Application of corn nitrogen utilization related gene ZmMAPK6

Abstract

The invention relates to the field of gene engineering and molecular biology, in particular to application of a corn ZmMAPK6 gene in corn nitrogen utilization, and by constructing a CRISPR / Cas9 material, performing directional and fixed-point editing on a target gene and quickly knocking out related genes in a wild type, it is detected that the low nitrogen resistance of a transgenic plant is improved compared with that of the wild type. According to the application of the ZmMAPK6 gene in corn nitrogen utilization, a new germplasm resource is provided for cultivating a new nitrogen-efficient corn variety, and meanwhile, a certain theoretical basis is laid for researching plant low-nitrogen response and improving the plant nitrogen utilization efficiency.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and molecular biology, and relates to the application of a maize nitrogen utilization-related gene ZmMAPK6. Background Art

[0002] With the rapid growth of the world's population and the continuous improvement of living standards, human food demand is rapidly increasing. The application of chemical fertilizers and the selection of high-quality varieties are key factors in achieving a qualitative leap in grain production. Corn is an important food crop, ranking first in China in terms of planted area and yield. However, corn requires a high level of soil nitrogen fertilizer and lacks the ability to biologically fix nitrogen. Excessive nitrogen fertilizer application not only increases economic costs but also causes serious environmental pollution. Nitrogen deficiency is also a major factor limiting corn growth and yield. Improving corn's nitrogen use efficiency and enhancing its tolerance to low-nitrogen soils are crucial for reducing agricultural costs and alleviating environmental pollution. Therefore, studying how corn tolerates low nitrogen from a molecular biological perspective and understanding the physiological and biochemical changes in corn under low-nitrogen environments are particularly important for breeding new low-nitrogen-tolerant corn varieties.

[0003] Studying the molecular mechanisms of corn's specific response to low nitrogen stress and the main factors affecting its main physiological and biochemical processes can effectively discover high-quality low nitrogen-related genetic resources, thereby providing a certain theoretical basis for further improving corn's tolerance to low nitrogen and cultivating 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 an application of the maize nitrogen utilization-related gene ZmMAPK6. By constructing CRISPR / Cas9 materials, the target gene is directed and site-specifically edited, the related gene is rapidly knocked out in the wild type, and the transgenic plant's ability to tolerate low nitrogen is detected compared to the wild type.

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

[0006] The application of reduced or disappeared expression of maize nitrogen utilization-related gene ZmMAPK6 in plant resistance to low nitrogen stress.

[0007] The application of reduced or absent expression of maize nitrogen utilization-related gene ZmMAPK6 in the breeding of nitrogen-efficient plants.

[0008] Method for enhancing plant resistance to low nitrogen stress,

[0009] The corn ZmMAPK6 gene is weakened by genetic engineering means; the weakening includes knocking out or reducing the expression of the ZmMAPK6 gene.

[0010] According to the above application or method, the nucleotide sequence is shown as SEQ ID NO.1.

[0011] According to the above application or method, the amino acid sequence of the protein encoded by the corn ZmMAPK6 gene is shown as SEQ ID NO.2.

[0012] The invention relates to a maize low nitrogen tolerance gene plant expression vector ZmMAPK6-pBUE411C, wherein the expression vector is obtained by connecting the fragment shown in SEQ ID NO. 1 to the vector pBUE411C.

[0013] Preferably, the CRISPR / Cas gene editing technology is used to mutate the maize ZmMAPK6 gene, so that the corresponding gene function of the gene is lost.

[0014] Preferably, the breeding method includes designing a gRNA sequence; amplifying, recovering, and constructing a vector; transferring into Agrobacterium; and infecting the plant.

[0015] Preferably, the sequence of the gRNA action site is 5'-TGATGCGACGCGGATACTC-3'.

[0016] Preferably, primers with sequences of SEQ ID NO. 6 and SEQ ID NO. 7 are used for amplification and construction of the vector.

