Rice low temperature germination gene nac23 and application thereof

Abstract

This invention discloses a rice low-temperature germination gene NAC23 and its applications, relating to the field of agricultural biotechnology. The transgenic plants containing the NAC23 gene constructed in this invention improve the low-temperature germination rate of rice seeds. Furthermore, through functional interpretation of the NAC23 gene, the genetic mechanism of low-temperature germination in rice is further elucidated, laying the foundation for improving low-temperature germination and sowing methods in rice.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and more specifically to the rice low-temperature germination gene NAC23 and its applications. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important cereal crops. Currently, rice cultivation in my country is mainly based on transplanting. In recent years, with the continuous rise in rural labor costs, the increasing scarcity of freshwater resources, and the growing severity of environmental problems, rice production is facing a transformation from traditional small-scale production to modern large-scale production methods characterized by mechanization, intelligence, standardization, and intensification. Direct-seeded rice, with its advantages of saving labor and time and high water and fertilizer utilization rates, is receiving increasing attention.

[0003] However, for a long time, the rice varieties cultivated in my country have been based on the seedling transplanting method. Directly converting existing varieties to direct seeding has significant drawbacks, primarily in their tolerance to low temperatures and waterlogging during seed germination. Rice is a warm-season crop and is highly sensitive to low temperatures during germination. Direct-seeded rice generally requires a germination rate of over 80% at 11-13℃, while most existing varieties have a germination rate of less than 50% at low temperatures. Currently, seed rot, uneven emergence, and drastic yield reduction caused by low temperatures during germination have become common factors limiting the development and application of direct-seeded rice worldwide.

[0004] Although numerous QTLs for low-temperature germination in rice have been successfully identified by previous researchers, only a few genes exhibiting tolerance to low-temperature germination have been cloned, and their molecular regulatory networks are incomplete. Currently, the major genes identified in rice that exhibit tolerance to low-temperature germination include qLTG3-1, OsSAP16, GF14h, and OsUBC12. While these functional genes have been cloned and provide valuable genetic resources for direct-seeding rice breeding, due to the complexity of the traits and limitations in multi-gene manipulation techniques, only a very small number of these genes have been successfully applied to molecular design breeding.

[0005] Therefore, conducting research on the mechanism of regulating rice seed germination at low temperatures can provide important theoretical basis and excellent gene resources for the breeding of high-vibration direct-seeded rice varieties, and also provide a reference for the molecular breeding of other gramineous crops. Summary of the Invention

[0006] In view of this, the present invention provides a rice low-temperature germination-related gene NAC23, its encoded protein, and its applications. NAC23 belongs to the NAC transcription factor family, and changes in its single-gene expression level alter the low-temperature germination ability of rice, exhibiting significant genetic effects. This provides new gene resources for rice low-temperature germination and molecular design breeding, laying the foundation for improving rice cultivation from traditional transplanting to direct seeding.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A rice low-temperature germination gene NAC23, wherein the NAC23 gene has the nucleotide sequence shown in SEQ ID NO.1.

[0009] Preferably, a sequence that encodes a protein having the same function is generated by adding and / or substituting and / or deleting one or more nucleotides in the nucleotide sequence defined in SEQ ID NO.1.

[0010] Another object of the present invention is to provide a rice low-temperature germination protein NAC23, wherein the NAC23 protein has the amino acid sequence shown in SEQ ID NO.2.

[0011] Preferably, a protein derived from (A) with one or more amino acids added and / or substituted and / or deleted in the amino acid sequence defined in (5) and having equivalent function.

[0012] Another object of the present invention is to provide a biomaterial that reduces the expression level of the NAC23 gene, wherein the biomaterial is any one of the following:

[0013] A. An expression cassette capable of suppressing the NAC23 gene;

[0014] B. A recombinant vector containing the expression cassette described in A;

[0015] C. Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

[0016] Preferably, the expression cassette is Cas9Pubi-H-NAC23 or Cas9Pubi-B-NAC23. The recombinant microorganism can be understood as the engineered bacteria or host cell used by those skilled in the art in the transgenic process. However, with technological advancements, the selection of the engineered bacteria and host cell may change, or the use of vectors and engineered bacteria may also be involved in non-transgenic applications. But as long as it contains the gene or vector described in this invention, it is within the scope of protection of this invention.

