Application of rice NAC028 gene in regulation and control of rice aluminum resistance

Abstract

The invention provides application of a rice NAC028 gene in regulation and control of rice aluminum resistance, and belongs to the technical field of gene engineering. The invention provides an application of an NAC028 protein and / or a gene for coding the NAC028 protein in regulating and controlling the tolerance of a plant to aluminum. A gene function-deleted mutant nac028 plant and an overexpression plant are created and are subjected to aluminum stress treatment together with wild Nipponbare, and the result shows that the gene-deleted mutant is super sensitive to aluminum, and the tolerance of the overexpression plant to aluminum is enhanced. The NAC028 protein or the coding gene of the NAC028 protein can be used for perfecting an upstream and downstream regulation and control network of rice responding to aluminum stress, and also provides an important gene resource and a new path for breeding of aluminum-resistant plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to rice. NAC028 Application of genes in regulating aluminum tolerance in rice. Background Technology

[0002] Acidic soils account for approximately 22.7% of my country's total land area, mainly distributed in the southern regions rich in water and heat resources. Aluminum toxicity is a recognized major limiting factor affecting crop growth in acidic soils. Aluminum toxicity severely affects crop root growth, thereby seriously hindering the absorption of nutrients and water by the roots, leading to reduced crop yields and, in strongly acidic soils, even total crop failure.

[0003] Developing and cultivating crops resistant to acidic aluminum toxicity soils is of paramount importance for improving land utilization and crop quality and yield in areas with acidic aluminum toxicity soils. In recent years, research has focused on plant mechanisms of aluminum detoxification and plant tolerance to aluminum toxicity, but reports on genes that can effectively enhance plant tolerance to aluminum toxicity are scarce. Summary of the Invention

[0004] In view of this, the present invention provides a NAC028 protein and / or a gene encoding said NAC028 protein (rice). NAC028 The application of the gene in regulating plant tolerance to aluminum: Knocking out the gene encoding the NAC028 protein significantly reduced aluminum tolerance in rice, while overexpression... NAC028 It can significantly increase the aluminum tolerance of rice.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of the NAC028 protein and / or the gene encoding the NAC028 protein in regulating plant tolerance to aluminum; The amino acid sequence of the NAC028 protein is shown in SEQ ID NO: 1.

[0006] Preferably, at least one of the NAC028 protein, the gene encoding the NAC028 protein, and a gene derivative containing the gene positively regulates and enhances the plant's tolerance to aluminum.

[0007] Preferably, the improvement of plant tolerance to aluminum includes alleviating the inhibition of plant root development caused by aluminum toxicity and / or reducing the aluminum content in the root tip.

[0008] Preferably, the plant includes grasses; the concentration of aluminum ions in the growth environment of the plant is above 40 μM.

[0009] This invention provides a targeted rice NAC028The sgRNA of the gene, wherein the nucleotide sequence of the target of the sgRNA is shown in SEQ ID NO: 3.

[0010] The present invention provides a derivative containing the sgRNA, including a recombinant expression vector and / or recombinant bacteria.

[0011] This invention provides the application of the sgRNA or its derivative in improving the sensitivity of rice to aluminum and / or creating aluminum-sensitive rice varieties.

[0012] This invention provides a method for regulating the tolerance of rice to aluminum, by enhancing the biological function of the NAC028 protein in rice and / or increasing the expression level of the gene encoding the NAC028 protein to improve the tolerance of rice to aluminum. Alternatively, inhibiting the biological function of the NAC028 protein in rice and / or reducing the expression level of the gene encoding the NAC028 protein can reduce the tolerance of rice to aluminum. The amino acid sequence of the NAC028 protein is shown in SEQ ID NO: 1.

[0013] Preferably, the method for enhancing the biological function of the NAC028 protein in rice and / or increasing the expression level of the gene encoding the NAC028 protein includes overexpressing the gene encoding the NAC028 protein; The method for inhibiting the biological function of NAC028 protein in rice includes mutating the gene encoding the NAC028 protein; The nucleotide sequence of the gene encoding the NAC028 protein is shown in SEQ ID NO: 2.