[0017] The beneficial effects of the present invention are:

[0018] The application of the maize nitrogen utilization-related gene ZmMAPK6 provided by the present invention provides new germplasm resources for breeding new nitrogen-efficient maize varieties, and at the same time lays a certain theoretical foundation for studying plant low-nitrogen response and improving plant nitrogen utilization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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:

[0020] Figure 1 ZmMAPK6 Crispr Schematic diagram of sequence alignment of materials

[0021] Figure 2 ZmMAPK6 Crispr Sand culture phenotypes of materials under normal nitrogen (NN) and low nitrogen (LN) treatments DETAILED DESCRIPTION

[0022] 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.

[0023] The main reagents in the following examples are: Taq DNA polymerase, T4 ligase, KOD, purchased from biological companies such as NEB and Toyobo; plasmid miniprep kit and agarose gel recovery kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Agrobacterium strain EHA105, agar powder, agarose, antibiotics such as ampicillin (Amp), kanamycin (Kan), and rifampicin (Rif), and glucose, BSA, LB medium, etc. purchased from companies such as Sigma and Bio-Rad; various other chemical reagents used in the examples were imported or domestic analytical grade reagents.

[0024] The present invention used 271 B73 maize inbred lines and conducted a genome-wide association study (GWAS) on the glutamate content of the aerial part of maize seedlings at LN (0.05 mM). The candidate gene ZmMAPK6 was found near the significant association site on chromosome 10. The seeds of wild-type and ZmMAPK6 CRISPR / Cas9 materials were germinated on moist filter paper in the dark. After germination for two days, they were cultured in sand culture to the V1 stage and provided with sufficient nitrogen. With low nitrogen The phenotype was observed after 6 days of treatment with nutrient solution, and it was found that the CRISPR / Cas9 material of ZmMAPK6 had low nitrogen Insensitive phenotype.

[0025] The sequence of the ZmMAPK6 gene in maize is shown in SEQ ID NO. 1, and its accession number in the maize genome database is GRMZM2G089484. The maize ZmMAPK6 gene consists of 8976 bases, and the reading frame of the T02 transcript is from base 1624 to base 7038 from the 5' end. The gene consists of 10 exons, including 10 coding exons, reading frames from base 1624 to base 2117, base 2505 to base 2913, base 3671 to base 3798, base 3883 to base 3942, base 4783 to base 4932, base 5011 to base 5166, base 5280 to base 5491, base 5848 to base 5949, base 6053 to base 6201, base 6755 to base 7038, and the rest are intron sequences.

[0026] The sequence involved in the present invention is:

[0027] 1) The nucleotide sequence of the ZmMAPK6 gene is shown in SEQ ID No. 1;

[0028] 2) The protein sequence encoded by the ZmMAPK6 gene is shown in SEQ ID No. 2;

[0029] Variants that differ from the present invention by one or more nucleotides or amino acids but have the same or similar functions are still within the scope of protection of this application.

[0030] SEQ ID No.1

[0031]

[0032] SEQ ID No. 2

[0033] MQHDQKKKAPSEMDFFTEYGEGSRYKIEEVIGKGSYGVVCSAVDTHTGEKVAIKKINDIFEHVSDATRILREIKLLRLLRHPDIVEIKHILLPPSRREFRDIYVVFELMESDLHQVIKANDDLTPEHYQFFLYQLLRGL KYIHTANVFHRDLKPKNILANADCKLKICDFGLARVAFNDTPTAIFWTDYVATRWYRAPELCGSFFSKYTPAIDIWSIGCIFAELLTGKPLFPGKNVVHQLDIITDLLGTPSPEAISRIRNEKARRYLSSMRRKKPIPF TQKFPNADPLALCLLERMLAFEPKDRPSAEEALADPYFKNIASVDREPSAQAVTKLEFEFERRRVTKEDIRELIYREILEYHPKMLREFLEGTESSGFMYPSAVDHFKKQFAYLEEHYAKGSTGTPPERQHNSLPRPSV VYSDNRSQTTANITEDLSKCMLRENTQKTHPYSASVASKFPPHVPQGDVARPGKAVGSVMQYSPCPAPAAERYEQRRIARHPAVAPNNIPSGSSYPRRSQTCKSETGDAESHMDANQARQPKPYAANKLPATVDSRGGHW

[0034] Example 1 Construction and detection of ZmMAPK6 gene CRISPR / Cas9 material

[0035] The method for constructing a ZmMAPK6 material comprises the following steps:

[0036] 1) Design and screen candidate target sequences using the CRISPR / Cas9 target design online website crispr-P (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR / CRISPR). Target sequences are typically required to be located within or upstream of a conserved functional domain within the CDS and have a GC content between 45% and 75%.

