Application of ZmZFP1 protein and coding gene thereof in regulation and control of plant root development

Abstract

The invention discloses an application of ZmZFP1 protein and a coding gene thereof in regulation and control of plant root development, and provides an application of the ZmZFP1 protein or related biological materials thereof in regulation and control of plant root development, and the related biological materials are nucleic acid molecules capable of expressing the ZmZFP1 protein or expression cassettes, recombinant vectors, recombinant bacteria or mutant cell lines containing the nucleic acid molecules. The ZmZFP1 protein is a protein as shown in SEQ ID No.2 or a protein which has one or more nucleotide or amino acid differences with the protein as shown in SEQ ID No.2 and has the same function as the protein as the protein as shown in SEQ ID No.2. By knocking out ZmZFP1 or inhibiting ZmZFP1 expression of a corn mutant, the growth of a main root under low nitrogen stress treatment is obviously inhibited, which indicates that the gene responds to low nitrogen stress by regulating the growth of the main root, and the application provides a new germplasm resource for cultivating a new low nitrogen resistant corn variety, and has a wide application prospect. And a theoretical basis is laid for researching a mechanism of plant response to stress signals and a molecular mechanism of plant tolerance to adverse environments.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of ZmZFP1 protein and its encoding gene in regulating plant root development. Background Art

[0002] Nitrogen is one of the major factors limiting crop growth. Global agricultural nitrogen use has increased sevenfold over the past half century. However, most crops only utilize 30%-40% of the applied nitrogen. Excessive nitrogen fertilizer application leads to serious environmental problems such as eutrophication, acid rain, soil acidification, increased greenhouse gas emissions, and atmospheric nitrogen deposition. Corn is one of the world's three major staple crops, accounting for approximately one-fifth of total nitrogen fertilizer use in agricultural production. Nitrogen is essential for corn growth and development and is crucial for increasing yields. Over the past few decades, large amounts of nitrogen fertilizer have been applied to boost crop yields. Improving nitrogen use efficiency in corn is crucial for ensuring food security and environmental sustainability. However, only 30%-40% of applied nitrogen fertilizer is absorbed and utilized by crops, with the majority lost to the environment, causing significant environmental pollution. Therefore, it is urgent to improve nitrogen use efficiency in crops while reducing inputs.

[0003] Different maize inbred lines exhibit significant differences in their responses to nitrogen stress, and the study of key genetic variation sites is of significant significance. Studying the molecular mechanisms underlying maize's specific response to low nitrogen stress and the major factors influencing its key physiological and biochemical processes can be accomplished by knocking out relevant genes in wild-type maize plants and using appropriate experimental methods to determine whether the mutant plants exhibit changes in their low nitrogen tolerance compared to the wild type. This effectively identifies high-quality low nitrogen-related genetic resources, providing a theoretical basis for further improving maize's low nitrogen tolerance and breeding new low nitrogen-tolerant varieties. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the present invention aims to provide the application of the ZmZFP1 protein related to plant root development and its encoding gene for regulating low nitrogen stress resistance in corn. By editing the ZmZFP1 gene in corn, the ZmZFP1 gene is mutated, the encoded protein loses its function, and the mutant exhibits a more sensitive phenotype than the wild type.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] Application of ZmZFP1 protein, its encoding gene or biological material containing its encoding gene in regulating plant root development, wherein the ZmZFP1 protein is any one of the following proteins:

[0007] (A1) a protein having an amino acid sequence of SEQ ID No. 2;

[0008] (A2) A protein that has one or more amino acid differences from the amino acid sequence defined in (A1) but has the same or similar functions.

[0009] Application of ZmZFP1 protein, its encoding gene or biological material containing its encoding gene in low nitrogen stress resistance plant breeding, wherein the ZmZFP1 protein is any one of the following proteins:

[0010] (A1) a protein having an amino acid sequence of SEQ ID No. 2;

[0011] (A2) A protein that has one or more amino acid differences from the amino acid sequence defined in (A1) but has the same or similar functions.

