CN115820672B - Corn nitrogen utilization related gene ZmNCRG and application thereof - Google Patents

Abstract

The application provides a corn nitrogen utilization related gene ZmNCRG with a nucleotide sequence of SEQ ID No.1. The application also provides application of the gene as a molecular marker of the nitrogen utilization capacity of corn and a method for enhancing the low nitrogen stress resistance of corn by using the gene.

Description

A maize nitrogen utilization-related gene ZmNCRG2 and its application

Technical Field

The invention belongs to the technical field of genetic engineering, and in particular relates to a maize nitrogen utilization-related gene ZmNCRG2 and a molecular marker and application thereof.

Background Art

Nitrogen, as a basic component of proteins, nucleic acids, chlorophyll and many secondary metabolites, is a limiting factor for plant growth and productivity. Therefore, a huge amount of nitrogen fertilizer (about 100 million tons) is used each year to increase crop yields (FAO, 2006), representing a major agricultural system cost (Masclax-Daubresse et al., 2010). However, large-scale application of chemical fertilizers, mainly nitrogen fertilizers, will damage the ecological environment, and low-nitrogen environments are widely present in our cultivated land. The lack of nitrogen nutrition will not only affect the growth and development of plants, but also be the main abiotic stress that restricts corn yield. In corn, improving nitrogen utilization and reducing agricultural production costs are research hotspots, mainly including research on the absorption, assimilation and redistribution of nitrogen in corn.

GWAS scans markers covering the entire genome and uses statistical analysis to find loci that are significantly associated with the target trait. Currently, GWAS is widely used in the study of human diseases and other complex traits. In 2008, the first genome-wide association analysis was reported in maize, and 8590 loci in 553 elite maize inbred lines were used to analyze genes that affect grain fatty acid content (Belo et al., 2008). With the publication of the B73 reference sequence, the number of GWAS in maize has increased dramatically, and many traits related to molecular metabolism, growth and development, yield, and stress resistance have been analyzed. Our previous studies found that the nitrogen response of different inbred lines varies greatly, and the responses of different inbred lines to low nitrogen stress are also different. The National Maize Improvement Center of our university constructed an association population consisting of 508 maize inbred lines (Li et al., 2013). By combining the 1.03 million gene-based SNP markers obtained by transcriptome sequencing analysis with the genotype data of the MaizeSNP50 BeadChip chip containing 56,110 SNP markers developed by Illumina, a high-density genetic linkage map of all materials was obtained (Ganal et al., 2011; Li et al., 2013), which served as the basis for the GWAS used in this study.

Summary of the invention

There have been some reports on genome-wide association analysis of maize using GWAS technology. We used nitrate content as a quantitative trait to explore factors related to nitrogen utilization efficiency.

On the one hand, the present application provides a corn nitrogen utilization-related gene ZmNCRG2, which encodes the protein shown in SEQ ID No.2.

Furthermore, the nucleotide sequence of the gene is SEQ ID No.1.

On the one hand, the present application provides a maize nitrogen utilization-related gene ZmNCRG2, and the use of an agent for knocking out ZmNCRG2 or reducing the expression of ZmNCRG2 in breeding maize with low nitrogen stress resistance.

On the other hand, the maize nitrogen utilization-related gene ZmNCRG2, and the application of reagents for knocking out ZmNCRG2 or reducing the expression of ZmNCRG2 in nitrogen-efficient maize breeding.

In another aspect, the present application provides a method for enhancing corn resistance to low nitrogen stress, comprising knocking out ZmNCRG2 or reducing ZmNCRG2 expression.

Furthermore, knocking out ZmNCRG2 or reducing ZmNCRG2 expression was performed using the CRISPR method.

Furthermore, the CRISPR method includes constructing a CRSPR-Cas9 knockout target vector pBUC411-ZmNCRG2, transferring the vector into Agrobacterium EHA105, and then transforming corn B73-329 to obtain transgenic plants.

