Application of rice transcription factor bZIP65 in improving nitrogen utilization efficiency of plants

Abstract

The invention discloses application of a rice transcription factor bZIP65 in improving the nitrogen utilization efficiency of plants, and relates to the technical field of biology. The construction of an overexpression plant of the bZIP65 gene proves that the overexpression of the gene can promote the transport and absorption of nitrate by the rice and improve the nitrogen fertilizer utilization rate, and the genetic mechanism of the nitrogen utilization rate of the rice is further clarified through the function interpretation of the bZIP65 gene. Meanwhile, a theoretical basis and a technical support can be provided for explaining and improving the nitrogen utilization efficiency of the rice and genetic improvement of varieties of the rice.

Description

Technical Field

[0001] The invention relates to the technical field of biotechnology, and more specifically to the application of rice transcription factor bZIP65 in improving plant nitrogen utilization efficiency. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important cereal crops in the world. In order to meet the needs of the growing population, rice production must be increased. Nitrogen is a component of many important compounds in plants, so nitrogen plays a multi-faceted role in maintaining and regulating plant physiological functions. However, in the natural soil environment, the content of nitrogen sources that can be directly absorbed and utilized by plants is low. Therefore, in order to promote crop yields, nitrogen fertilizers are used in large quantities in agriculture. However, less than 30% of the applied nitrogen fertilizers can be absorbed by crops, resulting in the large-scale use of nitrogen fertilizers that not only increases production costs, but also leads to serious soil acidification and eutrophication of water bodies. Therefore, improving the nitrogen fertilizer utilization efficiency of crops and crop yields will be a practical solution.

[0003] At present, some nitrogen utilization-related genes have been reported, such as NRT1.1B, which has nitrate transport activity under both high and low nitrogen conditions by triggering nitrate response, thereby promoting efficient nitrogen utilization and increasing yield; OsNRT2s are highly active nitrate transporters, responsible for the absorption of nitrate in the roots and the transportation to the aboveground parts, thereby increasing nitrogen utilization and promoting rice yield; OsAMTs are ammonium transporters in rice, which regulate the effective utilization of nitrogen by regulating the balance of rice carbon and nitrogen metabolism, thereby improving rice yield and quality. However, a large number of genes related to rice nitrogen utilization and their applications are still unknown and need to be explored to increase nitrogen utilization and provide excellent gene resources for high nitrogen metabolism for rice nitrogen metabolism. Summary of the invention

[0004] In view of this, the present invention provides an application of rice transcription factor bZIP65 in improving plant nitrogen utilization efficiency. The involved bZIP65 belongs to the bZIP transcription factor family, and the change of its single gene expression amount causes the change of rice nitrogen utilization rate, and the genetic effect is significant, which provides a new gene resource for improving rice nitrogen utilization rate and molecular design breeding.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] Application of rice transcription factor bZIP65 in plant breeding, the application being any one of the following:

[0007] A. Improve the nitrogen utilization efficiency of plants;

[0008] B. Increase the number of effective tillers;

[0009] C. Increase the number of grains per ear;

[0010] D. Increase production;

[0011] E. Increase photosynthetic rate;

[0012] The amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.2.

[0013] Preferably, the CDS nucleotide sequence of the rice transcription factor bZIP65 is shown in SEQ ID NO.1.

[0014] Another object of the present invention is to provide an application of a biological material for increasing the expression of rice transcription factor bZIP65, wherein the biological material is any one of the following:

[0015] A. an overexpression cassette comprising the transcription factor bZIP65;

[0016] B. a recombinant vector containing the overexpression cassette described in A;

[0017] C. A recombinant microorganism containing the overexpression cassette described in A or the recombinant vector described in B;

[0018] The amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.2.

[0019] Preferably, the CDS nucleotide sequence of the rice transcription factor bZIP65 is shown in SEQ ID NO.1.

[0020] More preferably, the application is any one of the following:

[0021] A. Improve the nitrogen utilization efficiency of plants;

[0022] B. Increase the number of effective tillers;

[0023] C. Increase the number of grains per ear;

[0024] D. Increase production;

[0025] E. Increase the photosynthetic rate.