[0012] Preferably,

[0013] The activity and / or expression level of the ZmZFP1 protein or its encoding gene in the plant is reduced.

[0014] The gene encoding the above-mentioned ZmZFP1 protein has a nucleotide sequence as follows:

[0015] (B1) nucleotide sequence is SEQ ID No. 1;

[0016] (B2) A nucleotide sequence that differs from the nucleotide sequence defined in (B1) by one or more nucleotides but encodes a protein having the same or similar function.

[0017] Preferably,

[0018] The relevant biological material is a nucleic acid molecule capable of expressing the ZmZFP1 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a mutant cell line containing the nucleic acid molecule.

[0019] A method for cultivating low nitrogen tolerant plant varieties,

[0020] Suppressing the expression and / or activity of ZmZFP1 protein in plants by genetic modification;

[0021] The ZmZFP1 protein is any one of the following proteins:

[0022] (A1) a protein having an amino acid sequence of SEQ ID No. 2;

[0023] (A2) A protein that has one or more amino acid differences from the amino acid sequence defined in (A1) but has the same or similar functions.

[0024] Preferably,

[0025] The CRISPR / Cas9 technology is used to knock out or reduce the expression of the gene encoding the ZmZFP1 protein in plants. The CRISPR method includes designing a sgRNA sequence; amplifying, recovering, and constructing a vector; transferring into Agrobacterium; infecting plants; and obtaining mutant corn with a loss of protein function.

[0026] Preferably,

[0027] The nucleotide sequence of the sgRNA action site is 5'-CACCGTGGTGAGTTCACGT-3'.

[0028] Preferably,

[0029] The plant is corn.

[0030] Preferably,

[0031] The reference sequence number of the ZmZFP1 gene is GRMZM2G430902.

[0032] The beneficial effects of the present invention are:

[0033] (1) The present invention provides genetic resources for breeding and improving new varieties of drought-resistant plants, and provides a theoretical basis for clarifying the molecular mechanism of gene ZmZFP1 in plant low-nitrogen response and improving plant nitrogen utilization efficiency.

[0034] (2) The application of the ZmZFP1 protein and its encoding gene provided by the present invention provides new germplasm resources for breeding new low-nitrogen tolerant corn varieties. Compared with traditional breeding methods, the breeding time is short, the purpose is strong, and the breeding cycle is significantly shortened. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0036] Figure 1 Comparison of maize ZmZFP1 CRISPR-Cas9 mutant and wild-type sequences

[0037] Figure 2 Comparison of plant growth between wild-type corn (control) and mutant lines after normal nitrogen and low nitrogen treatments. WT is wild-type corn, and ffp1-1 and zfp1-2 are T3 generation ZmZFP1 CRISPR-Cas9 mutant lines.

[0038] Figure 3Figure 2 shows the results of the detection of taproot length of wild-type maize ZmZFP1 mutant under normal nitrogen and low nitrogen conditions in Example 2; WT represents wild-type maize, and zfp1-1 and zfp1-2 are T3 generation ZmZFP1 CRISPR-Cas9 mutant lines.

[0039] Figure 4 RT-PCR results of ZmZFP1 gene expression in tissues DETAILED DESCRIPTION

[0040] The following describes preferred embodiments of the present invention in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0041] The ZmZFP1 gene in B73 maize is represented by the nucleotide sequence shown in SEQ ID NO. 1. Its accession number in the maize genome database is GRMZM2G430902. The maize ZmZFP1 gene consists of 2,372 bases and encodes three transcripts. The reading frame of the T01 transcript is from base 17 to base 2372 from the 5′ end, and its reading frame from base 11 to base 91, base 182 to base 596, base 665 to base 1920, base 1998 to base 2084 are exons, and the rest are intron sequences; the reading frame of the T02 transcript is from base 1 to base 2100 from the 5′ end, and its reading frame from base 26 to base 611, base 682 to base 1865, base 2014 to base 2100, and the rest are intron sequences; the reading frame of the T03 transcript is from base 1 to base 824 from the 5′ end, and its reading frame from base 26 to base 716, base 682 to base 1865, and there is no intron sequence.

