Application of potassium regulation gene NACs in crop potassium absorption and transport rate

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a potassium regulation gene NACs in potassium absorption and transportation of crops, the potassium regulation gene NACs disclosed by the invention comprises a TaNAC71 gene, an OsNAC71 gene or a ZmNAC48 gene, the potassium regulation gene NACs is influenced in different crops, the absorption and transportation rate of plants to potassium elements can be influenced, and the absorption and transportation rate of the plants to potassium elements can be influenced. Therefore, the adaptive capacity of the plant in the low-potassium environment is influenced. The invention provides a new production thought for improving the potassium absorption rate of crops for genetic breeding, and has a wide prospect in the field of crop breeding.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of potassium regulating gene NACs in crop potassium absorption and transport rate. Background Art

[0002] Potassium is one of the macroelements necessary for crop growth and development. However, in the field of planting, crops have low efficiency in utilizing potassium. In order to adapt to the ever-changing potassium ions in different soils, crops have formed corresponding tolerance mechanisms. However, according to statistics, the utilization efficiency of potassium in wheat in my country is less than 40% in the current season. Improving the utilization efficiency of potassium fertilizer is a major scientific issue that needs to be urgently addressed for the sustainable development of my country's agriculture.

[0003] At present, research on NAC family genes has shown that they have a certain promoting effect on wheat quality characteristics. For example, the prior art discloses that wheat NAC-A18 participates in the synthesis of grain storage proteins and starch, thereby affecting wheat grain weight; wheat NAC019 affects wheat grain quality by regulating globulin and starch accumulation; but there are no reports on the involvement of NACs genes in efficient potassium absorption. Summary of the invention

[0004] In order to achieve the above object, the present invention provides the application of potassium regulatory gene NACs in regulating the potassium absorption and transport rate of crops, which specifically includes the following technical solutions:

[0005] The invention discloses an application of potassium regulatory gene NACs in regulating the potassium absorption and transport rate of crops, wherein the NACs include TaNAC71 gene, OsNAC71 gene or ZmNAC48 gene.

[0006] Preferably, the application includes: application of biological materials that positively regulate the potassium regulatory gene NACs in increasing the potassium transport rate of crops or application of biological materials that negatively regulate the potassium regulatory gene NACs in reducing the potassium absorption and transport rate of crops.

[0007] The present invention also provides a biomaterial, which includes a biomaterial that positively regulates the potassium-regulated gene NACs as described above or a biomaterial that negatively regulates the potassium-regulated gene NACs as described above.

[0008] Preferably, the biological material that positively regulates the potassium-regulating gene NACs includes one or more of a primer set for amplifying the potassium-regulating gene NACs, a recombinant expression vector containing the potassium-regulating gene NACs, and a recombinant microorganism containing the potassium-regulating gene NACs.

[0009] Preferably, the primer set for amplifying the potassium-regulated gene NACs includes one or more of a primer set for amplifying the TaNAC71 gene, a primer set for amplifying the OsNAC71 gene, and a primer set for amplifying the ZmNAC48 gene.

[0010] Preferably, the sequences of the primer set for amplifying the TaNAC71 gene are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0011] The sequences of the primer set for amplifying the OsNAC71 gene are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0012] The sequences of the primer set for amplifying the ZmNAC48 gene are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0013] Preferably, the biological material for negatively regulating the potassium-regulating gene NACs includes one or more of the sgRNA of the potassium-regulating gene NACs, a recombinant expression vector containing the sgRNA, and a recombinant microorganism containing the sgRNA.

[0014] Preferably, the sgRNA for the potassium-regulated gene NACs includes the sgRNA of the TaNAC71 gene, the sgRNA of the OsNAC71 gene, and the sgRNA of the ZmNAC48 gene;

[0015] The sgRNA sequence of the TaNAC71 gene is shown in SEQ ID NO.7;

[0016] The sgRNA sequences of the OsNAC71 gene are shown in SEQ ID NO.8 and SEQ ID NO.9;

[0017] The sgRNA sequences of the ZmNAC48 gene are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0018] The present invention also provides the use of any of the above-mentioned biological materials in regulating the potassium transport rate of crops and / or creating low-potassium tolerant crop germplasm.

[0019] The present invention also provides a method for cultivating low-potassium tolerant crops, wherein the potassium regulating gene NACs as described above is introduced into target crops to obtain the low-potassium tolerant crops.