On the other hand, the present application provides a kit for identifying corn varieties tolerant to low nitrogen or varieties with high nitrogen utilization efficiency, which comprises a reagent for detecting ZmNCRG2-SNP6, wherein the sequence of ZmNCRG2-SNP6 is SEQ ID No.7.

Furthermore, the reagent for detecting the marker ZmNCRG2-SNP6 is a primer pair with the sequences of SEQ ID No.5 and SEQ ID No.6.

In another aspect, the present application provides a method for identifying corn varieties tolerant to low nitrogen or varieties with high nitrogen utilization efficiency, comprising:

Amplify the genomic DNA of the variety to be identified using the primer pair of SEQ ID No. 5 and SEQ ID No. 6;

The amplified product is detected to see whether it includes SEQ ID No.7. If it does, the variety to be identified is a low-nitrogen-tolerant variety; if it does not, the variety to be identified is a low-nitrogen-intolerant variety.

Furthermore, if the size of the amplified fragment is 950 bp, the variety to be identified is a low-nitrogen tolerant variety; if the size of the amplified fragment is 357 bp, the variety to be identified is a low-nitrogen intolerant variety.

On the other hand, the present application provides the use of the above-mentioned kit or method in the breeding of corn with low nitrogen tolerance or high nitrogen utilization efficiency.

The maize nitrogen efficient utilization gene ZmNCRG2 and the corresponding protein in the present application include not only the genes and proteins of SEQ ID No.1 and SEQ ID No.2, but also sequences with the same or similar functions formed by one or more substitutions, deletions and additions based on SEQ ID No.1 and SEQ ID No.2.

Beneficial effects:

At present, corn is the world's largest food crop and is widely planted all over the world. Lack of nitrogen nutrition not only affects the growth and development of plants, but is also the main abiotic adversity that restricts corn yield. The present invention uses GWAS analysis to mine a gene related to nitrogen utilization in corn, which can be applied to nitrogen efficient utilization breeding in corn, which is convenient and fast. In corn, improving nitrogen utilization and reducing agricultural production costs are research hotspots. The present invention uses GWA technology to mine genes related to nitrogen efficient utilization, which is helpful for research on hotspots.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a Manhattan plot of the ratio of NO ₃ ^- content in tissues of maize inbred lines under low nitrogen stress and high nitrogen conditions; the arrow indicates the main effect site (ZmNCRG2) of NO ₃ ^- concentration.

Figure 2 shows the CRISPR/Cas9 material identification results of ZmNCRG2.

FIG3 is a vector map after the ZmNCRG2 gene coding sequence is connected to the T7 vector.

Figure 4 shows the phenotype of corn materials of CRISPR/Cas9 material of ZmNCRG2 under high and low nitrogen conditions.

FIG. 5 shows that the protein encoded by ZmNCRG2 is localized in the cell membrane and cytoplasm; subcellular localization is observed in corn protoplasts.

FIG6 shows two haplotype promoters connected to a GFP vector and an empty GFP vector in protoplast transformation, and the expression levels were detected.

DETAILED DESCRIPTION

The following examples are provided to facilitate a better understanding of the present invention, but are not limited thereto. These examples are only used for illustrative purposes and in no way limit the scope of protection of the present invention.

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

Example 1 Identification of the main nitrogen efficient sites in corn

GWAS scans the markers covering the entire genome and finds sites significantly associated with the target trait through statistical analysis. The National Maize Improvement Center of our university constructed an association population consisting of 508 maize inbred lines (Li et al., 2013). By combining the 1.03 million gene-based SNP markers obtained by transcriptome sequencing analysis with the genotype data of the MaizeSNP50 BeadChip chip containing 56,110 SNP markers developed by Illumina, a high-density genetic linkage map of all materials was obtained (Ganal et al., 2011; Li et al., 2013). We selected 304 materials from them, and used the ratio of NO ^3- content in maize inbred line tissues under low nitrogen stress and high nitrogen stress as a quantitative trait, and used genome-wide association analysis (GWAS) to explore factors related to nitrogen use efficiency. The present invention locates a major effect site on chromosome 4 through analysis based on a mixed linear model, as shown in Figure 1, which is located at the 45Mb position of chromosome 4. A candidate gene was identified in this candidate region and named ZmNCRG2.