[0042] In the present application, the nucleotide sequence of ZmZFP1 is SEQ ID NO.1; the protein sequence encoded by ZmZFP1 is SEQ ID NO.2; mutants that differ from it by one or more nucleotides or amino acids but have the same or similar protein functions are still within the scope of protection of the present application.

[0043] SEQ ID NO.1

[0044]

[0045]

[0046] SEQ ID NO.2

[0047]

[0048]

[0049] The method of the present application is applicable to various corn varieties as long as they contain the ZmZFP1 gene.

[0050] The knockout or inhibition methods in this application, such as the CRISPR method, have operational details and reagents that are well known in the art or can be accomplished using existing kits.

[0051] The ZmZFP1 protein and its encoding gene provided by the present invention provide genetic resources for breeding new low-nitrogen tolerant corn varieties, and lay a theoretical foundation for studying the mechanism of plant response to adverse signals and the molecular mechanism of tolerance to adverse environments.

[0052] Unless otherwise specified, the equipment and reagents used in each example are commercially available.

[0053] The main reagents in the following examples are: Taq DNA polymerase, T4 ligase, and high-fidelity DNA polymerase were purchased from Novezan Biotechnology Co., Ltd. (Nanjing); plasmid extraction kit and agarose gel recovery kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; agar powder, agarose, antibiotics such as ampicillin (Amp), kanamycin (Kan), and rifampicin (Rif), as well as glucose, BSA, LB medium, etc. were purchased from Sigma, Bio-Rad, etc.; various other chemical reagents used in the examples were imported or domestic analytical grade reagents.

[0054] The primers used in the examples were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the related sequencing was performed.

[0055] Example 1 Construction and identification of ZmZFP1 mutant materials

[0056] 1) Based on the coding region sequence of the ZmZFP1 gene (numbered GRMZM2G430902 in the maize genome database), candidate target sequences were designed and screened using the website http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html. The target sequence was located within or upstream of the conserved functional domain of the CDS and had a GC content between 45% and 75%.

[0057] 2) Use the website CRISPR-RGEN-Tools (http: / / www.rgenome.net / cas-offinder / ) to perform off-target analysis on candidate target sequences, and further screen and determine the optimal specific target sequence with low potential off-target rate and no effect on other gene coding regions.

[0058] 3) Design specific primers upstream and downstream of the target sequence, amplify the target gene using the genomic DNA of the recipient material as a template, and verify the PCR product by sequencing. If there are any differences, correct the target sequence and re-perform the off-target analysis. Finally, select the optimal target sequence for vector construction.

[0059] The target site sgRNA sequence of the present invention is 5'-CACCGTGGTGAGTTCACGT-3' (SEQ ID NO. 3), and the vector used is pBUE411C from the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0060] 4) After the vector was constructed and sequenced, it was transformed into Agrobacterium strain EHA105, and maize immature embryos were infected with Agrobacterium to obtain mutant-positive seedlings. Homozygous mutant materials were obtained through self-pollination and sequencing.

[0061] Primers are designed according to the target site, and CRISPR / Cas9 targeting vectors (CRISPR / Cas9 gene editing vectors) are constructed through PCR, enzyme digestion, ligation and other operations.

[0062] The construction method of CRISPR / Cas9 gene editing vector is as follows:

[0063] The primers used are as follows:

[0064] SEQ ID NO.4

[0065] ID-1f: 5'-GGCG CACCGTGGTGAGTTCACGT -3' (the underlined sequence is the reverse complement of the underlined sequence in ID-1r);

[0066] SEQ ID NO.5

[0067] ID-1r: 5'-AAAC ACGTGAACTCACCACGGTG -3' (the underlined sequence is the reverse complement of the underlined sequence in ID-1f).

