Rice aminoacyl-trna synthetase encoding gene oshgz2 mutant and application thereof in increasing rice zinc content

Abstract

The application discloses a mutant of a rice aminoacyl-tRNA synthetase gene and application of the mutant in increasing zinc content in grains. OsHGZ2 The mutant is obtained by EMS mutagenesis, and the mutant is verified to have the 319th amino acid varied. OsHGZ2 It is found that the zinc content in grains of the mutant is about twice that of a wild type. OsHGZ2 The application is an excellent gene which can be applied to cultivating high-zinc rice grains. The application has very important significance for improving rice nutritional quality and human dietary health.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the gene encoding rice aminoacyl-tRNA synthetase. OsHGZ2 Mutants and their application in regulating zinc content in grains and in breeding. Background Technology

[0002] Zinc (Zinc, Zn) is an essential micronutrient crucial for both plants and animals. It participates in the formation of various enzymes and proteins within organisms, and is involved in many physiological and biochemical processes, playing an irreplaceable role in maintaining human health. The human body lacks zinc storage organs and must obtain zinc daily from food. However, zinc is primarily found in animal-based foods such as red meat and seafood, with generally low levels in other common foods, often failing to meet the body's normal needs. Nearly two billion people face health risks due to insufficient daily zinc intake, making it a global public health issue. Furthermore, with improved living standards, the nutritional needs of the public for agricultural products have shifted from a general shortage to a significant imbalance in nutritional supply and nutritional security. The low zinc content in agricultural products is a key manifestation of this nutritional imbalance.

[0003] Rice is one of the most important food crops for humankind, with approximately 50% of the world's population relying on it as a staple food. In Asian countries, rice accounts for 35-59% of the body's calories. However, recent surveys have revealed that with the continuous increase in grain consumption, zinc nutrition issues among grain-based populations are becoming increasingly apparent. The "Chinese Dietary Reference Intakes" recommends a daily zinc intake of 12.5 mg for adult men, but the zinc content in edible parts of grain crops and tubers falls far short of this standard. The International Crop Biofortification Project has proposed increasing the zinc content of rice from 16 mg / kg... -1 Increased to 28 mg / kg -1 The goal is to increase the zinc content of edible parts of crops and develop zinc-rich agricultural products to supplement the zinc nutrition needed by the human body through biofortification technology. This is an effective way to address insufficient zinc intake and solve the problem of zinc "hidden hunger".

[0004] Zinc biofortification of agricultural products refers to methods of increasing the zinc content of agricultural products through agronomic measures. Compared to nutritional supplements and food fortification, biofortification of agricultural products through breeding has many advantages, such as lower safety risks, no change in dietary habits, and greater public acceptance. It has been successfully implemented in some countries and regions in Africa, South Asia, and Southeast Asia. Zinc-enriched agricultural products obtained through this method do not require additional financial burdens and have advantages such as universality and sustainability. In 2013, Bangladesh approved its first zinc-enriched biofortified rice variety, "BRRI dhan62," which exhibits extremely early maturity and a grain zinc content of 20 mg / kg. -1Approximately. In addition, new strains such as BR7840-54-3-1, BR7840-54-2-5-1, and BR7840-54-1-2-5 also performed well in small-plot trials, with zinc content increasing by 6-8 mg / kg compared to the control. -1 It has great potential in zinc-enriched rice breeding.

[0005] Aminoacyl-tRNA synthetase is a key enzyme in protein synthesis, catalyzing the linking of amino acids to their corresponding tRNAs. Because its function is relatively conserved and crucial for cell development—gene mutations often have lethal effects—research on how it influences cell development is limited. Recent studies have revealed that aminoacyl-tRNA synthetase has other functions besides catalyzing amino acid-tRNA linking. Human methionine-tRNA synthetase (MetRS) can enter the cell nucleus during cell proliferation. When MetRS concentration decreases, rRNA synthesis is significantly inhibited, indicating that MetRS participates in rRNA synthesis in the cell nucleus. (Rice gene) WP1 (White panicle 1) encodes a valine-tRNA synthetase. WP1 The mutation leads to impaired chloroplast development. wp1 The mutant exhibits white stripes and an albinism phenotype on its leaves during the seedling stage, and the panicles turn white after heading. Mupirocin is a common example of an aminoacyl-tRNA synthetase inhibitor used clinically; it effectively inhibits the activity of leucine-tRNA synthetase in bacteria, thereby suppressing bacterial growth.

