Application of GE gene in regulation and control of quantity of regenerated buds of rice callus

Abstract

The invention discloses application of a GE gene in regulating and controlling the number of regenerated buds of rice callus, and belongs to the technical field of plant genetic engineering. The invention finds a key regulatory factor GE for regulating and controlling the regeneration of the rice callus. Through construction of the GE knockout mutant and the overexpression strain, compared with a wild type, the quantity of callus regeneration buds of the GE knockout mutant is remarkably reduced, and the quantity of callus regeneration buds of the overexpression strain is remarkably increased. Therefore, it is determined that the GE gene plays an important role in regulating and controlling the regeneration process of the callus. Therefore, the regeneration capacity of the callus can be improved by overexpressing the GE gene, so that the transgenosis efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and specifically to a GE Application of genes in regulating the number of regenerated shoots from rice callus. Background Art

[0002] Cultivated rice in Asia is divided into two subspecies: indica and japonica. Indica is primarily found in tropical and subtropical regions, while japonica is found across diverse latitudes. In rice genetic transformation, significant differences exist between indica and japonica. Most japonica varieties have high regeneration efficiency and are less susceptible to browning, making transgenic technology easier. However, many indica varieties face difficulties in regeneration or experience severe browning of the explants during regeneration, significantly hindering transgenic indica varieties. Therefore, improving the regeneration efficiency of rice, particularly indica, is crucial.

[0003] Rice callus formation and regeneration is a complex biological process regulated by multiple genes. Quantitative Trait Loci (QTL) or Genome-wide Association Study (GWAS) is a good method to study callus formation and regeneration. In recent years, a large number of QTLs related to rice callus formation and regeneration have been reported, but few genes have been cloned. OsNiR It plays an important role in the regeneration of rice callus. OsNiR Encodes ferredoxin nitrite reductase, which catalyzes the reduction of nitrite to ammonium and is a key enzyme in nitrate assimilation (Nishimura et al., 2005; Hellens et al., 2000). It can promptly remove nitrite and reduce its toxicity to cells. BOC1 Upregulation of gene expression can slow down callus browning and improve rice genetic transformation efficiency (Zhang et al., 2020).

[0004] Because indica rice transformation is highly genotype-dependent, identifying key factors regulating regeneration and browning is crucial. This work will help fundamentally address the significant variability in transgenic efficiency in rice and provide valuable guidance for improving the efficiency of transgenic grasses. Summary of the Invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a GE The application of genes in regulating the number of regenerated buds from rice callus tissue can improve the regeneration ability of rice callus tissue and thus improve the efficiency of rice genetic modification.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: provide a GEApplication of genes in regulating the number of regenerated shoots from rice callus.

[0007] further, GE The nucleotide sequence of the gene CDS region is shown in SEQ ID NO.1.

[0008] further, GE The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0009] Furthermore, the number of regenerated shoots from rice callus was regulated by knockout GE The number of regenerated shoots in rice callus tissue was reduced after gene addition.

[0010] Furthermore, regulating the number of regenerated shoots from rice callus is an important method for overexpression GE The number of regenerated shoots from rice callus increased after gene expression.

[0011] The present invention also provides a preparation for regulating the number of regenerated buds of rice callus, the preparation comprising the above GE Gene or the above GE Gene-encoded protein.

[0012] The present invention has the following beneficial effects: In the early stage of the present invention, the important sites involved in rice callus regeneration were located by RIL populations of NIP and HHZ, and candidate genes within the sites were screened and found. GE Genes play an important role in regulating callus regeneration. GE Knockout mutants and overexpression lines showed that compared with wild type, GE The number of regenerated shoots in the knockout mutant callus was significantly reduced. GE The number of regenerated shoots in the callus of the overexpression line increased significantly. GE Genes play an important role in regulating callus regeneration. GE The invention provides a new solution to the problem of significant differences in rice transgenic efficiency and has important guiding significance for improving the transgenic efficiency of gramineous crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the vector map of MH-OsR41 and pUN1301-GFP vector; Figure 2 for GE Identification diagram of the mutation type and callus regeneration phenotype of the knockout mutant; Figure A shows ... GESequencing results of knockout strains; Figure B shows the regeneration phenotypes of mutant strains and wild-type NIP under the NIP background; Figure C shows the number of regenerated buds in Figure B; Figure D shows the regeneration rate in Figure B; Figure 3 for GE Expression level detection and regeneration phenotype identification of overexpression lines; Figure A shows the regeneration phenotype of overexpression lines and wild-type NIP callus under NIP background; Figure B shows the regeneration phenotype of overexpression lines in leaves under NIP background. GE Relative expression level; Figure C shows the expression of the overexpression strain in callus under NIP background. GE Relative expression level; Figure D is a statistical diagram of the number of regenerated buds from callus tissue in Figure A. DETAILED DESCRIPTION