[0026] Another object of the present invention is to provide a method for regulating plant nitrogen utilization, by increasing the expression level of transcription factor bZIP65 through genetic engineering means, and then cultivating the transformed plant cells into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.2.

[0027] Another object of the present invention is to provide a method for increasing plant yield, by increasing the expression level of transcription factor bZIP65 by genetic engineering means, and then cultivating the transformed plant cells into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.2.

[0028] Another object of the present invention is to provide a method for increasing the number of effective tillers of plants, by increasing the expression level of transcription factor bZIP65 by genetic engineering means, and then cultivating the transformed plant cells into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.2.

[0029] Another object of the present invention is to provide a method for increasing the number of grains per panicle of a plant, by increasing the expression level of the transcription factor bZIP65 by genetic engineering means, and then cultivating the transformed plant cells into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.2.

[0030] Another object of the present invention is to provide a method for improving plant photosynthesis, by increasing the expression level of transcription factor bZIP65 by genetic engineering means, and then cultivating the transformed plant cells into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.2.

[0031] Beneficial effects: The present invention provides an application of rice transcription factor bZIP65 in improving plant nitrogen utilization efficiency. By constructing a bZIP65 gene overexpression plant, it is proved that the overexpression of the gene can promote the transport and absorption of nitrate by rice and improve the utilization rate of nitrogen fertilizer, providing a theoretical basis and technical support for explaining the improvement of rice nitrogen utilization efficiency and its variety genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 The attached figure is a plasmid map of the overexpression vector pU1301.

[0034] Figure 2 The attached figure shows the expression analysis of OsbZIP65 gene in overexpression plants.

[0035] Figure 3The attached figure shows the expression analysis of OsbZIP65 gene in different rice tissues.

[0036] Figure 4 The attached figure shows the expression analysis of OsbZIP65 gene under high and low nitrogen treatment conditions of rice.

[0037] Figure 5 The attached figure is a comparative analysis of the nitrogen utilization efficiency of wild-type Songjing 2 (SJ2) and bZIP65 overexpressing plants in hydroponic culture at the seedling stage.

[0038] Figure 6 The attached figure shows the comparative analysis of agronomic traits of wild-type Songjing 2 (SJ2) and bZIP65 overexpression lines under low nitrogen (LN) and high nitrogen (HN) field planting conditions. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The embodiments of the present invention disclose the application of rice transcription factor bZIP65 in improving nitrogen utilization efficiency of plants. The methods involved in the embodiments are conventional methods unless otherwise mentioned and will not be described in detail here.

[0041] Example 1

[0042] Construction of rice bZIP65 gene overexpression vector

[0043] 1. Target fragment amplification: Primers were designed based on the bZIP65 gene sequence (shown in SEQ ID NO.1) published on the Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ), and wild-type Nipponbare (NIP) cDNA was used as a template. KpnI and BamHI restriction sites were designed on the primers to amplify the full-length 543 bp cDNA of the target gene from the start codon to the stop codon.

[0044] cDNA sequence of bZIP65 gene:

[0045] ATGACCCTGTCAGGTGGGACCCTGTCAAGCGGGATAACCTCTGGCTCAAGCCATGGGACCCGGAGCTCAGGGTCAGAGGGTGATATCCAGCTGCAGGTCCAAATGGACCTGAAGCGGAAAAGAAGGAAGGAATCAAACCGAGAGTCGGCAAAGAGGTCTAGGTTACGGAAACAACAGCACCTCGATGACCTTACCTCACAGGTGAACCAGTTGAAGATGGAGAACCAGCAGCTCACCACAACATTGAACATGGTCACCCAAAGCTGTGCCTTCGCAGAGTCTCAGAACTCAGTGCTGCGCACTCAGATGATGGAGCTGGATAGCAGGCTATCAGCGCTCCGTGAGATAATCTTCTACATGAACAATGGAAACCAATCACCTTCTAATCCTACAATAGCAGCGACTACATACCCATCCACCACTACAATGACTGCAGCAACTACTGGCCATTACGATTATTATGATGTTTTCACTGCAAATGCATGGGGCTCAGGGATGCAAATGATGCAGCAGCCCATAGATCAGTTTCTGTACCAGTGCTAG, SEQ ID NO.1.