[0020] The beneficial effects of the above technical solution are:

[0021] The present invention provides a class of potassium regulatory genes NACs, and the potassium regulatory genes NACs include TaNAC71 gene, OsNAC71 gene and ZmNAC48 gene. The potassium regulatory genes NACs of the present invention play a regulatory function in the absorption and transport of potassium in crops. On this basis, the present invention also provides the protein encoded by the above gene and the application of the above gene in creating low potassium tolerant crop germplasm, which can create crops with high potassium utilization rate and tolerance to low potassium environment. The present invention provides important technical support for crop breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 This is a schematic diagram of the PCR amplification results for positive detection of the NAC71 gene in transgenic wheat in Example 4;

[0024] in, Figure 1 M is DL2000; plasmid is pWMB190-TaNAC71 plasmid; 1-8 represent wheat NAC71 transgenic lines; WT is Zhengmai 7698; H is negative control -H 2 O;

[0025] Figure 2 This is a schematic diagram of the comparison results of PCR sequences of wheat NAC71 gene in transgenic edited plants in Example 5;

[0026] Figure 3 This is a schematic diagram of the functional verification results of wheat NAC71 transgenic plants under potassium deficiency treatment in Example 6;

[0027] in, Figure 3 WT in the figure is the transgenic receptor Zhengmai 7698; TaNAC71-OE1, TaNAC71-OE2, and TaNAC71-OE3 represent different strains of overexpressed plants with Zhengmai 7698 as the receptor; tanac71-abd1, tanac71-abd2, and tanac71-abd3 represent different strains of edited plants with Zhengmai 7698 as the receptor;

[0028] Figure 4 This is a homology evolutionary tree diagram of the wheat NAC71 gene homology and species information in Example 7;

[0029] Figure 5 This is a schematic diagram of the sequence comparison and homology analysis results of wheat NAC71 gene and rice NAC71 gene in Example 7;

[0030] Figure 6This is a schematic diagram of the comparison results of the PCR sequences of the OsNAC71 gene in the rice NAC71 transgenic edited plants in Example 9;

[0031] Figure 7 This is a schematic diagram of the functional verification results of rice NAC71 edited plants under potassium deficiency treatment in Example 10;

[0032] in, Figure 7 ZhongCK: normal potassium concentration: 0.4mmol / L for 10 days; DK: potassium deficiency concentration: 0mmol / L for 10 days; WT is the editing receptor Zhonghua11; osnac71-1, osnac71-2, osnac71-3 represent different lines of editing plants with Zhonghua11 as the receptor;

[0033] Figure 8 This is a schematic diagram of the sequence comparison and homology analysis results of the wheat NAC71 gene and the corn NAC48 gene in Example 11;

[0034] Fig. 9 This is a schematic diagram of the comparison results of the PCR sequences of the ZmNAC48 gene in the maize NAC48 transgenic edited plants in Example 13;

[0035] Fig.10 This is a schematic diagram of the functional verification results of corn NAC48 edited plants under potassium deficiency treatment in Example 14; wherein, Fig.10 CK: normal potassium concentration: 6mmol / L for 10 days; DK: potassium deficiency concentration: 0mmol / L for 6 days; WT is the editing receptor: KN5585; zmnac48-1 and zmnac48-2 represent different lines of editing plants with KN5585 as the receptor. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0037] Application of potassium regulatory genes NACs in regulating the potassium transport rate of crops, wherein the potassium regulatory genes NACs include TaNAC71 gene, OsNAC71 gene or ZmNAC48 gene.

[0038] The NCBI accession number of the TaNAC71 gene described in the present invention is: KY461044.1.

[0039] The NCBI accession number of the OsNAC71 gene of the present invention is: XM_015761800.2.

[0040] The NCBI accession number of the ZmNAC48 gene of the present invention is: NM_001279678.2.

[0041] As an embodiment, the use of the potassium regulatory gene NACs of the present invention in regulating the potassium absorption and transport rate of crops includes: the use of biological materials that positively regulate the potassium regulatory gene NACs in improving the potassium absorption and transport rate of crops, or the use of biological materials that negatively regulate the potassium regulatory gene NACs in reducing the potassium transport rate of crops.

[0042] As an embodiment, the present invention also provides a biological material used for the application as described above, wherein the biological material includes a biological material that positively regulates the potassium-regulated gene NACs as described above or a biological material that negatively regulates the potassium-regulated gene NACs as described above.

[0043] As an embodiment, the biological material that positively regulates the potassium-regulating gene NACs of the present invention includes: one or more of a primer set for amplifying the potassium-regulating gene NACs, a recombinant expression vector containing the potassium-regulating gene NACs, and a recombinant microorganism containing the potassium-regulating gene NACs.

[0044] As an embodiment, the recombinant expression vector of the present invention includes an overexpression recombinant expression vector; as an embodiment, the overexpression recombinant expression vector is a pMWB190-TaNAC71 recombinant expression vector, and the pMWB190-TaNAC71 is constructed by inserting the NACs gene into the BamHI and Kpn I restriction sites based on pMWB190. As an embodiment, the recombinant microorganism of the present invention includes an overexpression recombinant microorganism; as an embodiment, the recombinant microorganism is based on Agrobacterium GV3101.

[0045] As an embodiment, the primer set for amplifying the potassium-regulated gene NAC71 of the present invention includes: a primer set for amplifying the TaNAC71 gene, a primer set for amplifying the OsNAC71 gene, or a primer set for amplifying the ZmNAC48 gene.