[0014] Preferably, the enhancement of the biological function of the NAC028 protein in rice and / or the increase of the gene encoding the NAC028 protein includes introducing a gene derivative containing the gene encoding the NAC028 protein into the recipient rice plant. Inhibiting the biological function of the NAC028 protein in rice and / or reducing the expression level of the gene encoding the NAC028 protein includes introducing the sgRNA or the derivative into the recipient rice plant.

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides the application of the NAC028 protein and / or the gene encoding the NAC028 protein in regulating plant tolerance to aluminum; the amino acid sequence of the NAC028 protein is shown in SEQ ID NO: 1. This invention identified a candidate gene controlling aluminum tolerance in rice by analyzing the responses of 353 core rice varieties worldwide to aluminum stress using a genome-wide association study (GWAS). NAC028 (LOC_Os12g32400), further using CRISPR / Cas9 gene editing technology to create a mutant with loss of gene function. nac028 When treated with aluminum along with wild-type Nipponbare (Nip), the mutant with this gene deletion was found to be extremely sensitive to aluminum. nac028 Root length was reduced compared to the wild type, while root tip aluminum content was increased. This invention further investigated the aluminum tolerance of the overexpressing transgenic plants; the results showed that the overexpressing lines… NAC028-OE The root length is longer than that of the wild type, and the aluminum content in the root tip is lower than that of the wild type. NAC028 Genes can not only be used to improve the upstream and downstream regulatory network of rice in response to aluminum stress, but also provide a new approach for breeding new aluminum-tolerant rice materials and ultimately increasing grain yield in acidic soils. Attached Figure Description

[0016] Figure 1 GWAS analysis results of relative root elongation in rice before and after aluminum treatment; Figure 2 For wild-type rice Nip and mutant materials nac028 Aluminum resistance phenotype analysis; Figure 3 For wild-type rice Nip and mutant materials nac028 Comparison of root length before and after aluminum treatment; Figure 4 For wild-type rice Nip and mutant materials nac028 A comparison chart of aluminum content in root tips after aluminum treatment; Figure 5 For wild-type rice Nip and overexpression lines NAC028-OE Aluminum resistance phenotype analysis; Figure 6 For wild-type rice Nip and mutant materials NAC028-OE Comparison of root length before and after aluminum treatment; Figure 7 For wild-type rice Nip and mutant materials NAC028-OE A comparison chart of aluminum content in root tips after aluminum treatment. Detailed Implementation

[0017] This invention provides a NAC028 protein and / or a gene encoding said NAC028 protein. NAC028 Application of NAC028 protein in regulating plant tolerance to aluminum; the amino acid sequence of the NAC028 protein is shown in SEQ ID NO: 1 (MGGATNLPPGFHFFPSDEELVVHFLRRKVSLLPCHPDIIPTLLPHRYNPWELNGKALQAGNQWYFFCHLTQSRTSSNGHWSPIGVDETVRSGGRNVGLKKTLLFSIGEPSEGIRTNWIMHEYHLLDGDCVAGGSSNLTSSSSNRRSHRKRGHSSMESNNWVLCRVFESSCGSQVSFHGEGTELSCLDEVFLSLDDYDEVSLPNK).

[0018] In this invention, the nucleotide sequence of the gene encoding the NAC028 protein is preferably as shown in SEQ ID NO: 2 (5'--3').

[0019] In this invention, at least one of the NAC028 protein, the gene encoding the NAC028 protein, and a gene derivative containing the gene preferably positively regulates and enhances the plant's tolerance to aluminum. This enhancement of plant tolerance to aluminum preferably includes alleviating the inhibition of root development caused by aluminum toxicity and / or reducing the aluminum content in the root tip. The plant preferably includes grasses, more preferably at least one of rice, maize, sorghum, and wheat, and even more preferably rice. In an embodiment of this invention, Nipponbare rice (…) is used. Oryza sativa The effects of the NAC028 protein and its encoding gene on plants are illustrated using an example. The concentration of aluminum ions in the plant's growth environment is above 40 μM, more preferably above 45 μM, and even more preferably 50-150 μM.