[0046] Amino acid sequence of bZIP65 protein:

[0047] MTLSGGTLSSGITSGSSHGTRSSGSEGDIQLQVQMDLKRKRRKESNRESAKRSRLRKQQHLDDLTSQVNQLKMENQQLTTTLNMVTQSCAFAESQNSVLRTQMMELDSRLSALREIIFYMNNGNQSPSNPTIAATTYPSTTTMTAATTGHYDYYDVFTANAWGSGMQMMQQPIDQFLYQC, SEQ ID NO.2.

[0048] pU1301-bZIP65-F: 5’-ACGATGATAAGGGCGGTACCATGACCCTGTCAGG TGGGAC-3’, SEQID NO.3;

[0049] pU1301-bZIP65-R: 5'-AGGCTACGTAGGATCCGCACTGGTACAGAAACTG-3', SEQ ID NO. 4.

[0050] 2. Construction of overexpression vector: The expression vector pU1301 (see Appendix) was double-digested with KpnI and BamHI. Figure 1 ) and the target gene fragment, the recovered product was connected with the homologous recombinase at 37°C for 30 minutes, and then transformed into Escherichia coli DH5α competent cells. After a single clone was grown, the positive bacteria were identified and screened, the plasmid was extracted, and the pU1301-bZIP65 overexpression vector was obtained after correct sequencing.

[0051] Example 2

[0052] Construction of transgenic plants

[0053] 1. Genetic transformation of rice callus: Take 10μL pU1301-bZIP65 plasmid and add it to the chemical competent state of Agrobacterium EHA105, mix it by blowing gently with a pipette, put it in an ice bath for 5min, freeze it in liquid nitrogen for 5min, and then put it on ice for 3-5min immediately after a 5min water bath at 37℃; add 800μL LB culture medium without antibiotics to the competent state, and culture it at 28℃ shaker at 120rpm for 3-4hrs; centrifuge it at 8000rpm for 1min, pour out the supernatant, and spread the remaining 100μL on the LB solid plate with corresponding resistance, and culture it upside down in a 28℃ incubator for 2-3days. After a single colony grows, screen and identify the positive engineering bacteria.

[0054] 2. Finally, the callus tissue of Songjing 2 (SJ2) was transformed by Agrobacterium-mediated transformation to obtain T0 transgenic plants, and the expression level of bZIP65 gene was analyzed using the primer set shown in SEQ ID NO.3 and SEQ ID NO.4.

[0055] The results showed that the expression level of bZIP65 gene in the overexpressing transgenic plants was significantly increased compared with that in the wild type (see Appendix Figure 2 ).

[0056] Example 3

[0057] Identification of the expression level of bZIP65 gene in various tissues of rice Songjing 2 (SJ2)

[0058] 1. Extraction of RNA from various tissues of rice SJ2: Take roots, leaves, leaf sheaths, panicles and other tissues, put them in RNase-free centrifuge tubes and immediately put them into liquid nitrogen, put in steel balls, and use a grinder to shake them into powder. Add 300μL SDS RNA extraction solution to the broken sample, mix well and let it stand on ice for 5 minutes; add 200μL water-saturated phenol: chloroform = 1:100 to the centrifuge tube, vortex for 30s to mix well; put it in a 4℃ centrifuge, centrifuge at 12000rpm for 10min, and draw 200μL supernatant into a new centrifuge tube; add 1mL Trizol, shake and mix well, and let it stand at room temperature for 5min; add 200μL chloroform, gently invert to mix, 4℃, 12000rpm, centrifuge for 10min; draw 500μL supernatant liquid into a new centrifuge tube; add 1mL Trizol, shake and mix well, and let it stand at room temperature for 5min; add 200μL chloroform, gently invert to mix, 4℃, 12000rpm, centrifuge for 10min; draw 500μL supernatant liquid into a new Add an equal volume of isopropanol to an RNase-free centrifuge tube to precipitate RNA, centrifuge at 4°C, 12000rpm, for 10 min, and discard the supernatant; add 500μL 70% anhydrous ethanol for washing, centrifuge at 4°C, 12000rpm, for 5 min, discard the supernatant, centrifuge again for 1 min, use a pipette to absorb the residual liquid, evaporate naturally for 1 min, add 30μL DEPC water to dissolve and measure the concentration, then identify the quality by gel electrophoresis, and use it for reverse transcription or directly freeze it at -80°C.