[0068] (1) Annealing: The above primers are diluted to 10 μM and then subjected to gradient annealing to anneal them into double strands, thereby obtaining double-stranded DNA fragments gRNA with sticky ends of enzyme cleavage sites.

[0069] (2) Enzyme digestion of vector: perform enzyme digestion according to the reaction system and reaction procedure shown in Table 1 to obtain the enzyme digestion vector.

[0070] Table 1

[0071]

[0072] (3) Ligation: Ligating the digested vector obtained in step (2) and the annealed fragment in step (1) according to the reaction system and reaction procedure shown in Table 2 to obtain the product of the digestion-ligation system;

[0073] Table 2

[0074]

[0075]

[0076] (4) Take 5 μl of the product of the enzyme digestion-ligation system obtained in step (3) and transform it into competent E. coli. Screen positive clones on LB plates containing 50 μg / mL kanamycin, identify single clones by colony PCR, select positive clones for sequencing, and obtain the CRISPR / Cas9 gene editing vector ZmZFP1-pBUE411C for ZmZFP1 gene mutation.

[0077] The primers used for colony PCR are as follows:

[0078] SEQ ID NO.6

[0079] ID-1f: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3';

[0080] SEQ ID NO.7

[0081] ID-1r: 5'-CTCACAAATTATCAGCACGCTAGTC-3'.

[0082] The sequencing primers are as follows:

[0083] SEQ ID NO.8

[0084] OsU3-FD3: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3'

[0085] Specifically:

[0086] The constructed and sequenced CRISPR / Cas9 gene editing vector ZmZFP1-pBUE411C was transformed into the competent Agrobacterium tumefaciens EHA105 strain by heat shock method. Positive clones were identified by colony PCR and selected for sequencing based on whether the size of the agarose gel band matched the ZmZFP1 gene. A single colony of Agrobacterium tumefaciens was inoculated into 2-3 mL of liquid culture medium containing 100 μg / mL kanamycin and 50 μg / mL rifampicin, cultured with shaking at 28°C overnight, and then transferred to 200 mL of liquid culture medium containing 100 μg / mL kanamycin and 50 μg / mL rifampicin and cultured with shaking, and resuspended to OD 600Between 0.8v1.0. After the B73 maize embryos dug out under sterile conditions were infected with the above bacterial solution, the maize callus tissue was obtained by dedifferentiation through callus induction technology, and the callus tissue was further induced into seedlings through redifferentiation technology to obtain mutant plants. The mutant-positive plants were determined by PCR amplification and sequencing of gene fragments including the target site. Finally, two knockout materials of the ZmZFP1 candidate gene were obtained (named zfp1-1 and zfp1-2). The sequencing results showed that two homozygous mutant strains were obtained. The sequences are as follows Figure 1 shown.

[0087] The primers used to identify mutant materials are:

[0088] SEQ ID NO.9

[0089] zfp1-F:5'-GCAAGGATTTCCAGGTGCTC-3'

[0090] SEQ ID NO.10

[0091] zfp1-R:5'-TCCCTGGCGATATGCAACAG-3'

[0092] The sequencing primer was zfp1-F.

[0093] Example 2 Low Nitrogen Response Detection of ZmZFP1 Gene CRISPR / Cas9 Materials

[0094] First, the ffp1-1 and zfp1-2 strains obtained by neutralizing wild-type corn were germinated on filter paper. Two days after germination, the seeds were rolled up with 30×30 cm filter paper and placed in 8 liters of pure water for vertical culture for one week. After removing the endosperm, the hydroponic seedlings were divided into normal nitrogen (4mM) and low nitrogen (0.04mM) treatments, and the phenotypes were observed after two weeks.