[0017] Another object of the present invention is to provide an application of reducing the expression level of the aforementioned NAC23 gene in rice breeding, wherein the application is to cultivate low-temperature resistant germination rice germplasm. Preferably, it is used as a screening marker in the rice breeding process for screening low-temperature resistant germplasm resources.

[0018] Another object of the present invention is to provide the application of inhibiting the activity of the aforementioned NAC23 protein in rice breeding, wherein the application is to cultivate low-temperature resistant germination rice germplasm. Preferably, it is used as a screening marker in the rice breeding process for screening low-temperature resistant germplasm resources.

[0019] Another objective of this invention is to provide the application of the aforementioned biomaterials that reduce NAC23 gene expression in rice breeding, specifically for cultivating low-temperature resistant germination rice germplasm. Preferably, these materials are used as screening markers in the rice breeding process for screening low-temperature resistant germplasm resources.

[0020] Another objective of this invention is to provide a method for regulating the low-temperature germination of grass plants by reducing the expression level of the aforementioned genes or inhibiting the activity of the aforementioned proteins through genetic engineering, and then cultivating the transformed grass plant cells into plants.

[0021] Preferably, the grass plant is rice.

[0022] Beneficial Effects: This invention discloses a rice low-temperature germination tolerance gene NAC23, its protein, and its applications. Through functional interpretation of the NAC23 gene, this invention further elucidates the genetic mechanism of low-temperature germination tolerance in rice, laying the foundation for improving low-temperature germination and sowing methods in rice. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The image of the pU1301 plasmid, the overexpression vector used in this invention.

[0025] Figure 2 The images show the knockout CRISPR / Cas vectors used in this invention; where A is pYLCRISPR / Cas9Pubi-H and B is the pYLCRISPR / Cas9Pubi-B plasmid map.

[0026] Figure 3The image shows the identification of NAC23 transgenic plants; where A is the identification site of the knockout line; B is the relative expression level of the NAC23 gene in the overexpression line; ns represents no statistically significant difference between the two groups, and * represents a statistically significant difference between the two groups (*p<0.05, **p<0.01, T-test).

[0027] Figure 4 This is a diagram showing the gene expression pattern of the low-temperature germination gene NAC23 under low-temperature conditions for 48 hours; where LT represents the low-temperature condition of 15℃ and CK represents the normal condition of 28℃.

[0028] Figure 5 Germination phenotypes of wild-type NIP, knockout line CrNAC23, and overexpression line OxNAC23 under low temperature conditions; where A represents the statistical differences in phenotypes among the materials, and B represents the germination criteria.

[0029] Figure 6 The graph shows the difference in germination rates of wild-type NIP, knockout line CrNAC23, and overexpression line OxNAC23 under low temperature conditions; * indicates statistical significance between the two groups (*p<0.05, **p<0.01, T-test). Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] Construction of rice NAC23 gene overexpression vector

[0033] 1. Amplification of the target fragment: Primers were designed based on the NAC23 gene sequence (as shown in SEQ ID NO.1) published on the Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ). Using wild-type Nipponbare (NIP) cDNA as a template, KpnI and BamHI restriction enzyme sites were designed on the primers to amplify the full-length cDNA of the target gene from the start codon to the stop codon, which is 759 bp.

[0034] OxNAC23-F:5'-cgcggatccATGGCGATGACACCGCA-3', SEQ ID NO.3;

[0035] OxNAC23-R: 5’-aaaactgcagcTAGCCACCATGGTTTCT-3’, SEQ ID NO.4.