[0006] Currently, the target for rice aminoacyl-tRNA synthetase encoding... OsHGZ2 Research on the regulation of rice traits by genes is still lacking. Summary of the Invention

[0007] To address the aforementioned technical problems in the existing technology, this invention provides a mutant of the rice aminoacyl-tRNA synthetase encoding gene OsHGZ2 and its application in increasing the zinc content of rice.

[0008] The first objective of this invention is to provide a rice aminoacyl-tRNA synthetase mutant, the amino acid sequence of which is shown in SEQ ID NO.4, wherein the amino acid position 319 of the wild-type rice aminoacyl-tRNA synthetase shown in SEQ ID NO.2 is changed from threonine to isoleucine.

[0009] The second objective of this invention is to provide a mutant of the rice aminoacyl-tRNA synthetase encoding gene, the sequence of which is shown in SEQ ID NO.3, wherein the mutant of the rice aminoacyl-tRNA synthetase encoding gene is a mutation of C to T at position 956 of the wild-type rice aminoacyl-tRNA synthetase encoding gene shown in SEQ ID NO.1.

[0010] That is, the CDS sequence of the rice aminoacyl-tRNA synthetase encoding gene (i.e., the wild-type rice OsHGZ2 gene) is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The mutant of the rice aminoacyl-tRNA synthetase encoding gene is the rice aminoacyl-tRNA synthetase encoding gene (i.e., the wild-type rice OsHGZ2 gene). OsHGZ2 The C at position 956 of the gene's CDS sequence region was mutated to T (SEQ ID NO.3), resulting in the amino acid at position 319 being changed from threonine to isoleucine (SEQ ID NO.4).

[0011] A third objective of this invention is to provide a knockout vector for the rice aminoacyl-tRNA synthetase encoding gene, wherein the target sites of the knockout vector are: T1:GCTGTTCGACACGAGGACGA (SEQ ID NO.5) and T2:TCGCCTTCGACGTCCTCTTC (SEQ ID NO.6).

[0012] Furthermore, the knockout primers are: hgz2 gRT1: CTGTTCGACACGAGGACGAgttttagagctagaaat (SEQ ID NO.7), hgz2 U6aT1: TCGTCCTCGTGTCGAACAGCggcagccaagccagca (SEQ ID NO.8), hgz2 gRT2: TCGCCTTCGACGTCCTCTTCgttttagagctagaaat (SEQ ID NO.9), and hgz2 U6bT2: GAAGAGGACGTCGAAGGCGACaacacaagcggcagc (SEQ ID NO.10).

[0013] A fourth objective of this invention is to provide the application of the wild-type rice aminoacyl-tRNA synthetase shown in SEQ ID NO.2, or the encoding gene of the wild-type rice aminoacyl-tRNA synthetase shown in SEQ ID NO.1, or the mutant of the rice aminoacyl-tRNA synthetase shown in SEQ ID NO.4, or the mutant of the encoding gene of the rice aminoacyl-tRNA synthetase shown in SEQ ID NO.3, or the aforementioned knockout vector, in increasing the zinc content in rice grains and / or maintaining the rice's ability to be passaged.

[0014] Furthermore, the application involves mutating the wild-type rice aminoacyl-tRNA synthetase shown in SEQ ID NO.2, or knocking out the gene encoding the wild-type rice aminoacyl-tRNA synthetase shown in SEQ ID NO.1, or transferring the aforementioned knockout vector into rice to increase the zinc content in rice grains.

[0015] The beneficial effects of this invention are as follows: Provides superior genes that can be applied to the breeding of high-zinc rice grains. OsHGZ2 Genes. Obtained through EMS mutagenesis. OsHGZ2 A mutant with a variation at position 319 of the threotype was found to have approximately twice the zinc content in its grains compared to the wild type. Knockout of the mutant further confirmed the reproducibility of this result. Furthermore, this invention can increase grain zinc content, the mutant can be propagated in the field, and the high-zinc phenotype is stably inherited. This invention has significant theoretical and practical implications for improving the nutritional quality of rice and enhancing human dietary health. Attached Figure Description

[0016] Picture 1 The rice aminoacyl-tRNA synthetase gene in Example 1 OsHGZ2 Results of zinc digestion content in grains of mutant and wild-type rice for two consecutive years; Picture 2 To implement the three rice aminoacyl-tRNA synthetase genes OsHGZ2 XRF scan results of zinc in grains of mutant and wild-type rice; Picture 3 In Example 1 OsHGZ2 The gene structure and location of mutations; Picture 4 The zinc content of mature grains in field growth of the knockout mutant (KO) and wild-type rice obtained in Example 2 is shown.