The CRSPR-Cas9 knockout target construction vector pBUC411-ZmNCRG2 was designed and transferred into Agrobacterium EHA105, and then transformed into corn B73-329 to obtain transgenic plants. The transgenic positive plants were identified by PCR sequencing of gene fragments including the target site, and the knockout material of the ZmNCRG2 candidate gene was obtained.

Example 2 Functional Analysis

MaizeGDB website predicts the structure of ZmNCRG2 gene, the full length of the gene (from start codon to stop codon) is 746bp, including 3 exons and 2 introns, of which the coding region sequence is 558bp in length. In order to clone the full-length coding sequence of ZmNCRG2, 10-day-old maize inbred line B73 seedlings were used as materials. Total RNA was extracted using the plant total RNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., and cDNA was obtained by reverse transcription using the reverse transcriptase of Polymer (Beijing) Biotechnology Co., Ltd. ZmNCRG2-specific primers (forward primer ZmNCRG2-gene-FATGGCCTGCACCAGGAGG, SEQ ID No.3; reverse primer ZmNCRG2-gene-R CTACTTGTACTCGTCCATGCC, SEQ ID No.4) for PCR amplification, and a product consistent with the expected size (558 bp) was obtained. The PCR amplification system was 50 μl, including: 2×Super Multiplex PCR Mix 25 μl; 10 μM Primer ZmNCRG2-F1 2 μl; 10 μM Primer ZmNCRG2-R1 2 μl; DNA 1 μl, ddH ₂ O 20 μl. PCR reaction conditions were: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 15 s, 34 cycles from denaturation to extension, and final extension at 72°C for 5 min).

The PCR product was recovered and purified using the gel recovery kit (Cat.#DP105-3) of Tiangen Biochemical Technology (Beijing) Co., Ltd., and the purified product was connected to the T vector using the pEASY-Blunt Cloning Kit of Beijing Quanshijin Biotechnology Co., Ltd. (see Figure 3 for the vector map), then transferred into Escherichia coli competent cells, single clones were picked, and colony PCR amplification was performed using universal primers T7/SP6. Colonies with target size bands detected by electrophoresis were positive recombinants, and the corresponding bacterial liquid was sent to Beijing Sanbo Yuanzhi Biotechnology Co., Ltd. for sequencing, and a 558bp ZmNCRG2 exon sequence was obtained, the nucleotide sequence of which is shown in SEQ ID No.1, and the corresponding protein sequence is shown in SEQ ID No.2.

The vector pCAMBIAsuper1300-GFP-ZmNCRG1 was constructed and transformed into corn protoplasts to observe subcellular localization. (1) Use enzymatic hydrolysis to obtain corn protoplasts (2) Resuspend the protoplasts with MMG solution to a final concentration of 5×10 ⁵ /mL (3) Add 30μg of plasmid and 400μL of protoplasts to a 5mL centrifuge tube and mix gently for transformation (4) After transformation, store at room temperature in the dark for 14-16h (5) Prepare slides and observe the localization using a laser confocal microscope. It is shown that the ZmNCRG2 protein is localized on the cell membrane and cytoplasm as shown in Figure 5.

Example 3 Phenotype of ZmNCRG2 CRISPR/Cas9 material.

First, wild-type maize and Zm NCRG2 ^crispr -1 and Zm NCRG2 ^crispr -2 strains were germinated in vermiculite. After germination for 6-7 days, the vermiculite at the root of the seedlings was washed off with deionized water, the endosperm was removed, and then the seedlings were grown in 1/2×Hoagland hydroponic solution for 2 days. After two days of seedling acclimatization, the hydroponic seedlings were divided into normal nitrogen and low nitrogen treatments, and the phenotypes were observed after two weeks. There was no significant difference between the wild-type material and the mutant material under normal nitrogen treatment, but under low nitrogen treatment, when the first leaf of the wild-type material turned yellow under low nitrogen conditions, the mutant materials Zm NCRG2 ^crispr -1 and Zm NCRG2 ^crispr -2 were green. The second leaf was continuously observed, and it was found that the wild-type material turned yellow faster than the mutant material, indicating that the knockout of ZmNCRG2 in maize can significantly improve the tolerance to low nitrogen environment.