[0014] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0015] 1. Experimental Materials (1) Plant material: wild-type NIP; (2) MH-OsR41 and pUN1301-GFP vectors (see vector map for details) Figure 1 ) is given as a gift by another person; (3) N6D medium: N6 medium containing 600 mg / L hydrolyzed casein, 500 mg / L proline, 500 mg / L glutamine, 100 mg / L inositol, 30 g / L sucrose, 2 mg / L 2,4-dichlorophenoxyacetic acid, and 3 g / L Phytagel; Callus screening medium: N6D medium containing 50 mg / L hygromycin and 400 mg / L timentin.

[0016] Regeneration screening medium: MS medium containing 2 g / L hydrolyzed casein, 50 mg / L hygromycin, 2 mg / L kinetin (KT), 0.2 mg / L naphthaleneacetic acid, 30 g / L sucrose, 20 g / L sorbitol and 3 g / L Phytagel.

[0017] (4) DNA lysis buffer: The components are shown in Table 1.

[0018] Table 1 Composition of DNA lysis buffer

[0019] 2. Experimental Methods (1) Plant DNA extraction 2 cm rice leaf tissue was cut, frozen in liquid nitrogen and then fully ground. 500 μL DNA lysis buffer was added and the mixture was shaken and then 12,000 g Centrifuge for 15 min; aspirate the supernatant and add an equal volume of isopropanol, mix well, and then g Centrifuge for 15 min, discard the supernatant and dry it, then add 100 μL of double-distilled water to dissolve it.

[0020] (2) PCR amplification The PCR amplification reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0021] Table 2 Reaction system (50 μL)

[0022] Table 3 Reaction procedure

[0023] (3) Plant total RNA extraction and reverse transcription ① RNA was extracted using the Omega kit. The specific steps were as follows: 3 pieces of callus tissue were quickly placed in a 2 mL grinding centrifuge tube and quickly frozen in liquid nitrogen. Then, the cells were fully ground in a cryogenic grinding instrument and 500 μL RNA Binding Buffer (RB) solution was added. After oscillation and mixing, 14,000 μL of RB solution was added. g Centrifuge for 1 min, transfer the lysate to a 2 mL collection tube fitted with a filter column, and incubate at room temperature at 14,000 g Centrifuge for 5 min, transfer the filtrate to a new 1.5 mL centrifuge tube, add 0.5 times the volume of ethanol, and mix well; draw the mixed solution into the adsorption column (the adsorption column is placed in the collection tube), 12,000 g Centrifuge for 1 min, remove the filtrate, add 400 μL of washing solution (RWF), 10,000 g Centrifuge for 30 s, remove the filtrate, and then add 500 μL of Wash Buffer II, 10,000 g Centrifuge for 30 s and repeat the washing process; place the adsorption column into the collection tube at 14,000 g Centrifuge for 2 min to remove residual liquid; place the adsorption column in a new EP tube, add 50 μL double distilled water (RNAase-free), 12,000 g RNA was collected after centrifugation for 1 min; ② A Takara reverse transcription kit was used for reverse transcription. The specific operation was as follows: according to the extracted RNA concentration, an appropriate amount of RNA was added, 1 μL of gDNA digester and 2 μL of 5× gDNA digester buffer were added, and double-distilled water (RNAase-free) was added to make up to 10 μL, and mixed; the reaction was carried out at 42°C for 2 min; then 4 μL of 5× buffer, 4 μL of double-distilled water (RNAase-free), 1 μL of RT Enzyme Mix, and 1 μL of RT Primer Mix were added and mixed; then the reaction was carried out at 37°C for 15 min, 85°C for 5 s, and 4°C for 2 min.

[0024] (4) Real-time fluorescence quantitative PCR Design primers according to the target gene; dilute the synthesized cDNA stock solution 10 times and set aside; prepare 15 μL reaction system, the reaction system and reaction procedure are shown in Table 4 and Table 5 respectively, and then use rice ACTIN3 The ΔΔCt method was used to calculate the relative expression of genes.