[0037] 2) Use the website CRISPR-RGEN-Tools (http: / / www.rgenome.net / cas-offfnder / ) 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.

[0038] 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.

[0039] The gRNA sequence used in the present invention is 5'-TGATGCGACGCGGATACTC-3' (SEQ ID No. 3), and the vector used is pXUE411C from the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0040] 4) After the vector was constructed and sequenced, it was transformed into Agrobacterium strain EHA105, and maize embryos were infected with Agrobacterium to obtain transgenic positive seedlings. Homozygous mutant materials were obtained through self-pollination and sequencing.

[0041] 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.

[0042] The construction method of CRISPR / Cas9 gene editing vector is as follows:

[0043] The primers used are as follows:

[0044] SEQ ID NO.4

[0045] ID-1f: 5'-GGCG TGATGCGACGCGGATACTC -3' (the underlined sequence is the reverse complement of the underlined sequence in ID-1r);

[0046] SEQ ID NO.5

[0047] ID-1r: 5'-AAAC GAGTATCCGCGTCGCATCA -3 (the underlined sequence is the reverse complement of the underlined sequence in ID-1f).

[0048] (1) Annealing: The above primers were diluted to 10 μM and then subjected to gradient annealing to anneal them into double strands, thereby obtaining double-stranded DNA fragments with sticky ends of enzyme cleavage sites and gRNA.

[0049] (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.

[0050] Table 1

[0051]

[0052] (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;

[0053] Table 2

[0054]

[0055] (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 ZmMAPK6-pBUE411C for ZmMAPK6 gene mutation.

[0056] The primers used for colony PCR are as follows:

[0057] SEQ ID NO.6

[0058] ID-1f: 5'-GGCGCACCGTGGGTGAGTTCACGT-3';

[0059] SEQ ID NO.7

[0060] ID-1r: 5'-AAACACGTGAACTCACCACGGTG-3'.

[0061] The sequencing primers are as follows:

[0062] SEQ ID NO.8

[0063] OsU3-3: 5'-GACAGGCGTCTTCIACTGGTGCTAC-3'

[0064] Specifically:

[0065] The constructed and sequenced CRISPR / Cas9 gene editing vector ZmMAPK6-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 the size of agarose gel bands. 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, transferred to 200 mL of liquid culture medium containing 100 μg / mL kanamycin and 50 μg / mL rifampicin, and resuspended to OD 600 The concentration of β-actin in maize was between 0.8 and 1.0. After the above-mentioned bacterial solution was used to infect the immature embryos of B73 maize dug out under sterile conditions, the maize callus was obtained by dedifferentiation through callus induction technology. The callus was further induced into seedlings through redifferentiation technology to obtain mutant plants. The mutant-positive plants were confirmed by PCR amplification and sequencing of gene fragments including the target site. Finally, two knockout materials of ZmMAPK6 candidate genes were obtained (named ZmMAPK6 crispr -1 and ZmMAPK6 crispr -2), the sequencing results showed that two homozygous mutant strains were obtained, the sequences of which are as follows Figure 1 shown.

[0066] Among them, the primer sequence for identifying the ZmMAPK6 gene is:

[0067] ZmMAPK6-F: GAAGTCGTGAATTTTAGCCTCGG (SEQ ID No. 9)

[0068] ZmMAPK6-R: ACCATAACGATCCATGTGAAAGC (SEQ ID No. 10)

[0069] PCR amplification and sequencing were performed using the above primers, and the PCR system was identified as follows:

[0070]

[0071] Perform PCR reaction according to the following procedure

[0072] Step 1: 95℃ 5min

[0073] Step 2: 95℃ 30sec

[0074] Step 3: 56℃ 30sec

[0075] Step 4: 72℃ 30sec

[0076] Step 5: 72℃ 5min

[0077] Step 6: 4℃ ∞

[0078] Steps 2-4 were cycled 37 times before sequencing.