[0059] 2. Reverse transcription reaction: The reverse transcription reaction kit used was from Shanghai Yisheng Biotechnology Co., Ltd. AdwanceFast 1st Strand cDNA. The specific steps are as follows: take 2 μg of total RNA and mix it with 2 μL 5×gDNA Digester Mix, and then use RNase-free H 2 Add 5 μL of 4×Hifair AdvanceFast SuperMix, 2 μL of Random Primers (for qPCR), and RNase-free H 2 Add 20 μL of HO and heat at 55°C for 5 min, 85°C for 5 s, and finally wash with RNase-free HO. 2 After diluting to 80 μL, use in subsequent molecular experiments or store at -20°C.

[0060] 3. Real-time fluorescence quantitative PCR: The kit used for RT-PCR was Zhongdao Life 2×SYBR Green HotstartqPCR Master M, and the internal reference gene for quantification was Ubiquitin (LOC_Os03g13170).

[0061] The primer sequences for quantitative detection of bZIP65 expression are:

[0062] bZIP65-qRT F: 5'-CTGTCAAGCGGGATAACCTC-3', SEQ ID NO.5;

[0063] bZIP65-qRT R: 5'-CAACTGGTTCACCTGTGAGG-3', SEQ ID NO. 6.

[0064] The internal reference primers are:

[0065] Ubi-qRT F:5'-TACCGTGCCCTTACTGTTC-3', SEQ ID NO.7;

[0066] Ubi-qRT R: 5'-CGGTGGAATGTCACAGACAC-3', SEQ ID NO. 8.

[0067] Amplification reaction system (10 μL): 2×SYBR Green Master Mix 5 μl, Forward primer 1 μl, Reverse primer 1 μl, cDNA 1 μl and ddH 2 O 2μl.

[0068] Amplification program: 95℃ pre-denaturation for 3min; 95℃ denaturation for 10s, 60℃ annealing for 30s, 72℃ extension for 15s (40 cycles); 65-95℃ (melting curve) 0.5℃ / 0.5s. Fluorescence signal collection was performed during the extension phase, and the final results were exported in Excel format using 2 -△△CT The relative expression was calculated by the method, and each sample was subjected to 3 biological replicates.

[0069] The results showed that the rice bZIP65 gene was expressed most highly in the panicle, followed by the leaf sheath. Figure 3 ).

[0070] Example 4

[0071] Analysis of bZIP65 expression in wild-type Songjing 2 (SJ2) rice under high and low nitrogen treatment hydroponic conditions

[0072] The root tissues of SJ2 rice were taken under high and low nitrogen treatment conditions (hydroponic conditions of 2 mM and 400 μM nitrate, i.e., high nitrogen (HN) and low nitrogen (LN)), respectively, and RNA was extracted and the expression level of bZIP65 was analyzed according to the experimental steps of Example 3.

[0073] The results showed that the expression of underground bZIP65 was significantly upregulated under low nitrogen conditions, indicating that underground bZIP65 was significantly induced under low nitrogen conditions (see Appendix Figure 4 ), thus affecting the nitrogen absorption of rice.

[0074] Example 5

[0075] Comparison of nitrogen use efficiency between wild-type Songjing 2 (SJ2) and bZIP65 overexpressing plants at the seedling stage in hydroponics

[0076] The wild-type Songjing 2 (SJ2) and bZIP65 overexpressing plants were placed under high and low nitrogen hydroponic conditions (see Example 4), and the growth status, chlorophyll content, photosynthetic rate and biomass of the wild-type Songjing 2 (SJ2) and bZIP65 overexpressing plants were measured.