[0095] Under normal conditions, the average taproot lengths of the wild type, zfp1-1, and zfp1-2 were 17.16 cm, 14.25 cm, and 13.05 cm, respectively. However, under low nitrogen conditions, the average taproot lengths of the wild type, zfp1-1, and zfp1-2 were 20.59 cm, 13.05 cm, and 13.39 cm, respectively, indicating that the growth of the taproots of zfp1-1 and zfp1-2 was significantly inhibited under low nitrogen treatment. Figure 2 , Figure 3 This shows that knockout of the maize ZmZFP1 gene can significantly inhibit the growth of the plant's taproot under low nitrogen conditions.

[0096] Example 3 ZmZFP1 gene expression and localization in tissues

[0097] Wild-type corn and the resulting mutant strains were germinated on filter paper. Two days after germination, the seeds were rolled up with 30x30 cm filter paper and placed vertically in 8 liters of pure water for one week. After removing the endosperm, the hydroponic seedlings were cultured in a normal nitrogen culture medium for two weeks. The aboveground and underground parts were collected separately for total RNA extraction.

[0098] The RNA of corn materials was extracted using the plant total RNA extraction kit provided by Beijing Meiji Biotechnology Co., Ltd. The specific procedures are as follows:

[0099] a. Add 4 volumes of anhydrous ethanol to Buffer RW2 and store at room temperature.

[0100] b. Aliquot an appropriate amount of Buffer RL before use, adding 20 μL of β-mercaptoethanol per mL of Buffer RL;

[0101] c. Grind the plant sample into powder using liquid nitrogen and weigh 50-300 mg of the powder into a 2.0 mL inlet centrifuge tube;

[0102] d. Immediately add 800 μL of mixed Buffer RL, vortex at high speed for 15-30 seconds to disperse the sample, and let it stand at room temperature for 3 minutes;

[0103] e. Centrifuge at 14,000 × g for 5 minutes at room temperature;

[0104] f. Place the gDNA Filter Column in a 2 mL collection tube, transfer 700 μL of the supernatant to the filter column, centrifuge at 14,000 × g for 2 minutes, and discard the gDNA filter column.

[0105] g. Add 0.5 times the volume (350 μL) of anhydrous ethanol to the filtrate and pipette 3 to 5 times;

[0106] h. Place the HiPure RNA Mini Column in a 2 mL collection tube, transfer 600 μL of the mixture to the column, and centrifuge at 12,000 × g for 60 seconds.

[0107] i. Discard the filtrate, return the column to the collection tube, transfer the remaining mixture to the column, and centrifuge at 12,000 × g for 60 seconds;

[0108] j. Discard the filtrate, return the column to the collection tube, add 500 μL of Buffer RW1 to the column, and centrifuge at 10,000 × g for 60 seconds.

[0109] k. Discard the filtrate, return the column to the collection tube, add 500 μL of Buffer RW2 to the column, and centrifuge at 12,000 × g for 60 seconds.

[0110] 1. Repeat step k;

[0111] m. Discard the filtrate, return the column to the collection tube, and centrifuge at 12,000 × g for 2 minutes;

[0112] n. Transfer the column to a 1.5 mL centrifuge tube, add 30–100 μL of RNase-free water to the center of the column membrane, incubate at room temperature for 2 minutes, and centrifuge at 12,000 × g for 60 seconds.

[0113] o. Discard the column and store the RNA at -80°C.

[0114] RNA reverse transcription was performed using the M5 Super qPCR RT Kit with gDNA remover kit from Beijing Polymer Biotechnology Co., Ltd. The specific steps are as follows:

[0115] a. Add 8.8 μL of gDNA remover to 4× DNA remover mix (440 μL) and mix thoroughly by inversion.

[0116] b. Calculate the volume of RNA template required for reverse transcription and incubate the RNA template at 65°C for 5 minutes;

[0117] c. Incubate on ice for 2 minutes;

[0118] d. To remove genomic DNA, add the following components on ice:

[0119]

[0120] The reaction solution was gently mixed and incubated at 37°C for 5 minutes;

[0121] e. For reverse transcription, add the following components on ice:

[0122]

[0123] f. Mix gently, centrifuge briefly, and incubate at 42°C for 20 minutes;

[0124] g. Heat at 96°C for 5 minutes to inactivate the enzyme;

[0125] h. Place on ice for subsequent experiments or freeze at -20°C.

