CN114644692B

CN114644692B - Method for creating drought-sensitive corn germplasm by site-directed mutagenesis and application thereof

Info

Publication number: CN114644692B
Application number: CN202011493445.XA
Authority: CN
Inventors: 巩志忠; 王瑜; 胡晓莹
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2023-08-11
Anticipated expiration: 2040-12-17
Also published as: CN114644692A

Abstract

The application discloses a method for creating drought-sensitive corn germplasm by site-directed mutagenesis and application thereof. The method comprises the following steps: the recombinant agrobacterium containing the recombinant plasmid pBUE411C-DNSP is adopted to transform the maize inbred line B73, DNSP genes can be edited, DNSP genes can be mutated after being edited by CRISPR/Cas9 endonuclease, and when the DNSP genes of two homologous chromosomes are mutated, the activity of protein DNSP can be lost; loss of protein DNSP activity results in reduced drought tolerance corn, i.e., drought sensitive corn. The amino acid sequence of the protein DNSP is shown as SEQ ID NO. 1. The DNSP gene of the corn can be edited by adopting the method of the application, and the drought-sensitive corn can be obtained. Therefore, the protein DNSP can regulate and control the drought resistance of corn, and the application has important application value.

Description

Method for creating drought-sensitive corn germplasm by site-directed mutagenesis and application thereof

Technical Field

The application belongs to the field of genetic engineering, and particularly relates to a method for creating drought-sensitive corn germplasm by site-directed mutagenesis and application thereof.

Background

Drought is one of the most serious abiotic stresses worldwide, and drought stress leads to the problems of plant growth and development resistance, short plant, crop yield and quality reduction, serious harm to agricultural production and influence on the world-wide agricultural production. Therefore, the genes involved in drought response of plants are searched, and the genes are mutated by utilizing the technologies of transgene overexpression or gene editing and the like, so that candidate gene resources can be provided for molecular breeding, germplasm improvement and changing drought resistance of crops.

CRISPR/Cas9 is a gene editing technology derived from the acquired immune system mediated by short palindromic repeats CRISPR (clustered regularly interspaced short palindromic repeats) based on regularly clustered intervals of bacteria or archaea. The technology firstly designs a segment of sgRNA gene, RNA transcribed by the gene can identify a target DNA sequence through base complementation pairing, cas9 nuclease is guided to cut the identified double-stranded DNA, homologous recombination (HDR, homologous directed repair) or non-homologous end-linked-joining (NHEJ) is induced, and then target DNA editing is realized. The gene editing technology developed based on the bacterial type II immune mechanism has great application prospect in genetic improvement of plants, especially crops. One of the basic requirements of this technology is that single molecule recognition RNA (sgRNA, single guiding RNA) is expressed in the receptor cell, which is responsible for recognizing specific gene editing sites, then mediating DNA cleavage activity by binding to Cas9 protein, introducing DNA double strand break damage at the designed site, introducing mutations via the intracellular NHEJ or HDR repair pathway. Thus, expression of sgrnas is an important component of this technology.

Disclosure of Invention

The application aims to create drought-sensitive corn germplasm.

The present application first protects the protein DNSP, which may be (a 1) or (a 2) or (a 3) or (a 4):

(a1) A protein shown in SEQ ID NO. 1;

(a2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in SEQ ID NO. 1 and is related to drought resistance of plants;

(a3) A fusion protein obtained by connecting a tag to the N-terminal or/and the C-terminal of the protein of (a 1);

(a4) A protein derived from maize and having more than 98% identity to (a 1) and being associated with drought resistance in plants.

The labels are specifically shown in table 1.

TABLE 1 sequence of tags

The protein of (a 2) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of (a 2) may be synthesized artificially or may be obtained by synthesizing the gene encoding the protein and then biologically expressing the gene.

The gene encoding the protein of (a 2) above can be produced by the method of producing a polypeptide of SEQ ID NO:3, and/or performing missense mutation of one or more base pairs, and/or ligating the coding sequence of the tag shown in table 1 at the 5 'end and/or the 3' end.

Nucleic acid molecules encoding any of the above-described protein DNSPs are also within the scope of the present application.