[0046] The sequences of the primer set for amplifying the TaNAC71 gene are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0047] SEQ ID NO.1: F: 5'-ATGGACCACGGCTTCGACG-3';

[0048] SEQ ID NO.2: R: 5'-TCAGAACGGTTTCTGCAGGTA-3';

[0049] The sequences of the primer set for amplifying the OsNAC71 gene are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0050] SEQ ID NO.3: F: 5'-ATGGAGTGCGGTGGTGCG-3';

[0051] SEQ ID NO.4: R: 5'-TTAGAACGGCTTCTGCAGGT-3';

[0052] The sequences of the primer set for amplifying the ZmNAC48 gene are shown in SEQ ID NO.5 and SEQ ID NO.6;

[0053] SEQ ID NO.5: F: 5'-ATGAGCGGCGCCGGTCCGGA-3';

[0054] SEQ ID NO. 6: R: 5'-TCAGAACGGTTTCTGCAGGTA-3'.

[0055] The biological material for negatively regulating the potassium regulatory gene NAC71 of the present invention comprises: one or more of the sgRNA of the potassium regulatory gene NAC71, a recombinant expression vector containing the sgRNA, and a recombinant microorganism containing the sgRNA.

[0056] As an embodiment, the recombinant expression vector containing the sgRNA described in the present invention is a gene editing recombinant expression vector; as an embodiment, the gene editing recombinant expression vector is based on pWMBX110-SpCas9, and a recombinant expression vector containing the sgRNA is inserted into the Cas9 site to construct the base vector.

[0057] As an embodiment, the recombinant expression vector of the present invention includes a gene editing recombinant plasmid; the recombinant expression vector is based on Agrobacterium GV3101.

[0058] The sgRNA for the potassium regulatory gene NAC71 of the present invention includes: sgRNA of the TaNAC71 gene, sgRNA of the OsNAC71 gene, and sgRNA of the ZmNAC48 gene;

[0059] The single target sgRNA sequence of the TaNAC71 gene is shown in SEQ ID NO.7;

[0060] SEQ ID NO.7: 5'-CCGAGGTCGACCTGTACAAGTTC-3';

[0061] The dual-target sgRNA sequences of the OsNAC71 gene are shown in SEQ ID NO.8 and SEQ ID NO.9;

[0062] SEQ ID NO.8: 5'-CCGACCGACGATGAATTGGTCGG-3';

[0063] SEQ ID NO.9: 5'-CCGAAGCGTGACCTGGAGTGGTT-3';

[0064] The dual-target sgRNA of the ZmNAC48 gene is shown in SEQ ID NO.9 and SEQ ID NO.11;

[0065] SEQ ID NO.10: 5'-TGGCAGCTGCAGATCCGGACCGG-3';

[0066] SEQ ID NO. 11: 5'-CCGCTGCGCCGGCCTGCCCATCG-3'.

[0067] The present invention also provides the use of any of the above-mentioned biological materials in regulating the potassium transport rate of crops or creating low-potassium tolerant crop germplasm.

[0068] The present invention also provides a method for cultivating low-potassium tolerant crops, wherein the potassium regulating gene NAC71 as described above is introduced into a target crop to obtain the low-potassium tolerant crop.

[0069] The present invention introduces the potassium regulatory gene NACs into the target crop. As an embodiment, the introduction of the potassium regulatory gene NACs into the target crop is achieved by Agrobacterium-mediated infection.

[0070] Unless otherwise specified, the raw materials involved in the present invention are all commercially available, and the methods involved in the present invention are all conventional operations in the art, which can be confirmed by those skilled in the art according to the prior art in the art. The present invention is further described below in conjunction with specific examples.

[0071] Example 1 Construction of wheat NAC71 overexpression vector

[0072] The winter wheat variety Zhengmai 7698 (Triticum aestivum L, AABBDD) was used as the wheat material. According to the map restriction sites (BamHI and Kpn I) of the overexpression vector pMWB190, the upstream and downstream primers of wheat NAC71 were designed;

[0073] The primers for wheat NAC71 are:

[0074] SEQ ID NO.12: Forward Primer F: 5'-GCAGGTCGACTCTAGAGGATCC ( Bam HI )ATGGACCACGGCTTCGACGG-3′;

[0075] SEQ ID NO.13: Reverse Primer R: 5'-CCAGTCATAGAAGCCATGGTACC( KpI )GAACGGTTTCTGCAGGTACGTGAG-3'.

[0076] The above primers were used to perform PCR amplification on the wheat NAC71 gene. The electrophoresis detection showed that the amplified fragment was consistent with the target gene (1005 bp) in size, and a single 1005 bp target fragment of the amplified band was recovered.

[0077] At the same time, the target fragment was inserted into the pMWB190 vector using the BamHI and KpnI double restriction enzyme digestion method to construct the pMWB190-TaNAC71 overexpression vector.

[0078] Example 2 Construction of wheat NAC71 gene editing vector

[0079] The winter wheat variety Zhengmai 7698 was used as the wheat material. Based on the homology of the three copies (4A, 4B, and 4D) of wheat NAC71 and in order to distinguish other homologous genes, primers were designed according to the wheat NAC71-specific sgRNA sequence, the sequence of which is shown in SEQ ID NO.7. The sgRNA sequence of TaNAC71 was synthesized by PCR reaction and inserted into the pWMBX110-SpCas9 vector to construct a wheat NAC71 gene editing vector.