[0025] Table 4 Reaction system (15 μL)

[0026] Table 5 Reaction procedure

[0027] Example 1: Transgenic vector pCRISPR- GE and pUN1301- GE Construction (1) Knockout vector pCRISPR- GE Construction ① GE Target sequence of the knockout vector: 5′-CCTCTTCCTCCTCCCGACGT-3′ (SEQ ID NO. 3); ②Synthetic single-strand F1: 5'-TGCACCTCTTCCTCCTCCCGACGT-3' (SEQ ID NO. 4); Synthetic single-strand R1: 5′-AAACACGTCGGGAGGAGGAAGAGG-3′ (SEQ ID NO. 5); ③Add 30 μL F1 (10 μM) and 30 μL R1 (10 μM) to the single-stranded tube, mix well, and place in a PCR instrument at 98°C for 5 minutes; ④MH-OsR41 plasmid digestion: 5 μL 10× Cutsmart, 1 μL BsaI, 1 μg plasmid, and double-distilled water to make up to 50 μL; ⑤ Gel recovery: Gel recovery was performed using a Thermo Scientific gel recovery kit. The specific steps were as follows: After the nucleic acid electrophoresis was completed, the gel of the target band was cut using a gel cutter, an appropriate amount of Binding Buffer was added, and the gel was dissolved at 55°C. The completely melted gel solution was transferred to an adsorption column (the adsorption column was placed in a collection tube) at 12,000 °C. g Centrifuge for 1 min; remove the filtrate and add 650 μL Wash Buffer, 12,000 g Centrifuge for 1 min and remove the filtrate; then put the adsorption column back into the collection tube and g Centrifuge for 2 min to completely remove the residual liquid; place the adsorption column in a new EP tube, add 50 μL double distilled water and g Centrifuge for 1 min to collect the nucleic acid solution; ⑥T4 ligase recombination ligation: 50 ng vector, 1 μL T4 ligase, 2 μL T4 Buffer, 6 μL template DNA, and double-distilled water to 20 μL; react at 25°C for 1 h; then transform the ligation product into DH5α competent culture, spread on LB medium with corresponding resistance, and culture at 37°C; select the strain with correct sequencing and save it for future use.

[0028] (2) Overexpression vector pUN1301- GE Construction The overexpression vector was constructed using homologous recombination. The primer pairs F2 and R2 were designed and synthesized based on the recombination ligation method. The rice cDNA was used as a template for PCR amplification. The plasmid pUN1301 was double-digested with restriction endonucleases KpnI and BamHI. The digestion product was recovered and purified by agarose gel electrophoresis. The purified PCR product and the linearized vector plasmid were recombinantly ligated using a recombination ligation kit. The resulting ligation product was transformed into Escherichia coli DH5α competent cells, and positive clones were obtained by screening on a resistance plate containing kanamycin. The recombinant plasmids in the positive clones were extracted and sequenced for verification. The vector plasmid with the correct sequence was named pUN1301- GE The nucleotide sequences of F2 and R2 are as follows: F2: 5'-GGATGACGACGATAAGggtaccATGGCGCTCTCCTCCATGGC-3' (SEQ ID NO. 6); R2: 5'-CAGTCTAGAGTCGACggatccTCAGGCCCTAGCCACGGCCTT-3' (SEQ ID NO. 7).

[0029] Example 2: GEObtaining genetically modified rice (1) The above pCRISPR- GE and pUN1301- GE The plasmid was transformed into Agrobacterium EHA105; (2) Shake the Agrobacterium culture solution to OD 600 The bacterial cell was resuspended in AAM medium and infect NIP callus for 20 minutes. After the bacterial solution was dried with filter paper, the callus was placed on N6-AS transformation medium containing a layer of filter paper and kept in the dark for 2 days. The infected callus was then washed 5 times with sterile water containing 400 mg / L timentin. The excess water was dried with sterile filter paper and the callus was blown in a clean bench for 60 minutes. Then, the callus was transferred to callus screening medium and cultured for 28 days. (3) Take the actively growing positive callus tissue and transfer it to the differentiation medium for 21 days. Then transfer the positive seedlings to the rooting medium for rooting. The culture conditions are: 12 h light / 12 h dark; light intensity is 8000 lux; temperature is 28°C. (4) When the seedlings grow to about 10 cm, open the container sealing film, harden the seedlings for 3 days, and then move the seedlings into the artificial climate chamber for cultivation.