[0020] The embodiments of the present invention demonstrate that treating mutants with CaCl2 solutions (pH 4.5) containing and without AlCl3... nac028 ( NAC028 (plants with gene loss of function) and NAC028 After gene overexpression in plants, mutants nac028 Root length was reduced compared to the wild type, while root tip aluminum content was increased; and overexpression plants NAC028-OE The roots are longer than those of the wild type, and the aluminum content in the root tips is lower than that of the wild type. This indicates that the NAC028 protein and its encoding gene can regulate the aluminum tolerance of plants. This invention provides an opportunity for the breeding of aluminum-tolerant rice varieties and provides genetic resources and technical support for the cultivation of aluminum-tolerant plants / crops.

[0021] In this invention, the gene derivative containing the gene preferably includes a recombinant expression vector and / or a recombinant bacterium. The backbone vector of the recombinant expression vector preferably includes the pCambia1300 vector, and the multiple cloning site of the backbone vector is preferably... Kpn 1 and BamH 1. The pCambia1300 vector described in this invention is a common plant expression vector. This invention does not have special requirements for the construction method of the recombinant expression vector; any construction method well-known to those skilled in the art can be used. The host bacteria of the recombinant bacteria preferably include Agrobacterium, more preferably Agrobacterium tumefaciens, and most preferably Agrobacterium strain EH105. The recombinant bacteria preferably include the recombinant expression vector and the host bacteria, obtained by transferring the recombinant expression vector into the host bacteria. The recombinant expression vector of this invention (… 35S:NAC028-GFP ) can be expressed NAC028 The gene, when used to prepare transgenic rice using this recombinant expression vector, can improve rice's tolerance to aluminum.

[0022] This invention also provides a targeted rice NAC028The sgRNA of the gene, wherein the nucleotide sequence of the target of the sgRNA is shown in SEQ ID NO: 3 (5'-CCCTGACATCATCCCGACGC-3').

[0023] This invention designs sgRNAs based on target sequences, wherein the sgRNAs preferably include sgRNA-S and sgRNA-A; the nucleotide sequence of sgRNA-S is preferably as shown in SEQ ID NO: 4 (5'-CAGCCCTGACATCATCCCGACGC-3'); the nucleotide sequence of sgRNA-A is preferably as shown in SEQ ID NO: 5 (5'-AACGCGTCGGGATGATGTCAGGG-3'). The sgRNAs provided by this invention can specifically knock out... NAC028 The gene, which was used to prepare transgenic rice, can reduce rice's tolerance to aluminum.

[0024] The present invention also provides a derivative containing the sgRNA, comprising a recombinant expression vector and / or recombinant bacteria.

[0025] In this invention, the backbone vector of the recombinant expression vector preferably includes a Cas9 / sgRNA vector, and the multiple cloning site of the backbone vector is preferably BspQ1. The Cas9 / sgRNA vector of this invention was purchased from Beijing Weishang Lide Biotechnology Co., Ltd., catalog number: VK005-01. This invention does not have special requirements for the construction method of the recombinant expression vector; any construction method well known to those skilled in the art can be used. The host bacteria of the recombinant bacteria preferably include Agrobacterium, more preferably Agrobacterium tumefaciens, and most preferably Agrobacterium strain EH105. The recombinant bacteria preferably include the recombinant expression vector and the host bacteria, obtained by transferring the recombinant expression vector into the host bacteria.

[0026] This invention provides the application of the sgRNA or its derivative in improving the sensitivity of rice to aluminum and / or creating aluminum-sensitive rice varieties.

[0027] In this invention, the enhancement of rice’s sensitivity to aluminum preferably includes inhibiting root development and / or increasing the aluminum content in the root tip.

[0028] In this embodiment of the invention, the sgRNA / 35S:NAC028-GFP Prepared transgenic rice mutants nac028 / overexpression strains NAC028-OE The mutant was discovered 24 hours later. nac028 Compared to the wild type, nac028 The mutant showed significantly reduced tolerance to aluminum; overexpression lines NAC028-OE Compared to the wild type, the overexpression lines NAC028-OE Its resistance to aluminum is significantly increased.

