Cas9 system are positions 437-456 of SEQ ID No. 4, positions 437-456 of SEQ ID No. 5, and / or positions 437-456 of SEQ ID No.

6.

6. The method according to any one of claims 3-5, characterized in that: The nucleic acid molecule capable of expressing the ZmHB77 protein is the DNA molecule shown in SEQ ID No. 2 or SEQ ID No. 3.

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