[0079] The results showed that ZmMAPK6 Crispr -1 material mutation was a T base inserted after the 207th base from the start codon, causing premature termination, while ZmMAPK6 Crispr The mutation of -2 material is that the 206th base A is deleted from the start codon, resulting in premature termination, such as Figure 1 shown.

[0080] Example 2 Phenotypic observation and low nitrogen tolerance test of ZmMAPK6 gene CRISPR materials

[0081] wild-type maize and ZmMAPK6 Crispr -1, ZmMAPK6 Crispr -2 strains were germinated on filter paper in the dark and cultured to the V1 stage in sand culture two days after germination, and sufficient nitrogen was provided. With low nitrogen The cells were treated with nutrient solution and the phenotypes were observed after 6 days.

[0082] Soak 40 corn seeds of the same size in saturated calcium sulfate for 7-8 hours. Pour out the saturated calcium sulfate solution and rinse five times with deionized water. Then, place a layer of filter paper on a tray and moisten the filter paper with water. Place the seeds on the filter paper and then place another layer of filter paper on top and moisten it. Use toilet paper to absorb excess water from the filter paper and place it in the dark for two days until it germinates. Then, place the germinated seeds in a seedling pot containing a 1:3 ratio of sand and vermiculite. After cultivating until two leaves and one heart are formed, the seedlings with the same growth are treated with sufficient nitrogen (4mM) and low nitrogen (0.05mM) until the first leaf shows the typical inverted "V" phenotype of nitrogen deficiency.

[0083] Under nitrogen-sufficient (NN) conditions, mutant ZmMAPK6 Crispr -1, ZmMAPK6 Crispr -2 and the wild type showed no phenotypic difference, and the first leaves did not show yellowing phenotype. However, under low nitrogen (LN) conditions, the first leaves of the wild type were significantly yellower than those of ZmMAPK6 Crispr -1, ZmMAPK6 Crispr -2 The first leaf turns yellow, such as Figure 2 As shown. It shows that mutant ZmMAPK6 Crisp r-1、ZmMAPK6 Crispr -2 Not sensitive to low nitrogen.

[0084] The above conclusions show that the gene ZmMAPK6 found through the GWAS method is insensitive to low nitrogen.

[0085] The contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0086] 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. The application of reduced or disappeared expression of maize nitrogen utilization-related gene ZmMAPK6 in plant resistance to low nitrogen stress.

2. The application of reduced or disappeared expression of maize nitrogen utilization-related gene ZmMAPK6 in the breeding of nitrogen-efficient plants.

3. A method for enhancing plant resistance to low nitrogen stress, characterized in that: The corn ZmMAPK6 gene is weakened by genetic engineering means; the weakening includes knocking out or reducing the expression of the ZmMAPK6 gene.

4. The use or method according to any one of claims 1 to 3, wherein the nucleotide sequence is shown as SEQ ID NO.

1.

5. The use or method according to claim 4, wherein the amino acid sequence of the protein encoded by the maize ZmMAPK6 gene is shown as SEQ ID NO.

2.

6. A maize low nitrogen tolerance gene plant expression vector ZmMAPK6-pBUE411C, which is obtained by connecting the fragment shown in SEQ ID NO. 1 to the vector pBUE411C.

7. The use or method according to any one of claims 1 to 3, characterized in that Using CRISPR / Cas gene editing technology, the corn ZmMAPK6 gene was mutated, resulting in the loss of the corresponding gene function.

8. The use or method according to claim 7, characterized in that The breeding method includes designing gRNA sequences; amplifying, recovering, and constructing vectors; transferring into Agrobacterium; and infecting plants.

9. The method according to claim 7, characterized in that The sequence of the gRNA action site is 5'-TGATGCGACGCGGATACTC-3'.

10. The method according to claim 7, characterized in that The vector was constructed by amplification using primers with sequences of SEQ ID NO.6 and SEQ ID NO.7.

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