Example 4 ZmNCRG2-SNP6 in ZmNCRG2 as a molecular marker

Because ZmNCRG2-SNP6 exists in nitrogen-efficient maize inbred lines, it causes a 593bp deletion in the ZmNCRG2 promoter region, and the deletion fragment is SEQ ID NO. 7, so ZmNCRG2-SNP6 can be used as a molecular marker to determine whether an individual is tolerant to low nitrogen. Among them, maize materials without SNP6 deletion are low nitrogen tolerant materials.

PCR amplification was performed using primer pair I based on the changes in the promoter region of ZmNCRG2-SNP6, and genomic DNA of maize inbred line GEMS12 and maize inbred line SY999 (deletion) as templates;

1.10μl PCR reaction system (2×Super Multiplex PCR Mix 5μl, 10μM PrimerZmNCRG1-F0.5μl, 10μM Primer ZmNCRG1-R 0.5μl, DNA 0.5μl, ddH2O 3.5μl)

2. PCR reaction conditions (pre-denaturation 95℃3min, denaturation 95℃30s, annealing 55℃30s, extension 72℃30s, 34 cycles from denaturation to extension, final extension 72℃5min), PCR products were delivered to Sanbo Polygala Company for sequencing analysis. The sequencing results showed that the two inbred lines had differences in the promoter region. The amplified fragment of the low-nitrogen-tolerant GEMS12 was larger than that of the low-nitrogen-intolerant SY999 variety. The amplified fragment size of GEMS12 was 950bp, while the amplified fragment size of SY999 was 357bp, with a difference of 593bp. Therefore, primer pair I can be used for nitrogen-efficient molecular-assisted breeding of maize. The sequence of the primer is: ZmNCRG2-PRO-F1:AGCTCCAGTGGACGGTTTAA (SEQ ID NO.5);

ZmNCRG2-PRO--R1:GAGCTGCTTGTTCGTACGTT (SEQ ID NO. 6).

Example 5 Detection of expression levels of two haplotype promoter regions connected to GFP tags

The promoter regions of the two haplotypes were constructed to connect sp1300-GFP, and two vectors were constructed, and then the vectors and the empty SP1300-GFP vector were transferred into protoplasts. The transformation method was as described in Example 2, and the protoplasts were collected after being cultured under high and low nitrogen conditions. CDNA was obtained by extracting RNA, reverse transcription, etc., and the expression was detected as a template. It was found that there was no difference in the expression of the three vectors under high nitrogen, while the expression of SNP6-GFP under low nitrogen was lower than that of the empty vector, and the expression of the vector without SNP6-GFP was higher than that of the empty vector, as shown in Figure 6, indicating that the expression of the two haplotypes of Zm NCRG2 under low nitrogen conditions was very different.

ZmNCRG2 gene sequence SEQ ID No.1:

ATGGCCTGCACCAGGAGGCAGTTGTGGTGGGCATGCCTGCTGGCGGCGTGGTGCTGCGCCGGCGCCGCCGCGGCCGCCCA

GCCGCCGGCGGCGGACGCGGACCCGCTGCAGAGCAGGTGCCAGGGGGACTTTGGGAAGCTGACGGACTGCATGGACTACG

CGACGGGGCACGCGGCGTCGCCCTCGTCCACCTGCTGCGGCGACGCCGGCGGCACGCAGAAGGCGCGCCCGGAGTGCCTC

TGCTACATCATCCAGCAGGTGCACGCGGGGCCGACCAGGTGCAGTCGCTCGGCCTGCGCTTCGACCGCCTCCTGGCGCT

CCCCGCCGCCTGCAGCCTCCCCAACGCCAACGTCTCGCTCTGCATCAACCTGCTGAACCTGAAGCCGGGCTCGCCAGACT

ACGCGCTGTTCGCCAACGCCTCCAAGATCACGCCATCAGCCGGAGGCAGCCCGGCGAGCGACACCGCCGCCGGCAGCCGTCGTTATCAGGCTCCAGGCTGGGGTCCGAGGCAGCGTCGCGCTCGCCGTGGTCTCTGCGATCGTCTCGTCAGTCTTCTGA; ZmNCRG2 amino acid sequence SEQ ID No. 2:

MACTRRQLWWACLLAAWCCAGAAAAQPPAADADPLQSRCQGDFGKLTDCMDYATGHAASPSSTCCGDAGGTQKARPECL

CYIIQQVHAGRDQVQSLGLRFDRLLALPAACSLPNANVSLCINLLNLKPGSPDYALFANASKITPSAGGSPASDTAAGSA VIRLQAGVRGSVALAVVSAIVSSVF.

ZmNCRG2 promoter region differential sequence SEQ ID No.7:

ATGGCCGGCCCTGAGGGTATGCAGGGTATGCGATCGCTCAGGGCCCCTAAGATCAGTAGGGCCTCCGTTTGTCCATATTA

TGTCCAAATACATATATGTTACAGGTAAATCTCTATTCTCGTTTCTTTGTTGCGTCTCTCTAATTTTCTGGAATCATCTC

TTCGAGGTCGTGCAGTTTACGACGAATACTAAATATTGATTGATGCAGGAAGGTCGTAAGGTGTTGTACACCTTTACCTA

ATAACCTACGTTGTTTAATCCAAGTTTTTTTTGCTGAAATTTGTTTTTCAGAAAATTTACTCTAATGTATTTAATTTTGGT

TTACTGTACATTTTAAGGTTTAGACTAGTTTGAAAAATTAAAAGTAAATACTCGCTCTCAAAGCAAATACAGAGTTATA

TTACATAATCTGTATAATTTTACAACTCATAAAATACTGTAACAATCTCTAGTCATATCATTTTGAAAAAAAATATGTAA

ATCATCAACTTTTTTTATTTTTAGTATCATATTTGAAAAAAATAGCAGAATCAAGCTTTCGTATAGAGCCTCTATATTGA

TTTTCGCTTAAGGCCCAAAATATCAGGACCGGC

Claims (5)

1. A method for enhancing low nitrogen resistance in corn, comprising knocking out corn nitrogen use related gene ZmNCRG or reducing expression of corn nitrogen use related gene ZmNCRG, wherein the nucleotide sequence of corn nitrogen use related gene ZmNCRG2 is SEQ ID No.1.

2. The method of claim 1, wherein knocking out corn nitrogen utilization related gene ZmNCRG or reducing corn nitrogen utilization related gene ZmNCRG2 expression is performed using a CRISPR method.

3. The method of claim 2, wherein the CRISPR method comprises CRSPR-Cas9 knockout target construction vector pBUC-ZmNCRG 2, the vector is transferred into agrobacterium EHA105, and then maize B73-329 transformation is performed to obtain a transgenic plant.

4. A method for identifying a low nitrogen tolerant variety of corn, said method comprising: amplifying the genome DNA of the variety to be identified by using primer pairs with sequences of SEQ ID No.5 and SEQ ID No. 6; detecting whether the amplified product comprises SEQ ID No.7, and if the amplified product comprises SEQ ID No.7, determining that the variety to be identified is a low nitrogen-resistant variety; if SEQ ID No.7 is not included, the variety to be identified is a low nitrogen intolerant variety.

5. The method according to claim 4, wherein the variety to be identified is a low nitrogen tolerant variety if the amplified fragment size is 950 bp; if the amplified fragment size is 357bp, the variety to be identified is an intolerant low nitrogen variety.