Specifically, the nucleic acid molecule encoding any of the above-described protein DNSPs may be a DNA molecule of (b 1) or (b 2) or (b 3) or (b 4) or (b 5) as follows:

(b1) A DNA molecule with a coding region shown as SEQ ID NO. 3;

(b2) A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 2;

(b3) A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 3;

(b4) A DNA molecule which hybridizes under stringent conditions to a DNA molecule defined in (b 1) or (b 2) or (b 3) and which encodes said protein DNSP;

(b5) A DNA molecule derived from maize and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to a DNA molecule as defined in (b 1) or (b 2) or (b 3) and encoding said protein DNSP.

The stringent conditions are hybridization and washing of the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing of the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein SEQ ID NO. 2 consists of 1509 nucleotides, SEQ ID NO.3 consists of 309 nucleotides, and the nucleotide shown in SEQ ID NO.3 encodes the amino acid sequence shown in SEQ ID NO. 1.

The nucleotide sequence encoding the protein DNSP of the present application can be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein DNSP isolated by the present application are all derived from and equivalent to the nucleotide sequence of the present application as long as the protein DNSP is encoded.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein DNSP of the present application which encodes the amino acid sequence set forth in SEQ ID NO. 1. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

Recombinant vectors, expression cassettes or recombinant bacteria containing any of the above-mentioned nucleic acid molecules are also within the scope of the application.

The application also protects the use of any of the above described protein DNSPs, which may be K1) or K2) or K3):

k1 Regulating drought resistance of the plant;

k2 Cultivating a transgenic plant with altered drought resistance;

k3 Cultivation of drought-sensitive plants.

The application also provides the use of any of the nucleic acid molecules described above, or of recombinant vectors, expression cassettes or recombinant bacteria comprising any of the nucleic acid molecules described above, which may be K1) or K2) or K3):

k1 Regulating drought resistance of the plant;

k2 Cultivating a transgenic plant with altered drought resistance;

k3 Cultivation of drought-sensitive plants.

In any of the above applications, the controlling plant drought resistance may be reducing plant drought resistance or increasing plant drought resistance.

In any of the above applications, the modulation is positive, i.e., inhibiting the protein DNSP reduces drought resistance in the plant.

In any of the above applications, the growing transgenic plant with altered drought resistance may be growing a transgenic plant with reduced drought resistance or growing a transgenic plant with increased drought resistance.

The application also protects S1), S2) or S3).

S1) a method I for cultivating transgenic plant A, which comprises the following steps: inhibiting the content and/or activity of any one of the above protein DNSP in the recipient plant to obtain transgenic plant A; the drought resistance of transgenic plant A is reduced compared to the recipient plant. The transgenic plant A can be a plant drought-sensitive mutant.

S2) cultivating drought-sensitive plants, comprising the following steps: inhibiting the content and/or activity of any one of the protein DNSP in the receptor plant to obtain the drought-sensitive plant.

S3) a second method for cultivating a transgenic plant B, which comprises the following steps: increasing the content and/or activity of any one of the above protein DNSP in the recipient plant to obtain transgenic plant B; compared with the receptor plant, the drought resistance of the transgenic plant B is improved.

In the above method, the method for inhibiting the content and/or activity of any of the above protein DNSPs in the recipient plant can achieve the purpose of inhibiting the content and/or activity of any of the above protein DNSPs in the recipient plant by RNA interference, homologous recombination, gene site-directed editing and other methods well known in the art.

In the above method, the inhibition of the content and/or activity of any of the above-mentioned protein DNSPs in a recipient plant may specifically be achieved by introducing into the recipient plant a plant genome editing vector;

the plant genome editing vector contains sgRNA coding genes;

the target DNA recognized by the sgrnas in plants is a DNA fragment encoding the protein DNSP.

The plant genome editing vector may also contain a gene encoding a Cas9 protein.

The target point recognized by the sgRNA can be SEQ ID No:3 from the 5' end at positions 188-206.

The vector edited by the plant genome can be specifically a recombinant plasmid pBUE411C-DNSP. The recombinant plasmid pBUE411C-DNSP may be a plasmid which encodes the sequence of SEQ ID No:4 into the recognition site of restriction enzyme BsaI of vector pBUE 411C.

The application also protects T1), T2) or T3).