[0017] Picture 5 The complement mutant (COM) obtained in Example 3 and the rice aminoacyl-tRNA synthetase gene obtained in Example 1. OsHGZ2 Zinc content in farmland growth of mutant and wild-type rice; Detailed Implementation

[0018] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0019] Example 1 Rice aminoacyl-tRNA synthetase gene OsHGZ2 Acquisition of mutants (1) Construction of the rice ethyl methanesulfonate (EMS) chemical mutagenesis library: Approximately 1 kg of rice seeds (Zhonghua 11) were washed with tap water and soaked at room temperature for 20 h to allow the seed coat to fully absorb water. After soaking, the seeds were washed several times with tap water and drained. All seeds were submerged in 0.8% (mass / volume concentration) EMS prepared in advance and mutagenized at room temperature for 12 h. After mutagenesis, the seeds were washed twice with tap water. 3 M NaOH was added to the EMS mutagenesis solution to a final concentration of 1 M and poured into a waste liquid container. The rinsed seeds could be rinsed again with running water in a regular water tank for 3 h. To avoid contamination, all operations after adding EMS were carried out in a fume hood. The EMS-mutated rice seeds were sown at the Nanjing base and transplanted to paddy fields after two weeks of seedling cultivation, one seedling per hole. Three months later, the M1 generation seeds (i.e., the M2 generation) were harvested. The M2 generation seeds were propagated at the base to obtain the M3 generation seeds. After drying, threshing, and storage, the construction of the rice mutant library was completed.

[0020] (2) Screening and gene cloning of rice mutants: Six plump, uniformly grown seeds were selected from the obtained M3 generation EMS-mutated seeds. The husks were removed, and the seeds were placed in centrifuge tubes and labeled for later use. The husks of the seeds were weighed and the grain weight was recorded. The weighed grains were transferred to digestion tubes, and 1 ml of analytical grade concentrated nitric acid was added to each tube. Digestion was performed using a graphite digestion furnace. In the third stage, when the digestion tube was clear and there was no obvious solid matter, with only a drop of oily liquid remaining, the digestion tube was removed, cooled, and then diluted to 10 ml with 2% nitric acid. 1 ml of the supernatant was centrifuged and transferred to a 2 ml centrifuge tube. The elemental content was determined using inductively coupled plasma mass spectrometry (ICP-MS).

[0021] (3) Using zinc content as the standard, the mineral element content data of 3200 rice EMS-mutated seeds were collected. The top 1% of rice plants with the highest zinc content were selected and recorded. These plants were then propagated for three consecutive years (all in Hainan winter planting) to verify the stability of the grain zinc content phenotype. Finally, the rice aminoacyl-tRNA synthetase gene was obtained. OsHGZ2 Mutant (hgz2).

[0022] Experimental results are as follows Picture 1 As shown: 12 mutant plants were planted in farmland. hgz2 From both the mutant and wild-type strains, 10 plants from each strain were selected for sampling. Six seeds from each plant were dried to constant weight, dehulled, and digested. The digestate was diluted with 2% nitric acid, and the mineral element content of the samples was determined by ICP-MS. The results showed that the mutant (… hgz2 The zinc content of the seeds is about twice that of the wild type, with a highly significant difference.

[0023] The results above indicate that the mutant can be propagated normally in the field, and that the high zinc phenotype can be stably inherited without being affected by generation.

[0024] The gene was located using molecular biology techniques, specifically as follows: The mutant was crossed with the wild type (Zhonghua 11) to obtain the F1 generation. After harvesting the F1 generation, the F2 generation was obtained, resulting in 400 F2 lines. Leaf samples were taken from the F2 seedlings, flash-frozen in liquid nitrogen, and stored at -80℃ for later use. Based on the grain element content determination results at maturity of the F2 generation, 10% of the F2 population lines with extremely high and 10% with extremely low zinc content were selected for pooled sequencing. Candidate genes were selected based on the results. Pooled sequencing was performed by Shanghai Ouyi Biomedical Technology Co., Ltd. Molecular biology techniques were used to determine the target gene. OsHGZ2 ( LOC_Os10g32570 The CDS sequence of the wild-type rice OsHGZ2 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The mutant is wild-type rice. OsHGZ2 The C at position 956 of the gene's CDS sequence region is mutated to T (SEQ ID NO.3), resulting in the amino acid at position 319 being changed from threonine to isoleucine (SEQ ID NO.4).