[0030] Example 3: GE Identification of knockout plant callus (1) Callus culture: The parameters of light-dark alternating culture are as follows: light intensity is 120 μmol·m -2 ·s -1 , the temperature was 28°C, the photoperiod was 16 h light / 8 h dark, and the specific operation was as follows: ① Mature rice seeds were hulled and sterilized, treated with 75wt% alcohol for 1 min, then treated with 20wt% NaClO solution for 30 min, and then rinsed with sterile water five times; ② After drying the rice seeds from step ①, place them on callus induction medium for 14 days; ③ Remove excess bud tissue and endosperm from the rice seeds in step ②, transfer the callus tissue to the regeneration medium and induce it for 25 days, then take pictures and count the number of regenerated buds in the callus tissue.

[0031] (2) GE Sequencing identification of knockout plants: primers F3 and R3 were used to identify knockout plants in the wild-type NIP background. GE The knockout plants were PCR amplified and sequenced for identification; the nucleotide sequences of F3 and R3 are shown below: F3: 5'-ACGTTCTCTCCTCCCAAGA-3' (SEQ ID NO. 8); R3: 5'-GGCTGACGAGGATCTCCCTC-3' (SEQ ID NO. 9).

[0032] Depend on Figure 2 As shown in Figure A, three mutation forms were found through sequencing and were named ge-1 , ge-2 and ge-3 .in, ge-1 An A base was inserted at 60 bp, causing a frame shift and premature termination of translation after the 20th amino acid; ge-2 A 2-base deletion (GA) at 60 bp resulted in a frameshift and premature termination of protein translation; ge-3 The deletion of 3 bases (ACG) at 60 bp resulted in the deletion of a threonine (Thr). Figure 2 As shown in Figure BC, compared with the wild-type NIP, the knockout mutant ge-1 、 ge-2 and ge-3 The number of regenerated buds from callus tissue was significantly reduced. Figure 2 As shown in Figure D, compared with the wild-type NIP, the knockout mutant ge-1、ge-2 and ge-3 The callus regeneration rates were reduced by 46.52%, 62.43%, and 21.7%, respectively (data are mean ± SD of three biological replicates, n = 40 for each replicate, one-way ANOVA was used for significance test, * indicates P < 0.05, **** indicates P < 0.0001).

[0033] Example 4: GE Identification of callus tissue of overexpression lines (1) The callus tissue culture method refers to step (1) of Example 3.

[0034] (2) Real-time fluorescence quantitative PCR: Transgenic lines overexpressing NIP in the background OE1 and OE2 Total RNA was extracted from T2 seedlings and callus tissues and reverse transcribed to synthesize cDNA. The reverse transcription product was diluted 10 times, and 2 μL was used as a template for real-time fluorescence quantitative PCR identification.

[0035] Primer F4: 5′-GACTCCGACATGATCGCTGTTCTT-3′ (SEQ ID NO. 10); Primer R4: 5′-ATCACCCACTCCATCAAGATCGCC-3′ (SEQ ID NO. 11).

[0036] Depend on Figure 3 As shown in Figures AC, the overexpression strain seedlings OE1 and OE2 middle GE The relative expression levels of GE in OE1 and OE2 calli were 40 and 60 times higher than those in NIP, respectively. The relative expression levels of GE in OE1 and OE2 calli were 4.7 and 4.1 times higher than those in NIP calli, respectively. Figure 3 As shown in Figure D, the number of regenerated shoots in OE1 and OE2 calli increased by 44.4% and 27.1%, respectively, compared with that in NIP calli (data are mean ± SD of three biological replicates, n = 20 per replicate, one-way ANOVA was used for significance testing, * indicates P < 0.05, ** indicates P < 0.01).

[0037] The above results showed that compared with the wild type, GE The regeneration rate and number of regenerated shoots of the mutant lines were significantly reduced; GE The number of regenerated shoots from callus of overexpression lines increased significantly, indicating that GE Genes play an important role in the regeneration process of rice callus.

[0038] In the present invention GE The nucleotide sequence of the CDS region of the gene and the amino acid sequence of its encoded protein are as follows: (2) Amino acid sequence of the encoded protein: (SEQ ID NO.2).

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. GE Application of genes in regulating the number of regenerated shoots from rice callus.

2. The use according to claim 1, characterized in that described GE The nucleotide sequence of the gene CDS region is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that described GE The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

4. The use according to claim 1, characterized in that The regulation of the number of regenerated shoots in rice callus is a knockout GE The number of regenerated shoots in rice callus tissue was reduced after gene addition.

5. The use according to claim 1, characterized in that The regulation of the number of regenerated shoots from rice callus is an overexpression GE The number of regenerated shoots from rice callus increased after gene expression.

6. A preparation for regulating the number of regenerated buds from rice callus, characterized in that: The preparation comprises the GE The gene or the one according to claim 2 GE Gene-encoded protein.

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