T1) method of plant breeding, comprising the steps of: reducing the content and/or activity of any of the above-mentioned protein DNSPs in the target plant, thereby reducing drought resistance of the plant.

T2) a method for breeding drought-sensitive plants, comprising the steps of: reducing the content and/or activity of any of the above-mentioned protein DNSPs in the plant of interest, thereby making the plant drought-sensitive.

T3) a second plant breeding method comprising the steps of: increasing the content and/or activity of any of the above-mentioned protein DNSPs in the plant of interest, thereby increasing drought resistance of the plant.

Any of the plants described above may be monocotyledonous or dicotyledonous.

The plant is Gramineae plant.

The plant is a maize plant.

The plant is corn.

The plant is maize inbred line B73.

As the same DNA segment sequence of corn can produce different transcripts and translate different proteins, the different transcripts produced by SEQ ID NO. 2 and the translated different proteins are all within the protection scope of the application.

Experiments prove that the recombinant agrobacterium containing the recombinant plasmid pBUE411C-DNSP is adopted to transform the maize inbred line B73, DNSP genes can be edited, DNSP genes can be mutated after being edited by CRISPR/Cas9 endonuclease, and when the DNSP genes of two homologous chromosomes are mutated, the activity of protein DNSP can be lost; loss of protein DNSP activity results in reduced drought tolerance corn, i.e., drought sensitive corn. The DNSP gene of the corn can be edited by adopting the method of the application, and the drought-sensitive corn can be obtained. Therefore, the protein DNSP can regulate and control the drought resistance of corn, and the application has important application value.

Drawings

FIG. 1 is a diagram of the detection of the mutation type of heterozygous mutants.

FIG. 2 shows the frame shift caused by the insertion of "G"1 nucleotide into DNSP gene on one homologous chromosome of a hybrid mutant.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Vector pBUE411C is described in the following literature: hui-Li Xing, li Dong, zhi-Ping Wang, hai-Yan Zhang, chun-Yan Han, bing Liu, xue-Chen Wang and Qi-Jun Chen. A CRISPR/Cas9 toolkit for multiplex genome editing in plants.BMC Plant Biology 2014,14:327. Vector pBUE411C contains the 3 XFLAG-NLS-zCas 9-NLS expression system and gRNA scaffold for insertion of target sequences.

Example 1 discovery of protein DNSP and genes encoding same

The inventors of the present application have found DNSP gene from maize inbred line B73 (hereinafter abbreviated as maize B73) through a large number of experiments. In the genome DNA of the maize B73, the nucleotide sequence of the DNSP gene is shown as SEQ ID NO. 2. In SEQ ID NO. 2, the transcripts are in frame with nucleotides 601 to 909 from the 5' end, without introns.

The nucleotide sequence of DNSP gene in cDNA of maize B73 is shown as SEQ ID NO. 3.

The DNSP gene encodes the protein DNSP. The amino acid sequence of the protein DNSP is shown as SEQ ID NO. 1.

Example 2 cultivation of drought-sensitive maize mutant and verification thereof

In this example, a target was selected for the experiment. The target sequence is as follows: 5'-CGAGCACTGAGCAGGTCGT-3' (i.e.SEQ ID No:3, positions 188-206 from the 5' end) corresponds to the DNSP gene.

1. Acquisition of recombinant plasmid pBUE411C-DNSP

1. Designing and synthesizing a primer ID-1f according to the target point: 5'-GGCGCGAGCACTGAGCAGGTCGT-3' and primer ID-1r:5'-AAACACGACCTGCTCAGTGCTCG-3'.

2. The ID-1f and the ID-1r are diluted and annealed to obtain the double-stranded DNA fragment gRNA with sticky ends.

3. The vector pBUE411C was taken and digested with restriction enzyme BsaI to recover the vector backbone.

4. And (3) connecting the vector skeleton recovered in the step (3) with the double-stranded DNA fragment gRNA with the sticky end obtained in the step (2) to obtain a recombinant plasmid pBUE411C-DNSP.

The recombinant plasmid pBUE411C-DNSP was sequenced. Sequencing results show that the recombinant plasmid pBUE411C-DNSP is prepared by the method of SEQ ID No:4 into the recognition site of restriction enzyme BsaI of vector pBUE 411C.