[0025] OsHGZ2 Gene structure and mutation location, such as Picture 3 As shown: OsHGZ2 The gene has 9 exons and 8 introns. The mutation protected by this patent occurs in the sixth exon, where the amino acid at position 319 changes from threonine to isoleucine (SEQ ID NO.4), resulting in an increase in zinc content in the mutant grains.

[0026] Example 2 Rice aminoacyl-tRNA synthetase gene OsHGZ2 Obtaining knockout mutants The rice aminoacyl-tRNA synthetase gene was studied using the CRISPR / Cas9 system. OsHGZ2Knockout was performed, with the target sites selected as follows: T1: GCTGTTCGACACGAGGACGA (SEQ ID NO.5), T2: TCGCCTTCGACGTCCTCTTC (SEQ ID NO.6); the corresponding knockout primers were as follows: hgz2 gRT1: CTGTTCGACACGAGGACGAgttttagagctagaaat (SEQ ID NO.7), hgz2 U6aT1: TCGTCCTCGTGTCGAACAGCggcagccaagccagca (SEQ ID NO.8), hgz2 gRT2: TCGCCTTCGACGTCCTCTTCgttttagagctagaaat (SEQ ID NO.9), hgz2 U6bT2: GAAGAGGACGTCGAAGGCGACaacacaagcggcagc (SEQ ID NO.10).

[0027] The above OsHGZ2 The specific construction process of gene knockout mutants is as follows: CRISPR / Cas9 gene editing was performed using the universal editing system for rice monocot and dicot plants established by Academician Liu Yaoguang's team at South China Agricultural University. The complete set of pYL-CRISPR / Cas9 vectors (kanamycin resistance, Kan) and sgRNA intermediate vectors (pYLsgRNA-OsU6a and pYLsgRNA-OsU6b vectors, ampicillin resistance, Amp) used is documented in Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., et al. (2015). A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant 8(8), 1274-1284.

[0028] OsHGZ2 Gene target site design was completed using the CRISPR-GE online tool (http: / / skl.scau.edu.cn / targetdesign / ), based on the Nipponbare rice MSU7.0 reference genome, and retrieved from the Rice Genome Annotation Project database. OsHGZ2Full-length genome sequences were used, prioritizing target sites near the start codon region in the gene's CDS region. The selection criteria were: target sequence length 20 nt, PAM sequence 5'-NGG-3', GC content between 50% and 75%, no cross-exon regions, and extremely low off-target effects. Off-target risks were verified by BLAST alignment, and off-scores were calculated. Ultimately, two specific targets (T1 and T2) were identified for dual-target knockout. Editing of either target or deletion of the segment between the two targets could lead to off-target effects. OsHGZ2 Loss of gene function.

[0029] The CRISPR / Cas9 knockout vector was constructed using conventional molecular biology techniques, as follows: (1) Culture medium preparation and plasmid preparation: Prepare LB liquid culture medium and solid plate culture medium. Pick the pYL-CRISPR / Cas9 vector, pYLsgRNA-OsU6a, and pYLsgRNA-OsU6b vector strains respectively and inoculate them into the corresponding resistant LB solid plates. Incubate overnight at 37°C with the plates inverted. The next day, pick a single colony and inoculate it into LB liquid culture medium containing the corresponding resistance. Incubate overnight at 37°C with shaking at 200 rpm. Purify and recover the plasmid using the OMEGA plasmid extraction kit and store at -20°C for later use.

[0030] (2) First round of PCR amplification: Using pYLsgRNA-OsU6a and pYLsgRNA-OsU6b plasmids as templates, the sgRNA expression cassette was amplified using Vazyme's Phanta Max high-fidelity enzyme. The single reaction system consisted of 50 μL of the following components: 1.0 μL of Phanta Max high-fidelity enzyme, 0.5 μL each of 10 μM specific target primers (hgz2 gRT1 / hgz2 U6aT1, hgz2 gRT2 / hgz2 U6bT2), 1.0 μL each of 10 μM universal primers (UF: CTCCGTTTTACCTGTGGAATCG, SEQ ID NO.11; gR-R: CGGAGGAAAATTCCATCCAC, SEQ ID NO.12), 1.0 μL of plasmid template, 25.0 μL of 2×Phanta Max Buffer, and 1.0 μL of 10 mM dNTP Mix. μL, add ddH2O to 50 μL. PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 20 s, 72℃ extension for 30 s, 40 cycles; 72℃ final extension for 5 min, incubate at 10℃ for later use.