[0080] SEQ ID NO.7: 5'-CCGAGGTCGACCTGTACAAGTTC-3'.

[0081] Example 3 Obtaining wheat NAC71 gene overexpression or gene editing transgenic plants

[0082] The overexpression vector of pMWB190-TaNAC71 in Example 1 or the wheat NAC71 gene editing vector described in Example 2 was transferred into the immature embryos of Zhengmai 7698 wheat by Agrobacterium (GV3101)-mediated infection method, and the agar at the root of the differentiated seedlings in the agar was washed with clean water, and then transferred to moist nutrient soil, placed in a light incubator with a day and night of 14h / 10h, a day temperature of 16°C, a night temperature of 12°C, and a humidity of 60%, and cultured for two weeks to strengthen the seedlings. After two weeks, it was transferred to a light incubator with a day and night of 16h / 8h, a day temperature of 26°C, a night temperature of 20°C, and a humidity of 60% for culture, and the wheat NAC71 gene overexpression transgenic plants or wheat NAC71 gene editing transgenic plants were obtained.

[0083] Example 4 Identification of wheat NAC71 gene overexpressing transgenic plants

[0084] Using the method described in Example 3, 8 wheat NAC71 gene overexpressing transgenic plants were prepared, which were recorded as transgenic wheat lines 1 to 8. The genomic DNA of the transgenic wheat lines 1 to 8 to be detected was extracted by CTAB method for overexpression detection;

[0085] Primers were designed based on the wheat NAC71 gene DNA sequence and the overexpression vector, as shown in SEQ ID NO.14 and SEQ ID NO.15. SEQ ID NO.14 and SEQ ID NO.15 were used to detect whether the wheat NAC71 gene was expressed in the transgenic plants overexpressing TaNAC71.

[0086] SEQ ID NO.14: F: 5'-GATATACTTGGATGATGGCATATGCA-3';

[0087] SEQ ID NO. 15: R: 5'-AGCGCGTTGTTGGACTTCTTGCGG-3'.

[0088] PCR amplification was performed using the 10 μL system shown in Table 1, and the PCR amplification program is shown in Table 2.

[0089] Table 1 PCR reaction system

[0090] Reaction components Dosage 2XTaqMix 5μL F-Primer 0.1μL R-Primer 0.1μL DNA template 0.5μL <![CDATA[ddH 2 The]]> 4.5μL Total 10μL

[0091] Table 2 PCR reaction conditions

[0092]

[0093] After PCR amplification, the PCR products were subjected to agarose gel electrophoresis to detect the PCR products. Figure 1 shown.

[0094] Depend on Figure 1 It can be seen from the content that, taking the wild-type variety Zhengmai 7698 as the control, relative to the control plants, the transgenic wheat lines 1 to 8 all detected bands of the target gene and vector combination, indicating that the TaNAC71 gene was overexpressed in these transgenic wheat lines.

[0095] Three transgenic wheat lines were selected from transgenic wheat lines 1 to 8, named TaNAC71-OE1, TaNAC71-OE2 and TaNAC71-OE3, for subsequent research.

[0096] Example 5 Identification of wheat transgenic plants with gene editing of wheat NAC71 gene

[0097] Using the method described in Example 3, wheat NAC71 gene-edited transgenic plants were obtained, which were recorded as three wheat gene-edited lines, namely tanac71-abd1, tanac71-abd2 and tanac71-abd3. Specific detection primers for the conserved segment were designed using each copy of TaNAC71 and the editing restriction site Sal I. The primer sequences are shown in Table 3. The TaNAC71-4A / B / D gene coding region fragments were amplified. The PCR amplification system and program refer to Tables 1 and 2. After the PCR amplification was completed, the PCR amplification products were subjected to agarose gel electrophoresis to detect the PCR products.

[0098] Table 3 Primers for wheat edited plant detection

[0099] Primer name Primer sequence 5'-3' TaNAC71-4A-Edit-F SEQ ID NO.16: 5'-GCCGATCCAACCAATCTCCCGAGCGCCCGC-3' TaNAC71-4A-Edit-R SEQ ID NO.17: 5'-CCTTTACTAAATGCGCA-3' TaNAC71-4B-Edit-F SEQ ID NO.18: 5'-CCTCCGAGCTCAACCCCCCGTTCACCGGA-3' TaNAC71-4B-Editor-R SEQ ID NO.19: 5'-GGCGTGGGCAGGCAGGCA-3' TaNAC71-4D-Edit-F SEQ ID NO.20: 5'-TCGAACCAGCCCCCTCACT-3' TaNAC71-4D-Edit-R SEQ ID NO.21: 5'-CTCTTTTTAAATGA-3'

[0100] Through sequence comparison, strains in which all three copies of TaNAC71-4A / B / D were edited were screened out. It was found that base mutations in the gene-edited strains could lead to changes in protein translation. The seeds of the detected strains were propagated for generations, and PCR amplification and sequencing were performed in each generation until stable inheritance. Wheat NAC71 gene-edited transgenic plants were obtained. Three gene-edited wheat strains, named tanac71-abd1, tanac71-abd2 and tanac71-abd3, were selected for follow-up research.