SEQ ID No：4：5’-GGCGCGAGCACTGAGCAGGTCGTGTTT-3’。

2. Acquisition of the Crispr-DNSP mutant

Since maize is a diploid plant, when Cas9 functions to begin cleaving a particular gene, both alleles on two homologous chromosomes within the same cell are likely to be edited, producing the same type or different types of mutations, so both alleles in one plant are considered two gene editing events. A homozygous mutant refers to a plant in which the DNSP genes of two homologous chromosomes have undergone the same mutation. A biallelic mutant refers to a plant in which the DNSP genes of both homologous chromosomes are mutated but the mutated forms are different. The heterozygous mutant number refers to that the DNSP gene of one homologous chromosome in two homologous chromosomes of the plant is mutated and the DNSP gene of the other homologous chromosome is not mutated. Wild type means that no mutation of the DNSP gene occurs in both homologous chromosomes of the plant.

1. The recombinant plasmid pBUE411C-DNSP is transferred to agrobacterium tumefaciens EHA105 by a liquid nitrogen freezing method to obtain recombinant agrobacterium.

2. Transformation of recombinant Agrobacterium into maize inbred line B73 (described in Schable, P.S. et al, B73 main genome: compatibility, diversity, and dynamics.science 326, 1112-1115 (2009)), screening, differentiation and rooting to obtain T ₀ For the generation of transgenic maize, the following references are referred to for specific methods: wei Xiaoyu, shao Shidi, sun Su, etc., establishment of agrobacterium-mediated maize immature embryo genetic transformation system, university of gillin journal, 2017 (06) 640-647; huang Lu, wei Zhiming, agrobacterium-mediated maize genetic transformation [ J]Experimental biologies, 1999 (04).

3. Respectively by T ₀ Genomic DNA of the generation of the quasi-transgenic corn leaves is used as a template, and primers OsU3-FD3 are adopted: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' and primer ID-1r: and 5'-AAACACGACCTGCTCAGTGCTCG-3', performing PCR amplification to obtain corresponding PCR amplification products. The PCR amplified products were individually subjected to Sanger sequencing. The sequencing result is compared with a target sequence of the DNSP gene (shown as SEQ ID No. 3) Cas9, and the mutation type is counted.

1 heterozygous mutant strain was obtained. The insertion of "G"1 nucleotide into the coding region of DNSP gene (i.e., 1G from position 203 to 204 from the 5' -end of SEQ ID No: 3) occurred on one homologous chromosome of the heterozygous mutant, thereby causing a frame shift (see FIGS. 1 and 2).

4. Selfing the heterozygous mutant strain obtained in the step 3 to obtain seeds which are T ₁ Seed generation, T ₁ The plant grown from the seed generation is T ₁ Generating plants; will T ₁ The seed obtained by selfing the plant is T ₂ Seed generation, T ₂ The plant grown from the seed generation is T ₂ And (5) replacing plants.

5. Respectively by T ₂ Genomic DNA of leaves of the plant is taken as a template, and primers OsU3-FD3 are adopted: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' and primer ID-1r: and 5'-AAACACGACCTGCTCAGTGCTCG-3', performing PCR amplification to obtain corresponding PCR amplification products. The PCR amplified products were individually subjected to Sanger sequencing. The sequencing result is compared with a target sequence of the DNSP gene (shown as SEQ ID No. 3) Cas9, and the mutation type is counted.

1 homozygous mutant was obtained. The same mutation is generated in DNSP genes of two homologous chromosomes of the homozygous mutant strain, specifically, 1 nucleotide insertion of ' G ' is generated in a coding region of the DNSP genes on the two homologous chromosomes (namely, 1G is inserted from the 203 th to 204 th positions from the 5' tail end of SEQ ID No. 3), thereby causing frame shift and causing the loss of functions of protein DNSP.

6. Selfing the homozygous mutant strain obtained in the step 5 to obtain a seed which is T ₃ Seed generation, T ₃ The plant grown from the seed generation is T ₃ And (5) replacing plants.