[0029] The present invention also provides a method for regulating the tolerance of rice to aluminum, by enhancing the biological function of the NAC028 protein in rice and / or increasing the expression level of the gene encoding the NAC028 protein to improve the tolerance of rice to aluminum. Alternatively, inhibiting the biological function of the NAC028 protein in rice and / or reducing the expression level of the gene encoding the NAC028 protein can reduce the tolerance of rice to aluminum. The amino acid sequence of the NAC028 protein is shown in SEQ ID NO: 1.

[0030] In this invention, the method for enhancing the biological function of NAC028 protein in rice and / or increasing the expression level of the gene encoding NAC028 protein includes overexpressing the gene encoding NAC028 protein, more preferably introducing a gene derivative containing the gene encoding NAC028 protein into the recipient rice plant. The method for inhibiting the biological function of NAC028 protein in rice includes mutating the gene encoding the NAC028 protein, more preferably introducing the sgRNA or the derivative into the recipient rice plant. The preferred nucleotide sequence of the gene encoding the NAC028 protein is shown in SEQ ID NO: 2.

[0031] In this embodiment of the invention, sgRNA / 35S:NAC028-GFP After being introduced into recipient rice plants, mutants were obtained. nac028 / overexpression strains NAC028-OE mutant nac028 The gene for the NAC028 protein was mutated, including the deletion of a T base at 189 bp or the insertion of a T base between 189 bp and 190 bp in the gene fragment with the nucleotide sequence shown in SEQ ID NO: 2; overexpression lines NAC028-OE Detected by Western blotting and real-time quantitative polymerase chain reaction, respectively. NAC028 Gene expression levels. Compared to wild-type plants, mutants... nac028 Increased sensitivity to aluminum, overexpression lines NAC028-OE Its resistance to aluminum is significantly increased.

[0032] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 After germinating 353 core rice varieties from around the world in the dark for 3 days in a 0.5 mM CaCl2 solution (pH 4.5), their root lengths were measured. They were then placed in CaCl2 solutions containing and without AlCl3 for an additional 24 hours in the dark. The CaCl2 solutions contained 0.5 mM CaCl2 at pH 4.5; the AlCl3 solutions contained 50 μM AlCl3. After measuring the root length and calculating the relative root elongation, a GWAS analysis was performed based on these measurements. The results are shown below. Figure 1 .

[0034] A candidate gene controlling aluminum tolerance in rice was identified. NAC028 The rice NAC028 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1; the rice NAC028 The nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0035] Example 2 A type of knockout or overexpression rice NAC028 Recombinant gene expression vector 1. A kind NAC028 Methods for constructing gene knockout and recombination expression vectors A complementary DNA sequence derived from the target sequence shown in SEQ ID NO: 3 was synthesized, the complementary DNA sequence consisting of sgRNA-S1 and sgRNA-A1; The nucleotide sequence of the sgRNA-S1 is shown in SEQ ID NO: 4; The nucleotide sequence of the sgRNA-A1 is shown in SEQ ID NO: 5; The synthesized sgRNA-S1 and sgRNA-A1 were diluted to 10 μM, and mixed at a volume ratio of sgRNA-S1:sgRNA-A1:ddH2O = 5:5:15. The mixture was then incubated in a 95℃ metal bath for 3 min and allowed to cool naturally at room temperature for 10 min to obtain Oligo dimers for later use. The Cas9 / sgRNA vector was digested with BspQ1 restriction endonuclease at 50°C for 1 hour. After digestion, the Cas9 / sgRNA was recovered using a gel extraction kit (Easy Gel Extraction & Clean-up Kit, DR0101250, purchased from Easy Biotech). The specific recovery procedure was as follows: 3 volumes of Buffer A1 were added and transferred to a centrifuge column. After centrifugation at 12000 rpm for 1 minute, the filtrate was discarded. 500 μL of Buffer A2 solution was added, and the mixture was centrifuged at 12000 rpm for 1 minute. The filtrate was discarded, and the mixture was centrifuged again at 12000 rpm for 1 minute to remove any residual liquid. The centrifuge column was then placed in a new 1.5 mL centrifuge tube, and 30 μL of preheated Elution 2.0 (at 65°C) was added. After standing at room temperature for 1 minute, the mixture was centrifuged at 12000 rpm for 1 minute to obtain the Cas9 / sgRNA digestion product. The vector was ligated using the T4 enzyme. The Cas9 / sgRNA digestion product: Oligo dimer: T4 enzyme: T4 buffer = 1:7:1:1 was mixed and incubated at room temperature for 4 hours to obtain the knockout recombinant expression vector. The constructed knockout recombinant expression vector was transformed into DH5α competent cells, and positive single-clone screening was performed using kanamycin-resistant LB solid medium. Single colonies were picked and sent to the company for sequencing to obtain the successfully constructed knockout recombinant expression vector. The kanamycin-resistant LB solid medium consisted of the following components at the following concentrations: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, kanamycin 0.5 g / L, and agar powder 20 g / L.