[0077] The results showed that under low nitrogen conditions, the bZIP65 overexpressing plants showed better growth, higher chlorophyll content, photosynthetic rate and biomass than the wild type Songjing 2 (SJ2) (see Appendix). Figure 5 ).

[0078] Example 6

[0079] Comparison of agronomic traits between wild-type Songjing 2 (SJ2) and bZIP65 overexpressing lines under low nitrogen (LN) and high nitrogen (HN) field conditions

[0080] The obtained wild type Songjing 2 (SJ2) and bZIP65 overexpression lines OE-bZIP65-1 and OE-bZIP65-2 were planted in the experimental field of Fuyang Base of China National Rice Research Institute, respectively. Single plants were planted in low nitrogen field (LN: nitrogen fertilizer application rate 10kg / mu) and high nitrogen field (HN: nitrogen fertilizer application rate 30kg / mu), with row spacing of 19.8cm and plant spacing of 16.5cm. All field experiments were uniformly managed in accordance with normal field production methods. Yield trait investigation: After the seeds matured, 15 to 20 plants in the middle of the inner row were taken as the investigation objects, and 10 times were repeated. The number of tillers per plant, the number of grains per panicle, the yield per plant and the nitrogen use efficiency were investigated.

[0081] The results showed that, whether under low nitrogen (LN) or high nitrogen (HN) conditions, the bZIP65 overexpression line showed significant increases in plant type, tillering number per plant, grain number per panicle, yield per plant, and nitrogen use efficiency compared with the wild type SJ2, and the performance was more obvious under low nitrogen (LN) conditions (see Appendix). Figure 6 ).

[0082] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0083] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of rice transcription factor bZIP65 in plant breeding, characterized in that: The application is any of the following: A. Improve the nitrogen utilization efficiency of plants; B. Increase the number of effective tillers; C. Increase the number of grains per ear; D. Increase production; E. Increase photosynthetic rate; The amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.

2.

2. The use of the rice transcription factor bZIP65 in plant breeding according to claim 1, characterized in that: The CDS nucleotide sequence of the rice transcription factor bZIP65 is shown in SEQ ID NO.

1.

3. Application of biological materials for increasing the expression of rice transcription factor bZIP65, characterized in that: The biological material is any one of the following: A. an overexpression cassette comprising the transcription factor bZIP65; B. a recombinant vector containing the overexpression cassette described in A; C. A recombinant microorganism containing the overexpression cassette described in A or the recombinant vector described in B; The amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.

2.

4. The use of the biomaterial for increasing the expression of rice transcription factor bZIP65 according to claim 3, characterized in that: The CDS nucleotide sequence of the rice transcription factor bZIP65 is shown in SEQ ID NO.

1.

5. The use of the biomaterial for increasing the expression of rice transcription factor bZIP65 according to claim 3 or 4, characterized in that: The application is any of the following: A. Improve the nitrogen utilization efficiency of plants; B. Increase the number of effective tillers; C. Increase the number of grains per ear; D. Increase production; E. Increase the photosynthetic rate.

6. A method for regulating plant nitrogen utilization, characterized in that: The expression level of transcription factor bZIP65 is increased by genetic engineering, and the transformed plant cells are then cultured into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.

2.

7. A method for increasing plant yield, characterized in that: The expression level of transcription factor bZIP65 is increased by genetic engineering, and the transformed plant cells are then cultured into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.

2.

8. A method for increasing the number of effective tillers of a plant, characterized in that: The expression level of transcription factor bZIP65 is increased by genetic engineering, and the transformed plant cells are then cultured into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.

2.

9. A method for increasing the number of grains per ear of a plant, characterized in that: The expression level of transcription factor bZIP65 is increased by genetic engineering, and the transformed plant cells are then cultured into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.

2.

10. A method for improving plant photosynthesis, characterized in that: The expression level of transcription factor bZIP65 is increased by genetic engineering, and the transformed plant cells are then cultured into plants; the amino acid sequence encoded by the rice transcription factor bZIP65 gene is shown in SEQ ID NO.2.

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