[0031] (3) Second round of PCR amplification: 1 μL of the first round PCR product was mixed and used as the template for the second round of amplification. The universal primers for the corresponding target sites were selected, and the same high-fidelity enzyme was used for amplification. The purpose was to introduce BsaI restriction sites at both ends of the sgRNA expression cassette. The reaction system was 50 μL: Phanta Max enzyme 1.0 μL, 10 μM universal primers for the second round 2 each, diluted template 1.0 μL, 2×Phanta Max Buffer 25.0 μL, 10 mM dNTP Mix 1.0 μL, and ddH2O was added to 50 μL. The reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ final extension for 5 min. The amplified products were detected by 1% agarose gel electrophoresis, purified using an OMEGA gel extraction kit, and the concentration was determined before being used for subsequent enzyme digestion and ligation reactions.

[0032] (4) Enzyme digestion and ligation reaction: Construct a 15 μL enzyme digestion and ligation system with the following components: 1.5 μL 10×CutSmart Buffer, 1.5 μL 10 mM ATP, 60-80 ng pYL-CRISPR / Cas9 plasmid, and 10-15 ng sgRNA expression cassette mixture. Bsa 10 U of I-HF endonuclease and 10 U of T4 DNA ligase were added, and sterile ddH2O was used to make up the volume. PCR reaction program: 37℃ for 5 min, 10℃ for 5 min, 20℃ for 5 min, 12 cycles; incubate at 37℃ for 5 min for complete digestion, and store at 4℃ for later use.

[0033] (5) E. coli transformation and positive clone screening: Take 1 μL of the above enzyme digestion and ligation product, add 100 μL of ice bath thawed product. E. coli DH5α competent cells were gently mixed and then incubated on ice for 30 min; heat-shocked in a 42℃ water bath for 45 s, followed by rapid cooling on ice for 3 min; 700 μL of antibiotic-free LB liquid medium was added to a clean bench and the cells were thawed at 37℃ and 200 rpm for 45 min; the cells were concentrated by centrifugation and spread on LB solid plates containing 50 μg / mL Kan, and incubated upside down at 37℃ for 12-16 h to obtain single colonies.

[0034] (6) Colony PCR and sequencing verification: The above single colonies were picked and colony PCR was performed using Vazyme 2×Rapid TaqMaster Mix with SP-L1 / SP-R as identification primers to screen positive clones; positive clones were inoculated into LB liquid medium containing Kan and cultured at 37℃ with shaking for 12 h, plasmids were extracted and sent for sequencing, and the results were confirmed by sequence alignment. OsHGZ2The CRISPR / Cas9 knockout vector for the gene was constructed correctly.

[0035] (7) Rice genetic transformation and mutant identification: The correctly sequenced knockout vector was sent to a biotechnology company for Agrobacterium-mediated genetic transformation using Zhonghua 11 as the recipient material, resulting in T0 generation positive transformed plants. Genomic DNA was extracted from the leaves of T0 generation seedlings using the CTAB method. Specific amplification primers were designed before and after the target site. The PCR amplification products were sequenced and compared with... OsHGZ2 Gene reference sequence alignment was used to screen for homozygous / heterozygous mutants with frameshift mutations and base deletions / insertions. T0 generation mutants were transplanted to the field for seed production to obtain T1 generation seeds. The genotypes of the T1 generation lines were identified using Sanger sequencing. After two consecutive generations of screening, [the following was observed]... OsHGZ2 The homozygous knockout mutant lines were designated KO1 and KO2, respectively, for subsequent gene function analysis.

[0036] Two types of edited rice knockout plants were obtained through identification. hgz2KO (KO1, KO2), the zinc content in the seeds of transgenic plants (KO1, KO2) was also significantly increased. Picture 4 ).