[0036] 2. A kind NAC028 Methods for constructing gene overexpression recombinant expression vectors Synthesize upstream and downstream complementary DNA sequences consisting of the nucleotide sequence shown in SEQ ID NO: 2, wherein the complementary DNA sequences are composed of NAC028-OE-F and NAC028-OE-R; The nucleotide sequence of NAC028-OE-F is shown in SEQ ID NO: 6 (5'-CGAACGATAGCCGGTACCATGGGAGGAGCTACAAACTT-3'); The nucleotide sequence of NAC028-OE-R is shown in SEQ ID NO: 7 (5'-GCGGACTCTAGAGGATCCTTTATTCGGCAAACTTACTT-3'); Using Nipponbare cDNA dilution as a template, KOD FX enzyme was used for amplification. NAC028Genes. The PCR reaction system consisted of 1 µL cDNA, 1 µL NAC028-OE-F / R primers, 10 µL 2× PCR Buffer, 4 µL dNTPs, 0.4 µL KOD FX, and 2.6 µL ddH2O. The PCR program was based on the KOD FX instruction manual.

[0037] The pCambia1300:35S:GFP vector was digested with Kpn1 and BamH1 restriction endonucleases at 37°C for 1 hour in a water bath. After digestion, the vector was recovered from the gel using the same method as above. Homologous recombination was performed using the homologous recombinase (ClonExpress Ultra One Step Cloning Kit V3, catalog number: C117) purchased from Novizan. The digestion products of the vector were obtained at a volume ratio of pCambia1300:35S:GFP. NAC028 Gene cloning gel recovery product: homologous recombinase = 2.5:2.5:5, mixed and reacted at 50℃ for 15 min to obtain the overexpression recombinant expression vector ( 35S:NAC028-GFP The constructed overexpression recombinant expression vector was transformed into DH5α competent cells and positive single-clone screening was performed using kanamycin-resistant LB solid medium. Single colonies were picked and sent to the company for sequencing to obtain the successfully constructed overexpression recombinant expression vector.