[0036] cDNA sequence of NAC23 gene:

[0037] ATGGCGATGACACCGCAGCTAGCATTTTCTCGCATGCCTCCAGGGTTTCGGTTCCAGCCGACGGACGAGCAGCTTGTCGTCGACTACTTGCAGAGGCGTACCGCTGCGCAGCCATGCGTTACTCCCGACATCACCGATATCGACGTTTACAACGTCGACCCGTGGCAGCTTCCAGCCATGGCGATGTATGGATCGGATCATGACCGGTACTTCTTCACGATGGCGGCCCGAGAGGCGCAGGCCAGACGAACGACACCGTCGGGTTTCTGGAAGCCCACCGGCACAAAGAAGACGATCTTCGTCGTCGCCGGTGGGCATGAGGTGCCCACCGCCGTCAAGAGGAGGTTCGTCTTCTACCTCGGCCACCACCAACCATCGGGCAGCAACAACAACAACAAAACATCATGGATCATGCATGAGTACCGTCTCATGAACTCTCCAAGAGCGGCAGTGCCGTCGTCTTCTTCGGTGAATCGTCTTCCCACTGATGATCTCACGGAAGAGATGGTGCTGTGTAGGATCTCCAACAAGGACCTGCCTAAACCACCCTTCATCCACAACAGCTTGTTGCAGTTCTCTTCAGTGGGGTTGAATGGTGATGGGTATAATTACTTGATCCTTGATCACCTTGAGCCTCCAGCAATGGAGTATCCTAATGTTGGCATTGGTAATGTTGATGATGCTGCTGCTGGTACTGATGATCCGGGTGACCTTGATGAGGAGATTGATGATAGCATGCAAAGAAACCATGGTGGCTAG, SEQ ID NO.1.

[0038] Amino acid sequence of NAC23 protein:

[0039] MAMTPQLAFSRMPPGFRFQPTDEQLVVDYLQRRTAAQPCVTPDITDIDVYNVDPWQLPAMAMYGSDHDRYFFTMAAREAQARRTTPSGFWKPTGTKKTIFVVAGGHEVPTAVKRRFVFYLGHHQPSGS NNNNKTSWIMHEYRLMNSPRAAVPSSSSVNRLPTDDDLTEEMVLCRISNKDLPKPPFIHNSLLQFSSVGLNGDGYNYLILDHLEPPAMEYPNVGIGNVDDAAAGTDDPGDLDEEIDDSMQRNHGG, SEQ ID NO.2.

[0040] 2. Overexpression vector construction: The expression vector pU1301 was double-digested with KpnI and BamHI (see Appendix). Figure 1 The target gene fragment was recovered, and the product was ligated overnight at 16°C using T4 ligase. The ligation was then performed on E. coli DH5α competent cells. After single clones grew, positive bacteria were identified and screened. Plasmids were extracted, and after correct sequencing, the pU1301-Ubiquitin-NAC23 overexpression vector was obtained.

[0041] Example 2

[0042] Construction of rice NAC23 gene knockout vector

[0043] 1. Construction of the knockout vector: Based on the principles of the CRISPR / Cas9 system, the online gene knockout target site and primer design website CRISPR-GE (http: / / skl.scau.edu.cn / home / ) was used to select target sites and design primers on the NAC23 gene. Two different CRISPR / Cas9 vectors, pYLCRISPR / Cas9Pubi-H and pYLCRISPR / Cas9Pubi-B (see Appendix) were constructed. Figure 2 The target gene fragment was introduced into an sgRNA expression cassette initiated by OsU3 through two rounds of PCR amplification, and finally ligated into the CRISPR / Cas9 binary vectors pYLCRISPR / Cas9Pubi-H and pYLCRISPR / Cas9Pubi-B initiated by the maize ubiquitin promoter via a cleavage-while-ligating method. After successful sequencing, the knockout vectors Cas9Pubi-H-NAC23 and Cas9Pubi-B-NAC23 were obtained. The pYLCRISPR / Cas9Pubi-H vector contains a hygromycin resistance selection gene, and the pYLCRISPR / Cas9Pubi-B vector contains a glufosinate resistance selection gene.

[0044] OsNAC23-U3-F: 5'-ggcaTGCGAGAAAATGCTAGCTG-3', SEQ ID NO.5;

[0045] OsNAC23-U3-R: 5'-aaacCAGCTAGCATTTTCTCGCA-3', SEQ ID NO. 6.