[0101] The PCR sequence comparison results of TaNAC71 gene in transgenic edited plants are as follows Figure 2 shown.

[0102] Example 6 Role of wheat NAC71 transgenic plants in wheat potassium uptake and transport

[0103] The transgenic wheat lines TaNAC71-OE1, TaNAC71-OE2 and TaNAC71-OE3 in Example 4 and the gene-edited wheat lines tanac71-abd1, tanac71-abd2 and tanac71-abd3 in Example 5 were used to investigate the function of wheat transgenic plants under potassium stress.

[0104] Through indoor hydroponics, the absorption and utilization of K by plants was strictly controlled, and CK and DK groups were set up;

[0105] The wheat in the CK group was treated with Hoagland's nutrient solution containing 2 mM K, and the wheat in the DK group was treated with Hoagland's nutrient solution containing 0 mM K.

[0106] Wheat NAC71 transgenic plants and wild-type plants were treated under CK and DK conditions, and the phenotypes and related physiological indexes were statistically analyzed. Figure 3 shown.

[0107] Depend on Figure 3 It can be seen that after 8 days of treatment, under the DK group treatment, the transgenic wheat lines TaNAC71-OE1, TaNAC71-OE2 and TaNAC71-OE3 did not show obvious potassium deficiency phenotypes, such as root shortening and leaf wilting, compared with the wild type WT, indicating that they are more tolerant to potassium deficiency. In contrast, the gene-edited wheat lines tanac71-abd1, tanac71-abd2 and tanac71-abd3 all grew worse than WT, showing obvious potassium deficiency symptoms such as root shortening and leaf wilting, indicating that they are more sensitive to potassium deficiency.

[0108] The plant height, root length, aboveground dry weight, underground dry weight, potassium content in different tissues and other related indicators were measured. The plant physiological indicators are shown in Table 4.

[0109] Table 4 Physiological parameters of wheat NAC71 transgenic plants

[0110]

[0111] Note: Different letters in the table represent multiple comparisons of ANOVA.

[0112] As shown in Table 4, the plant height, root length, aboveground dry weight and underground dry weight of transgenic wheat lines TaNAC71-OE1, TaNAC71-OE2 and TaNAC71-OE3 were significantly higher than those of the wild type, especially under potassium deficiency conditions, which were 20.90%, 23.88% and 22.24% higher; 29.31%, 27.59% and 22.42% higher; 62.07%, 70.69%, 53.45% and 36.84%, 15.53% and 11.58% higher than those of the wild type, respectively. At the same time, it was found that the plant height, root length, aboveground dry weight and underground dry weight of the gene-edited wheat lines tanac71-abd1, tanac71-abd2 and tanac71-abd3 were significantly lower than those of the wild type, especially under potassium deficiency conditions, which were 35.82%, 7.46%, 1.49% lower than those of the wild type; 53.45%, 18.97%, 14.66% lower than those of the wild type; 37.81%, 26.42%, 25.78% lower than those of the wild type; and 25.82%, 15.41%, 18.30% lower than those of the wild type. The potassium content of the aboveground and underground parts of the transgenic wheat lines TaNAC71-OE1, TaNAC71-OE2 and TaNAC71-OE3 was significantly higher than that of the wild type, which were 288.34%, 541.24%, 445.61% and 216.07%, 320.15%, 220.90% respectively. It was found that the potassium content in the aboveground and underground parts of the gene-edited wheat lines tanac71-abd1, tanac71-abd2 and tanac71-abd3 were significantly lower than those of the wild type, which were 63.99%, 62.92%, 48.59% and 45.06%, 51.85%, respectively.

[0113] In summary, overexpression of the wheat NAC71 gene improved the plant's tolerance to potassium deficiency stress, and editing of the wheat NAC71 gene significantly affected the plant's growth, making the plant more sensitive to potassium deficiency stress. In addition, the wheat NAC71 gene can affect the plant's absorption and transport of potassium and play an important role in it.

[0114] Example 7 Homologous gene comparison

[0115] Comparison of homologous genes of wheat NAC71 in rice:

[0116] To verify the function of wheat NAC71 homologous genes in rice, the wheat NAC71 sequence was input into NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and the rice NAC71 with the closest homology was found based on homology relationship and species information, such as Figure 4 shown.

[0117] The results of sequence comparison and homology analysis are as follows Figure 5 shown.

[0118] according to Figure 5 It can be seen that the bases in the dark part are the same bases. There are many identical bases in the sequences of TaNAC71 and OsNAC71, and the two are located in the same branch of the evolutionary tree, indicating that TaNAC71 and OsNAC71 have high similarity and homology.

[0119] This shows that OsNAC71 and TaNAC71 are homologous genes.