[0038] Example 3 mutant nac028 / overexpression strains NAC028-OE Preparation method The positive clone bacterial culture obtained in Example 2 was subjected to recombinant plasmid extraction using a plasmid extraction kit (EasyPlasmid Miniprep Kit, DR0201250, purchased from EasyBio). The extracted recombinant plasmid (knockout recombinant expression vector or overexpression recombinant expression vector) was transformed into Agrobacterium EH105 and then infected rice Nipponbare ( Oryza sativa (Nip) callus tissue was screened to obtain positive transgenic mutants. nac028 The specific method is as follows: Agrobacterium EH105 transformed with the recombinant plasmid was inoculated into LB solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin resistance, and cultured at 28°C to obtain single colonies. These single colonies were then picked from the kanamycin and rifampicin resistant LB solid medium and inoculated into 20 mL of LB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and cultured at 28°C until late logarithmic growth to obtain a bacterial suspension. 0.5 mL of this bacterial suspension was then transferred to 50 mL of LB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and cultured at 28°C until OD (dark cycle) was reached. 600 The pH was 0.5; after centrifuging the cultured Agrobacterium at 4000g for 10 min, the precipitate was resuspended in an equal volume of AAM-AS medium to obtain the AAM-AS resuspension; the AAM-AS medium was based on AAM medium (purchased from Solarbio, catalog number LA8580), containing only 100 μmol / L acetylsylgenone; the pH of the AAM-AS medium was 5.2; Mature Nip seeds were collected, and after removing the glumes, they were disinfected with 70% ethanol for 1 min and rinsed 3 times with sterile water. Then, they were disinfected with a 2% sodium hypochlorite solution with available chlorine and shaken for 60 min, rinsed 5 times with sterile water, and then the embryos were isolated under sterile conditions and inoculated onto N6D2S2 medium. The embryos were cultured in the dark at 28℃ for 4 days to induce callus tissue. The N6D2S2 medium was based on N6 medium (purchased from Haibo Biotechnology, catalog number HBZ0601-2) and also contained only hydrolyzed casein 500 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, plant gel 2.5 g / L, hygromycin 50 mg / L, and cephalosporin 300 mg / L. The pH of the N6D2S2 medium was 5.8. The callus tissue was immersed in the AAM-AS resuspension for 20 min, blotted dry with sterile filter paper, and then transferred to N6D2C medium. It was incubated in the dark at 25°C for 3 days to obtain the infected callus tissue. The N6D2C medium was based on N6 medium and also contained only hydrolyzed casein 500 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, plant gel 2.5 g / L, glucose 10 g / L, and acetylsyringone 100 μmol / L. The pH of the N6D2C medium was 5.2. The infected callus tissue was washed five times with sterile water containing 300 mg / L cephalosporin, blotted dry with sterile filter paper, and then transferred to N6D2S1 medium for screening. The N6D2S1 medium was based on N6 medium and also contained only hydrolyzed casein 500 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, plant gel 2.5 g / L, hygromycin 25 mg / L, and cephalosporin 600 mg / L. The pH of the N6D2S1 medium was 5.8. Two weeks later, the tissue was transferred to N6D2S2 medium for second-generation selection. One generation was selected every two weeks. Vigorous growth of resistant callus tissue after three generations of selection was transferred to pre-differentiation medium and cultured for 7 days in a differentiation incubator at a 12-hour photoperiod and 28°C during the day and 25°C at night. Then, the tissue was transferred to differentiation medium and cultured in a differentiation incubator at a 12-hour photoperiod and 28°C during the day and 25°C at night until regenerated seedlings were produced. The formulation of both the pre-differentiation and differentiation media was: MS medium as the basal medium, containing only hydrolyzed casein 300 g / L, hygromycin 50 mg / L, 6-BA 3 mg / L, 6-KT 3 mg / L, 6-ZT 0.2 mg / L, and plant gel 2.5 g / L. The pH of both the pre-differentiation and differentiation media was 5.8. The regenerated seedlings were transferred to a rooting and seedling strengthening medium and rooted and strengthened under a 12-hour photoperiod, daytime temperature of 28°C and nighttime temperature of 25°C. The rooting and seedling strengthening medium consisted of 1 / 2 MS medium as the basal medium with 10 g / L sucrose added. When the seedlings grow to 10 cm, open the container seal for 3 days, then transfer the seedlings into an artificial climate chamber for cultivation to obtain the plants for later use. The plant leaves were ground into powder using liquid nitrogen, and 600 μL of TPS DNA extraction solution was added. The mixture was thoroughly mixed, and the mixture was incubated in a water bath at 65°C for 15 min, during which it was inverted and mixed 3 times. The mixture was then centrifuged at 12,000 rpm at room temperature for 10 min. The TPS DNA extraction solution consisted of the following components: 1 M Tris-HCl, 0.5 M EDTA, and 5 M KCl. Next, 300 μL of the supernatant was added to an equal volume of isopropanol, mixed and shaken well, and allowed to stand for 10 min. Then, it was centrifuged at 12000 rpm at room temperature for 10 min and the supernatant was discarded. 500 μL of 70% ethanol was added to wash the precipitate, and the mixture was centrifuged at 12000 rpm at room temperature for 5 min and the supernatant was discarded. The precipitate was dried in a clean bench. 50 μL of ddH2O was added to dissolve the DNA to obtain plant gDNA. Genomic PCR amplification was performed using hygromycin sequence primers Hyg-F and Hyg-R via 2×Rapid Taq Master Mix (purchased from Vazyme, catalog number P222-01) to obtain PCR products; the nucleotide sequence of Hyg-F is shown in SEQ ID NO: 8 (5'-TTTCTTTGCCCTCGGACGAGT-3'); the nucleotide sequence of Hyg-R is shown in SEQ ID NO: 9 (5'-ATGAAAAAGCCTGAACTCACC-3'); The PCR amplification system consisted of: 10 μL 2×Rapid Taq Master Mix, 0.5 μL Hyg-F (10 µM), 0.5 μL Hyg-R (10 µM), 1 μL sample DNA, and 8 μL ddH2O.