7. Respectively by T ₃ Genomic DNA of leaf of plant as template, zCAS9-IDF:5'-CGGCCTCGATATTGGGACTAACTCT-3' and zCAS9-IDR:5'-CTTATCTGTGGAGTCCACGAGCTTC-3', performing PCR amplification to obtain corresponding PCR amplification products; then, the following judgment is made: if the PCR amplification product contains a DNA fragment of 569bp in size, the T ₃ The generation plants contain Cas9 protein; if the PCR amplification product does not contain a DNA fragment of 569bp in size, the T ₃ The generation plants did not contain Cas9 protein.

T without Cas9 protein ₃ The plants of the generation are the crispr-DNSP mutant.

3. Drought resistance identification of crispr-DNSP mutant

Test seed: t of the Crispr-DNSP mutant ₃ Seed of generation or seed of maize inbred line B73.

1. The seeds to be tested are respectively sown in small basins filled with nutrient soil and are cultivated for 7 days at 25 ℃.

2. After the step 1 is completed, transplanting seedlings with consistent growth vigor into rectangular big pots filled with 2500g of nutrient soil, planting 15 crispr-DNSP mutants in one half area of each pot, planting 15 maize inbred line B73 plants in the other half area, watering normally and culturing for 7 days. Three replicates were set, 5 pots per replicate.

3. After completion of step 2, watering was continued for 20 days, at which time the phenotype of the crispr-DNSP mutant and maize B73 plants was observed.

The results show that the phenotype of the maize B73 plant and the crispr-DNSP mutant are obviously different; leaf wilting degree of the crispr-DNSP mutant was significantly increased compared to maize B73 plants.

4. After the step 3 is completed, normal watering and cultivation are resumed for 7 days, and then survival rate is counted.

Survival is the percentage of surviving plants in the total number of plants.

The survival rate of the crispr-DNSP mutant was 36% + -2% and the survival rate of maize inbred line B73 was 48% + -3%.

Thus, the drought resistance of corn can be reduced by inhibiting protein DNSP.

Corn is a diploid plant, DNSP genes can cause DNSP gene mutation after being edited by CRISPR/Cas9 endonuclease, and when the DNSP genes of two homologous chromosomes are mutated, the DNSP activity of protein can be lost; loss of protein DNSP activity results in reduced drought tolerance maize, i.e., drought sensitive maize germplasm.

Based on the current state of research, inhibiting a gene causes a trait, and over-expression of the gene will generally cause the opposite trait. Therefore, based on the above experimental results, it is presumed that the overexpression of the protein DNSP can improve drought resistance of corn.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> Chinese university of agriculture

<120> method for creating drought-sensitive corn germplasm by site-directed mutagenesis and application thereof

<160>4

<170> PatentIn version 3.5

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<213>Zea mays L.

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aagggtggca tcttcgtcca aggatatgca ggcctcagtg caaggcagaa aatggcggcc 780

actgcaacga gcactgagca ggtcgtcggc ggcggcggcg aaggcatgcc ggcgaccacc 840

acggactccc ggcctacggc tcccggcaac agccccggta ttggcaacaa agggaagatc 900

aacaactagt ttaattgccg tgtgtgttct tgtatgcgtt cttatctaag taataagcat 960

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Claims

1. A method of breeding transgenic corn comprising the steps of: suppressing the content of protein DNSP in the starting corn to obtain transgenic corn; compared with the starting corn, the drought resistance of the transgenic corn is reduced;

the protein DNSP is the protein shown in SEQ ID NO. 1.

2. The method according to claim 1, characterized in that: the content of the protein DNSP in the starting corn is inhibited by mutating the coding gene of the protein DNSP in the corn;

the coding gene of the protein DNSP in the mutant corn is as shown in SEQ ID No:3 is shown in the figureDNSPThe gene mutation is DNSP/+1bp; the DNSP/+1bp is set forth in SEQ ID No:3 from the 5' end between nucleotides 203 to 204, and keeping the other nucleotide sequence of SEQ ID No.3 unchanged.

3. The method according to claim 1, characterized in that: the coding gene of the protein DNSP in the mutant corn is realized by introducing a CRISPR/Cas9 system into the corn; the CRISPR/Cas9 system comprises a recombinant expression vector; the recombinant expression vector contains a DNA molecule that expresses a gRNA that targets the gene encoding the protein DNSP.

4. A method according to claim 3, characterized in that: the target sequence of the gRNA is SEQ ID No:3 from the 5' end at positions 188-206.