[0120] Example 8 Construction of Rice NAC71 Gene Editing Vector

[0121] The japonica rice material Zhonghua No. 11 (Oryza sativa L.) was used as the recipient plant. The NAC71 genome sequence in Zhonghua No. 11 was used to design the gene editing dual-target sgRNA, the dual-target sgRNA sequences are shown in SEQ ID NO.8 and SEQ ID NO.9, and the vector was constructed according to the CRISPR / Cas9 requirements, which was recorded as the rice NAC71 editing vector.

[0122] SEQ ID NO.8: 5'-CCGACCGACGATGAATTGGTCGG-3';

[0123] SEQ ID NO.9: 5'-CCGAAGCGTGACCTGGAGTGGTT-3'.

[0124] Example 9 Obtaining and identifying rice NAC71 gene-edited transgenic plants

[0125] Obtaining transgenic rice NAC71 gene-edited plants:

[0126] The rice NAC71 editing vector constructed in Example 8 was transferred into the embryonic callus of Zhonghua No. 11 by Agrobacterium-mediated infection. After resistant callus screening and differentiation induction, the seedlings were grown. The root agar of the differentiated seedlings in the agar was washed with clean water, and then transferred to moist nutrient soil, placed in a light incubator with a day and night of 14h / 10h, a day temperature of 16°C, a night temperature of 12°C, and a humidity of 60%, and cultured for two weeks to strengthen the seedlings. After two weeks, it was transferred to a light incubator with a day and night of 16h / 8h, a day temperature of 28°C, a night temperature of 22°C, and a humidity of 60%, and different types of rice NAC71 gene-edited transgenic plants were obtained.

[0127] Identification of transgenic rice NAC71 gene-edited plants:

[0128] The genomic DNA of the transgenic rice NAC71 gene-edited plants to be tested was extracted in the same manner as in Example 4. The rice NAC71 gene coding region fragment was amplified and sequenced using the specific detection primers for the rice NAC71 dual targets shown in Table 5. The PCR system and procedure were the same as in Tables 1 and 2.

[0129] Table 5 Primers for rice editing plant detection

[0130]

[0131]

[0132] After the PCR amplification is completed, the PCR amplification products are subjected to agarose gel electrophoresis and sent for testing.

[0133] Through sequence comparison, three strains in which both target sites of rice NAC71 were edited were screened out. It was found that base mutations could lead to changes in protein translation. The seeds of the detected strains were propagated for generations, and PCR amplification and sequencing were performed in each generation until stable inheritance. Rice NAC71 gene-edited transgenic plants were obtained. Three different types of gene-edited rice strains were selected and recorded as osnac71-1, osnac71-2 and osnac71-3.

[0134] Edit the sequence alignment results as Figure 6 shown.

[0135] Depend on Figure 6 It can be seen that there are multiple types of base editing at the target sites of rice NAC71 transgenic lines, and it was found that base mutations can lead to changes in protein translation.

[0136] Example 10 Role of Rice NAC71 Gene in Rice Potassium Uptake and Transport

[0137] The function of rice NAC71 gene in potassium stress was explored using osnac71-1, osnac71-2 and osnac71-3 in Example 9.

[0138] Through indoor hydroponics, the absorption and utilization of K by plants was strictly controlled, and CK and DK groups were set up;

[0139] The CK group was treated with Yoshida rice nutrient solution containing 0.4 mM K, and the DK group was treated with Yoshida rice nutrient solution containing 0 mM K.

[0140] Rice NAC71-edited plants and wild-type plants were treated under CK and DK conditions, and the phenotypes and related physiological indexes were statistically analyzed. Figure 7 shown.

[0141] As shown in Table 5, 10 days after the gene-edited rice plants and wild-type rice were treated, under CK and DK treatments, the growth of the three strains of rice NAC71 gene edited, osnac71-1, osnac71-2 and osnac71-3, was worse than that of WT, and the potassium deficiency traits were obvious, indicating that they were more sensitive to potassium deficiency conditions.

[0142] The plant height, root length, aboveground dry weight, underground dry weight, potassium content in different tissues and other related indicators of rice NAC71 edited plants and wild-type plants under different treatments were measured. The results are shown in Table 6.

[0143] Table 6 Physiological indicators of rice NAC71 gene-edited transgenic plants

[0144]

[0145]

[0146] According to Table 6, the plant height, root length, aboveground dry weight and underground dry weight of rice NAC71 gene-edited transgenic plants osnac71-1, osnac71-2 and osnac71-3 were significantly lower than those of the wild type, especially under potassium deficiency conditions, which were 42.86%, 29.37%, 32.54% lower than those of the wild type; 45.10%, 35.29%, 25.49% lower than those of the wild type; 41.54%, 35.88%, 33.96% lower than those of the wild type; and 34.13%, 35.93%, 25.49% lower than those of the wild type. The potassium content of the aboveground and underground parts of rice NAC71 gene-edited transgenic plants osnac71-1, osnac71-2 and osnac71-3 was significantly lower than that of the wild type, which were 24.56%, 24.75%, 28.90% and 19.02%, 6.81%, 9.54% lower than those of the wild type, respectively.