[0039] The PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 15 s, repeated for 32 cycles; 72℃ complete extension for 3 min.

[0040] The PCR products were analyzed by agarose gel electrophoresis. If the target band was present, the mutant was preliminarily identified as a positive transgenic mutant, and the mutant was screened to obtain the mutant. nac028 / overexpression strains NAC028-OE ; mutant nac028 Identification, further utilizing target location sequence primers NAC028 -F and NAC028 -R, genomic PCR amplification was performed using 2×Rapid Taq Master Mix; the PCR amplification system and reaction procedure were the same as above, except that Hyg-F was replaced with NAC028 -F replaces Hyg-R with NAC028 -R; the NAC028 The nucleotide sequence of -F is shown in SEQ ID NO: 10 (5'-TAGTTGTAGCATGGGAGGAG-3'); NAC028 The nucleotide sequence of -R is shown in SEQ ID NO: 11 (5'-TTCTATCAGCACTCAGCATG-3'); the PCR product was sequenced to identify the mutant. nac028 Specific mutation type, nac028-1 There is a 1bp insertion at 190bp in the sequence shown in SEQ ID NO:2. nac028-2 There is a 1bp deletion at 189bp in the sequence shown in SEQ ID NO: 2. Figure 2 (A)

[0041] Overexpression lines NAC028-OE Identification was performed, and total plant protein and RNA were further extracted. The total protein was detected using Western blotting to identify the NAC028-GFP fusion protein. RNA was further reverse transcribed into cDNA, and the cDNA was analyzed using real-time quantitative polymerase chain reaction. NAC028 Gene expression was detected, and overexpression lines were screened. NAC028-OE The protein immunoblotting technique and real-time quantitative polymerase chain reaction involved in this method are routine research methods in this field and will not be described in detail here. Figure 5 China A and Figure 5 Line B is an overexpression strain. NAC028-OE The identification results showed that the overexpression strain NAC028-OE Successfully built.

[0042] Example 4 mutant nac028 / Overexpression lines NAC028-OE Its tolerance to aluminum mutant nac028 / Overexpression lines NAC028-OE After the wild-type *Nipponbare* (Nip) germinated in the dark in a 0.5 mM CaCl2 solution (pH 4.5), seedlings with uniform growth (bud length 0.5 mm) were selected and placed in CaCl2 solutions containing and without AlCl3 for one day. Root length was then observed and photographed. The CaCl2 concentration in the CaCl2 solution was 0.5 mM; the pH of the CaCl2 solution was 4.5; and the AlCl3 concentration in the CaCl2 solution containing AlCl3 was 50 μM. Figure 2 B in the figure (scale bar is 5 mm) represents wild-type rice Nip and mutant materials. nac028 Aluminum resistant surface Figure 5 C (scale bar 5 mm) wild-type rice Nip and overexpression lines NAC028-OE Aluminum resistant surface.