[0126] RT-PCR was performed by Beijing Rambolide Trading Co., Ltd.

[0127] PCR reaction system:

[0128]

[0129] PCR reaction conditions:

[0130]

[0131] The primers are:

[0132] SEQ ID NO.11

[0133] ZmZFP-qF: 5'-CGACAAGACGTACGTCGAG-3'

[0134] SEQ ID NO.12

[0135] ZmZFP-qR:5'-GGAGCCGCAGCATATTCTTC-3'

[0136] Amplify the maize ACTIN gene as an internal reference using the following primers:

[0137] SEQ ID NO.13

[0138] ACTIN-F: 5'-CGACAAGACGTACGTCGAG-3'

[0139] SEQ ID NO.14

[0140] ACTIN-R: 5'-CCCCCACTGAGGACAACG-3'

[0141] The results are as follows Figure 4 As shown, the ZmZFP1 gene is only expressed in the underground part of maize but not in the aerial part.

[0142] Therefore, inhibiting ZmZFP1 protein expression or knocking out the ZmZFP1 gene can inhibit root development of maize under low nitrogen conditions.

[0143] The contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0144] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of a ZmZFP1 protein, a gene encoding the same, or a biological material containing the gene encoding the same in regulating plant root development, wherein the ZmZFP1 protein is any one of the following proteins: (A1) a protein having an amino acid sequence of SEQ ID No. 2; (A2) A protein that has one or more amino acid differences from the amino acid sequence defined in (A1) but has the same or similar functions.

2. Application of a ZmZFP1 protein, a gene encoding the same, or a biological material containing the gene encoding the same in breeding plants resistant to low nitrogen stress, wherein the ZmZFP1 protein is any one of the following proteins: (A1) a protein having an amino acid sequence of SEQ ID No. 2; (A2) A protein that has one or more amino acid differences from the amino acid sequence defined in (A1) but has the same or similar functions.

3. The use according to any one of claims 1 to 2, characterized in that: The activity and / or expression level of the ZmZFP1 protein or its encoding gene in the plant is reduced.

4. A gene encoding the ZmZFP1 protein according to any one of claims 1 to 2, wherein the nucleotide sequence of the gene is: (B1) nucleotide sequence is SEQ ID No. 1; (B2) A nucleotide sequence that differs from the nucleotide sequence defined in (B1) by one or more nucleotides but encodes a protein with the same or similar function.

5. The use according to any one of claims 1 to 2, characterized in that: The relevant biological material is a nucleic acid molecule capable of expressing the ZmZFP1 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a mutant cell line containing the nucleic acid molecule.

6. A method for cultivating low nitrogen tolerant plant varieties, characterized in that: Suppressing the expression and / or activity of ZmZFP1 protein in plants by genetic modification; The ZmZFP1 protein is any one of the following proteins: (A1) a protein having an amino acid sequence of SEQ ID No. 2; (A2) A protein that has one or more amino acid differences from the amino acid sequence defined in (A1) but has the same or similar functions.

7. The method for cultivating low nitrogen tolerant plant varieties according to claim 6, characterized in that: The CRISPR / Cas9 technology is used to knock out or reduce the expression of the gene encoding the ZmZFP1 protein in plants. The CRISPR method includes designing a sgRNA sequence; amplifying, recovering, and constructing a vector; transferring into Agrobacterium; infecting plants; and obtaining mutant corn with a loss of protein function.

8. The method for cultivating low nitrogen tolerant plant varieties according to claim 7, wherein: The nucleotide sequence of the sgRNA action site is 5'-CACCGTGGTGAGTTCACGT-3'.

9. The method for cultivating low nitrogen tolerant plant varieties according to claim 6, wherein: The plant is corn.

10. The use according to any one of claims 1 to 2, characterized in that: The reference sequence number of the ZmZFP1 gene is GRMZM2G430902.

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