[0046] Example 3

[0047] Construction of transgenic plants

[0048] 1. Genetic transformation of rice callus: 10 μL of pU1301-Ubiquitin-NAC23, Cas9Pubi-H-NAC23, and Cas9Pubi-B-NAC23 plasmids were added to Agrobacterium EHA105 chemocompetent cells, respectively. The cells were gently mixed by pipetting, incubated on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, and then incubated in a 37℃ water bath for 5 min, followed immediately on ice for 3–5 min. 800 μL of antibiotic-free LB medium was added to the competent cells, and the cells were incubated at 28℃ and 120 rpm for 3–4 hours. After centrifugation at 8000 rpm for 1 min, the supernatant was discarded, and the remaining 100 μL was spread onto LB agar plates of the corresponding antibiotic resistance. The plates were incubated upside down at 28℃ for 2–3 days. Positive engineered bacteria were screened and identified after single bacterial colonies grew.

[0049] 2. Finally, T0 generation transgenic plants were obtained by transforming Nipponbare callus tissue using Agrobacterium-mediated transformation, and the expression level of the NAC23 gene was analyzed (see Appendix). Figure 3 ).

[0050] The primer sequences for amplification and sequencing to identify the OxNAC23 sequence are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0051] The primer sequences for amplification and sequencing to identify the CrNAC23 sequence are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0052] Note: All culture media (induction medium, screening medium, differentiation medium) involved in this embodiment are conventional culture media.

[0053] The results showed that, compared with the wild type, the expression level of NAC23 gene in the overexpression transgenic plants was significantly increased, while the expression level of NAC23 gene in the knockout transgenic plants remained unchanged. Further sequencing verified the homozygosity of the knockout transgenic plants.

[0054] Example 4

[0055] Identification of NAC23 gene expression pattern

[0056] 1. Seed RNA Extraction: Plump and healthy NIP seeds were selected and germinated under 15℃ low-temperature conditions and 28℃ normal conditions. Samples were taken at 0, 3, 6, 9, 12, 24, and 48 h, peeled, and placed in RNase-free centrifuge tubes, which were immediately immersed in liquid nitrogen. A steel bead was added, and the mixture was pulverized with a grinder to form a powder. 300 μL of SDS RNA extraction buffer was added to the pulverized sample, mixed well, and incubated on ice for 5 min. 200 μL of water-saturated phenol:chloroform = 1:100 was added to the centrifuge tube, and the mixture was vortexed for 30 s. The mixture was then centrifuged at 12000 rpm for 10 min at 4℃. 200 μL of the supernatant was transferred to a new centrifuge tube. 1 mL of the supernatant was added to the centrifuge tube. After mixing with Trizol by vortexing, let stand at room temperature for 5 min. Add 200 μL of chloroform, gently invert to mix, and centrifuge at 12000 rpm for 10 min at 4 °C. Transfer 500 μL of the supernatant to a new RNase-free centrifuge tube, add an equal volume of isopropanol to precipitate the RNA, centrifuge at 12000 rpm for 10 min at 4 °C, and discard the supernatant. Wash with 500 μL of 70% anhydrous ethanol, centrifuge at 12000 rpm for 5 min at 4 °C, discard the supernatant, centrifuge again for 1 min, aspirate the remaining liquid with a pipette, allow to evaporate naturally for 1 min, add 30 μL of LEPC in water to dissolve and measure the concentration, then run gel electrophoresis to check the quality. This solution can be used for reverse transcription or frozen directly at -80 °C.

[0057] 2. Reverse transcription reaction: The kits used for the reverse transcription reaction were from Shanghai Yisheng Biotechnology Co., Ltd. AdwanceFast 1st Strand cDNA. The specific steps are as follows: Mix 2 μg total RNA with 2 μL of 5×g DNA Digester Mix, add RNase-free H2O to a final volume of 10 μL, mix gently, and incubate at 42°C for 3 min. Then add 5 μL of 4×Hifair AdvanceFast SuperMix, 2 μL of Random Primers (for qPCR), and RNase-free H2O to a final volume of 20 μL. Incubate at 55°C for 5 min, then at 85°C for 5 s. Finally, dilute to 80 μL with RNase-free H2O for subsequent molecular experiments or store at -20°C.

[0058] 3. Real-time 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).