[0043] Measuring mutants nac028 / Overexpression lines NAC028-OE The root length and root tip aluminum content of its wild-type Nipponbare were shown in the following figures. Figure 3 and Figure 4 For wild-type rice Nip and mutant materials nac028 Comparison of root length before and after aluminum treatment, and comparison of aluminum content in root tips after aluminum treatment. Figure 6 and Figure 7 Wild-type rice Nip and mutant materials NAC028-OE Comparison of root length before and after aluminum treatment, and comparison of root tip aluminum content after aluminum treatment. Among them, after one day of growth in a CaCl2 solution without AlCl3, Nip... nac028-1and nac028-2 The root lengths were 2.35±0.25cm, 2.33±0.18cm, and 2.35±0.19cm, respectively; Nip, NAC028-OE-1 and NAC028-OE-2 The root lengths were 2.36±0.13 cm, 2.34±0.16 cm, and 2.35±0.16 cm, respectively; after growing in a CaCl2 solution containing AlCl3 for one day, Nip, nac028-1 and nac028-2 The root lengths were 1.66±0.17cm, 1.07±0.15cm, and 1.05±0.16cm, respectively; Nip, NAC028-OE-1 and NAC028-OE-2 The root lengths were 1.63±0.13 cm, 1.92±0.13 cm, and 1.91±0.14 cm, respectively. After growing in a CaCl2 solution containing AlCl3 for one day, Nip, nac028-1 and nac028-2 The aluminum contents of the root tips were 225.73±17.47 μg / g, 458.96±28.87 μg / g, and 469.55±18.43 μg / g, respectively; Nip, NAC028-OE-1 and NAC028-OE-2 The aluminum contents of the root tips were 225.73±17.47μg / g, 458.96±28.87μg / g, and 469.55±18.43μg / g, respectively.

[0044] Depend on Figures 2-4 It can be seen that, compared with the wild-type rice variety Nip, the mutant material nac028 Increased sensitivity to aluminum.

[0045] Depend on Figures 5-7 It can be seen that, compared with the wild-type rice variety Nip, the overexpression material NAC028-OE Enhanced resistance to aluminum.

[0046] In summary, the present invention provides NAC028 The gene is a key gene that can affect aluminum tolerance in rice; NAC028 Genes can provide genetic resources and technical support for the breeding of aluminum-tolerant rice varieties.

[0047] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a NAC028 protein and / or a gene encoding said NAC028 protein in regulating plant tolerance to aluminum; The amino acid sequence of the NAC028 protein is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, At least one of the NAC028 protein, the gene encoding the NAC028 protein, and a gene derivative containing the gene positively regulates and enhances plant tolerance to aluminum.

3. The application according to claim 2, characterized in that, The improvement of plant tolerance to aluminum includes alleviating the inhibition of plant root development caused by aluminum toxicity and / or reducing the aluminum content in root tips.

4. The application according to any one of claims 1 to 3, characterized in that, The plants include grasses; the concentration of aluminum ions in the growth environment of the plants is above 40 μM.

5. A type of targeted rice NAC028 The sgRNA of a gene is characterized by, The nucleotide sequence of the target of the sgRNA is shown in SEQ ID NO:

3.

6. A derivative containing the sgRNA of claim 5, characterized in that, This includes recombinant expression vectors and / or recombinant bacteria.

7. The use of the sgRNA of claim 5 or the derivative of claim 6 in improving the sensitivity of rice to aluminum and / or creating aluminum-sensitive rice varieties.

8. A method for regulating the tolerance of rice to aluminum, characterized in that, Enhancing the biological function of the NAC028 protein in rice and / or increasing the expression level of the gene encoding the NAC028 protein to improve the tolerance of rice to aluminum; Alternatively, inhibiting the biological function of the NAC028 protein in rice and / or reducing the expression level of the gene encoding the NAC028 protein can reduce the tolerance of rice to aluminum. The amino acid sequence of the NAC028 protein is shown in SEQ ID NO:

1.

9. The method according to claim 8, characterized in that, The method for enhancing the biological function of NAC028 protein in rice and / or increasing the expression level of the gene encoding the NAC028 protein includes overexpressing the gene encoding the NAC028 protein; The method for inhibiting the biological function of NAC028 protein in rice includes mutating the gene encoding the NAC028 protein; The nucleotide sequence of the gene encoding the NAC028 protein is shown in SEQ ID NO:

2.

10. The method according to claim 8 or 9, characterized in that, The enhancement of the biological function of the NAC028 protein in rice and / or the increase of the gene encoding the NAC028 protein includes introducing a gene derivative containing the gene encoding the NAC028 protein into the recipient rice plant. Inhibiting the biological function of the NAC028 protein in rice and / or reducing the expression level of the gene encoding the NAC028 protein includes introducing the sgRNA of claim 5 or the derivative of claim 6 into the recipient rice plant.

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