[0147] As can be seen from the above, editing the rice NAC71 gene significantly affected the growth of the plant, making the plant more sensitive when suffering from potassium deficiency nutritional stress; editing the rice NAC71 gene also significantly reduced the transport of potassium to the aboveground part of wheat, especially significantly reducing the utilization efficiency of potassium under potassium deficiency conditions.

[0148] Example 11 Obtaining the Homologous Gene of Wheat NAC71 in Maize

[0149] To verify the function of wheat NAC71 homologous genes in maize, the wheat NAC71 sequence was input into NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and the following was obtained based on the homology relationship and species information: Figure 4The homology phylogenetic tree diagram shown in the figure was used to find the closest homology to maize NAC48 (NP_001266607.2), and its sequence was compared. The results are as follows Figure 8 shown.

[0150] according to Figure 8 It can be seen that the bases in the dark part are the same bases. The TaNAC71 and ZmNAC48 sequences contain many identical bases, and the two are located in the same branch in the evolutionary tree, indicating that TaNAC71 and ZmNAC48 have a high homology.

[0151] This shows that ZmNAC48 and TaNAC71 are homologous genes.

[0152] Example 12 Construction of Maize NAC48 Gene Editing Vector

[0153] Maize KN5585 (Zea mays L.) was used as the genetic transformation material. The NAC48 gene in KN5585 was used to design the gene editing dual-target sgRNA sequence, as shown in SEQ ID NO.10 and SEQ ID NO.11, and the vector was constructed according to the CRISPR / Cas9 requirements to obtain the maize NAC48 editing vector.

[0154] The sgRNA sequence is as follows:

[0155] SEQ ID NO.10: 5'-CCGGTCCGGATCTGCAGCTGCCA-3';

[0156] SEQ ID NO.11: 5'-CGCTGCGCCGGCCTGCCCATC-3';

[0157] Example 13 Obtaining and identifying maize NAC48 gene-edited transgenic plants

[0158] Obtaining transgenic plants of maize NAC48 gene editing

[0159] The maize NAC48 editing vector obtained in Example 12 was transferred into the immature embryos of KN5585 by Agrobacterium-mediated infection method, and induced to differentiate into seedlings. The root agar of the differentiated seedlings in the agar was washed with clean water, and then transferred to moist nutrient soil, placed in a light incubator with a day and night of 14h / 10h, a day temperature of 16°C, a night temperature of 12°C, and a humidity of 60%, and cultured for two weeks to strengthen the seedlings. After two weeks, it was transferred to a light incubator with a day and night of 16h / 8h, a day temperature of 26°C, a night temperature of 20°C, and a humidity of 60% and cultured until the female ears matured, and then the T0 generation seeds were obtained by test cross with the receptor pollen, and then the homozygous strain of the maize NAC48 gene-edited transgenic plants was obtained by self-pollination.

[0160] Identification of transgenic plants edited with the NAC48 gene in maize

[0161] The genomic DNA of the maize plant to be tested was extracted in the same manner as in Example 4, and the coding region of the maize NAC48 gene was amplified using the specific detection primers for the dual targets of maize NAC48 as shown in Table 7 for sequencing. The PCR system and procedure are shown in Tables 1 and 2.

[0162] Table 7 Primers for detecting edited maize plants

[0163] Primer name Primer sequence 5'-3' ZmNAC48-target1-F SEQ ID NO.26: 5'-TGTCGTGGTCAACCTGTCA-3' ZmNAC48-target1-R SEQ ID NO.27: 5'-CGATTAGCTCGCCATCGT-3' ZmNAC48-target2-F SEQ ID NO.28: 5'-TGTCGTGGTCAACCTGTCA-3' ZmNAC48-target 2-R SEQ ID NO.29: 5'-CGATTAGCTCGCCATCGT-3'

[0164] After PCR amplification, the PCR amplification products were subjected to agarose gel electrophoresis, and the PCR amplification products were sent for testing. The editing sequence comparison results were as follows Fig. 9 .

[0165] Depend on Fig. 9 It can be seen that through sequence comparison, strains with both NAC48 targets edited were screened, and it was found that base mutations can lead to changes in protein translation. The detected strains were propagated for generations, and PCR amplification and sequencing were performed in each generation until stable inheritance, and transgenic plants edited with the NAC48 gene were obtained. Two types of gene-edited strains of corn were selected, recorded as zmnac48-1 and zmnac48-2.

[0166] Example 14 Effect of Maize NAC48 Gene on Maize Potassium Uptake and Transport

[0167] The zmnac48-1 and zmnac48-2 obtained in Example 13 were used to explore the function of the maize NAC48 gene under potassium stress.

[0168] Through indoor hydroponics, the absorption and utilization of K by plants was strictly controlled, and CK and DK groups were set up;

[0169] The CK group was treated with Hoagland's nutrient solution containing 6 mM K, and the DK group was treated with Hoagland's nutrient solution containing 0 mM K.

[0170] Phenotypic and related physiological index statistical analysis was performed on maize NAC48 edited plants and wild-type plants under different treatments. Fig.10 shown.