[0059] The primer sequences for quantitative detection of NAC23 expression are as follows:

[0060] NAC23-qRT F: 5'-TTCGGTGAATCGTCTTCCCA-3', SEQ ID NO.7;

[0061] NAC23-qRT R: 5'-TAGGCAGGTCCTTGTTGGAG-3', SEQ ID NO. 8.

[0062] The internal control quantitative primers are:

[0063] Ubi-qRT F:5'-TACCGTGCCCTTACTGTTC-3', SEQ ID NO.9;

[0064] Ubi-qRT R: 5'-CGGTGGAATGTCACAGACAC-3', SEQ ID NO. 10.

[0065] Amplification reaction system (10 μL): 2×SYBR GreenMasterMix 5 μL, Forwardprimer 1 μL, Reverse primer 1 μL, cDNA 1 μL and ddH2O 2 μL.

[0066] Amplification program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 15 s (40 cycles); 65-95℃ (melting curve) 0.5℃ / 0.5 s. Fluorescence signal was acquired during the extension phase. The final results were exported in Excel format and analyzed using 2... -△△CT The relative expression level was calculated using the method with three biological replicates per sample (see Appendix). Figure 4 Under normal conditions, the expression level of NAC23 did not change significantly, but under low temperature conditions, it showed a trend of first decreasing and then increasing, indicating that NAC23 was significantly induced under low temperature conditions, thus affecting seed germination at low temperatures.

[0067] Example 5

[0068] Verification of low-temperature germination tolerance of seeds from knockout and overexpression lines

[0069] Thirty seeds each from plump and healthy knockout and overexpression lines were selected. Dormancy was broken by soaking in 1% dilute nitric acid for 16 hours, followed by soaking in 75% ethanol for 1 minute. Then, the seeds were sterilized with 2.5% (v / v) NaClO for half an hour, and washed five times with ddH2O. After cleaning, the seeds were placed in 9 cm petri dishes lined with two layers of filter paper, and 10 mL of distilled water was added. The dishes were then placed in a 15℃ incubator for low-temperature germination treatment, with three replicates (see Appendix). Figure 5-6 ).

[0070] As attached Figure 5 As shown, with the germination standard being that the bud length is greater than half the seed length, the wild-type NIP had 5 ungerminated seeds, the knockout line had only 3 ungerminated seeds, and the overexpression line had 15 ungerminated seeds.

[0071] As attached Figure 6 As shown, the germination rate of wild-type NIP was 76.25%, the germination rate of knockout lines was 92.5%, and the germination rate of overexpression lines was 50%.

[0072] Low-temperature germination rate = (Number of germinated seeds at low temperature / Total number of seeds) / (Number of germinated seeds at room temperature / Total number of seeds)

[0073] Example 6

[0074] Application of the rice low-temperature germination gene NAC23 in rice breeding

[0075] First, in production practice, the aforementioned genes can be transformed into plant cells, and then the transformed plant cells can be cultivated into plants. Through this transgenic method, plant expression vectors can be used to transform plant cells to influence the low-temperature germination of rice seeds, thereby improving the resistance and sowing methods of rice or other gramineous plants. Second, in production practice, the aforementioned genes can also be used through molecular marker-assisted selection breeding methods to improve the yield, quality, and resistance of rice or other gramineous plants.

[0076] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0077] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Knocking out NAC23 application of genes in rice breeding, characterized in that, The application is for cultivating rice germplasm that can germinate at low temperatures. NAC23 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Use of inhibition of NAC23 protein activity in breeding of rice, characterized in that, The application is to cultivate low-temperature germination rice germplasm, and the application NAC23 The amino acid sequence of the protein is shown as SEQ ID NO.

2.

3. Knockout NAC23 The use of biological material of the gene in rice breeding, characterized in that, The NAC23 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the application is for breeding rice germplasm that can germinate at low temperatures; The biomaterial is any one of the following: A. expression cassette capable of knocking out NAC23 a gene; B. A recombinant vector containing the expression cassette described in A; C. Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

4. A method for regulating the low-temperature germination of grass plants, characterized in that, The gene described in claim 1 is knocked out or the activity of the protein described in claim 2 is inhibited by genetic engineering, and then the transformed gramineous plant cells are cultured into plants; the gramineous plant is rice.

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