[0171] Depend on Fig.10 It can be seen that after 6 days of treatment of maize NAC48 gene-edited transgenic plants and wild-type maize, the growth of maize NAC48 gene-edited transgenic plants zmnac48-1 and zmnac48-2 was worse than that of WT under CK and DK treatments, which suggests that they are more sensitive to potassium deficiency conditions.

[0172] The plant height, root length, aboveground dry weight, underground dry weight, potassium content in different tissues and other related indicators of the gene-edited corn plants and wild-type corn were measured, and the results are shown in Table 8.

[0173] Table 8 Physiological parameters of maize NAC48 transgenic plants

[0174]

[0175] As shown in Table 8, the plant height, root length, aboveground dry weight and underground dry weight of maize NAC48 gene-edited transgenic plants zmnac48-1 and zmnac48-2 were significantly lower than those of the wild type, especially under potassium deficiency conditions, which were 10.67%, 9.00% lower; 14.79%, 12.45% lower; 49.64%, 25.64% lower and 62.63%, 58.03% lower than those of the wild type. The potassium content of the aboveground and underground parts of maize NAC48 gene-edited transgenic plants zmnac48-1 and zmnac48-2 was significantly lower than that of the wild type, which were 61.33%, 53.33% lower and 77.69%, 58.04% lower, respectively.

[0176] The above content shows that editing the corn NAC48 gene significantly affects the growth of the plant, making the plant more sensitive when suffering from potassium deficiency nutritional stress. In addition, editing the corn NAC48 gene also significantly reduces the transport of potassium to the aboveground part of wheat, especially significantly reducing the utilization efficiency of potassium under potassium deficiency conditions.

[0177] In summary, the potassium regulatory gene NACs provided by the present invention plays a similar function in crop potassium transport. Positive regulation of wheat NAC71 and its homologous genes can improve crop potassium utilization efficiency and transport rate, and can also promote crop growth; knocking out wheat NAC71 and its homologous genes will reduce crop potassium transport and utilization efficiency, and will also affect crop growth. The potassium regulatory gene NAC71 and its homologous genes provided by the present invention can be used to create crops with high potassium utilization rate and tolerance to low potassium environments, providing important technical support for crop breeding.

[0178] The above description is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements without departing from the principle of the present invention, and these improvements should also be regarded as within the protection scope of the present invention.

Claims

1. Application of potassium regulatory genes NACs in crop potassium absorption and transport rate, wherein the NACs include TaNAC71 gene, OsNAC71 gene or ZmNAC48 gene.

2. The use according to claim 1, characterized in that The application includes: application of biological materials that positively regulate the potassium regulatory gene NACs in improving the potassium absorption and transport rate of crops, or application of biological materials that negatively regulate the potassium regulatory gene NACs in reducing the potassium absorption and transport rate of crops.

3. A biomaterial, characterized in that: The biological material includes a biological material that positively regulates the potassium-regulating gene NACs of claim 1 or a biological material that negatively regulates the potassium-regulating gene NACs of claim 1.

4. The biomaterial according to claim 3, characterized in that The biological material that positively regulates the potassium-regulating gene NACs includes one or more of a primer set for amplifying the potassium-regulating gene NACs, a recombinant expression vector containing the potassium-regulating gene NACs, and a recombinant microorganism containing the potassium-regulating gene NACs.

5. The biomaterial according to claim 4, characterized in that The primer set for amplifying the potassium-regulated gene NACs includes one or more of a primer set for amplifying the TaNAC71 gene, a primer set for amplifying the OsNAC71 gene, and a primer set for amplifying the ZmNAC48 gene.

6. The biomaterial according to claim 5, characterized in that The sequences of the primer set for amplifying the TaNAC71 gene are shown in SEQ ID NO.1 and SEQ ID NO.2; The sequences of the primer set for amplifying the OsNAC71 gene are shown in SEQ ID NO.3 and SEQ ID NO.4; The sequences of the primer set for amplifying the ZmNAC48 gene are shown in SEQ ID NO.5 and SEQ ID NO.

6.

7. The biomaterial according to claim 3, characterized in that The biological material for negatively regulating the potassium-regulating gene NACs includes one or more of the sgRNA of the potassium-regulating gene NACs, a recombinant expression vector containing the sgRNA, and a recombinant microorganism containing the sgRNA.

8. The biomaterial according to claim 7, characterized in that The sgRNA for the potassium-regulated gene NACs includes the sgRNA of the TaNAC71 gene, the sgRNA of the OsNAC71 gene, and the sgRNA of the ZmNAC48 gene; The sgRNA sequence of the TaNAC71 gene is shown in SEQ ID NO.7; The sgRNA sequences of the OsNAC71 gene are shown in SEQ ID NO.8 and SEQ ID NO.9; The sgRNA sequences of the ZmNAC48 gene are shown in SEQ ID NO.10 and SEQ ID NO.

11.

9. Use of the biological material according to any one of claims 3 to 8 in regulating the potassium absorption and transport rate of crops and / or creating low-potassium tolerant crop germplasm.

10. A method for cultivating low potassium tolerant crops, characterized in that: The potassium regulating gene NACs as claimed in claim 1 is introduced into the target crop to obtain the low potassium tolerant crop.

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