[0037] Example 3 Rice aminoacyl-tRNA synthetase gene OsHGZ2 Obtaining the complement mutant To confirm LOC_Os10g32570 It is the gene that controls high zinc levels in grains. HGZ2 ,Will LOC_Os10g32570 The entire genome was ligated into a complementary vector, along with the promoter and termination regions (7358 bp in total). pCAMBIA1301 of Eco R1 and Hin Between dⅢ restriction sites, mutants ( hgz2 Callus tissue was cultured and transformed. LOC_Os10g32570 The complete genome sequence, including the promoter and termination regions, is shown in SEQ ID NO.13 (7358 bp).

[0038] Ten independent transgenic positive rice plants were obtained through PCR and electrophoresis. hgz2COM (COM), the transgenic plants (COM) restored the zinc content of wild-type grains, and the zinc concentration returned to normal. Picture 5 This confirms LOC_Os10g32570 It is the gene that controls high zinc levels in grains. HGZ2 .

[0039] Example 4: XRF scanning of zinc in mutant grains Take the dried wild type and mutant ( hgz2Seeds were collected, the seed coats were removed by hand, and the seeds were evenly attached to Cape Town tape (2 cm × 30 m Kapton Tape, 3M) using plastic tweezers. The samples were then scanned on an X-ray fluorescence spectrometer (Bruker, M4 TORNADO PLUS).

[0040] Experimental results are as follows Picture 2 As shown, there were significant differences in zinc content among the three selected wild-type and mutant lines. The zinc content in the grains of the mutants was relatively stable, while the zinc content in the rice embryos increased significantly.

[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0042] SEQ ID NO.1 Wild-type CDS sequence SEQ ID NO. 2 Wild-type amino acid sequence MAEEGRSVGEEEPRGKRPAAELRLFDTRTKAAVPFRPRVEGKVAMYVCGVTPYDYSHVGHARAYVAFDVLFRYLKYLGYEVNYVRNFTDIDDKIIKRANEAGEDALSLSSRFIDEFHRDMYELQCLPPTHEPRVTKHIEQIIDLITKIMDNGKAYTIEGDVYFSVDNFPDYLSLSGRKVDQNRPGTRVAVDARKRNPADFALWKSAKEGEPSWESPWGRGRPGWHIECSAMSAHYLGNIFDIHGGGKDLIFPHHENELAQSQAAYPESEIISLYHPMALRLFLMRTHYRSDVNHSDTALQFASGRLYYIYQTLHDGEETVSLYGEHKLENSIPADDQKLIEENRSNFLEKMSDDLHTTAALDHLMKLQQKLEHQKKKQSQKQQQQQQKKPEDYIQALVALQNEVTDKLSILGLMPMSSLAEQLKDKALKRAGMTAEQLQQMIEQRTLARKNKDFAESDRIRTELSALGIALMGEPTGTLWRPSEPELAEGS* SEQ ID NO. 3 CDS sequence of the mutated gene Amino acid sequence of the mutated gene (SEQ ID NO.4) MAEEGRSVGEEEPRGKRPAAELRLFDTRTKAAVPFRPRVEGKVAMYVCGVTPYDYSHVGHARAYVAFDVLFRYLKYLGYEVNYVRNFTDIDDKIIKRANEAGEDALSLSSRFIDEFHRDMYEL QCLPPTHEPRVTKHIEQIIDLITKIMDNGKAYTIEGDVYFSVDNFPDYLSLSGRKVDQNRPGTRVAVDARKRNPADFALWKSAKEGEPSWESPWGRGRPGWHIECSAMSAHYLGNIFDIHGGG KDLIFPHHENELAQSQAAYPESEIISLYHPMALRLFLMRTHYRSDVNHSDTALQFASGRLYYIYQTLHDGEEIVSLYGEHKLENSIPADDQKLIEENRSNFLEKMSDDLHTTAALDHLMKLQQ KLEHQKKKQSQKQQQQQQKKPEDYIQALVALQNEVTDKLSILGLMPMSSLAEQLKDKALKRAGMTAEQLQQMIEQRTLARKNKDFAESDRIRTELSALGIALMGEPTGTLWRPSEPELAEGS* SEQ ID NO.13 LOC_Os10g32570 The entire genome and connecting promoter and termination region sequences

Claims

1. A rice aminoacyl-tRNA synthetase mutant, characterized in that, The amino acid sequence of the rice aminoacyl-tRNA synthetase mutant is shown in SEQ ID NO.

4. The rice aminoacyl-tRNA synthetase mutant is the rice aminoacyl-tRNA synthetase shown in SEQ ID NO.2, in which the amino acid position 319 is changed from threonine to isoleucine.

2. A mutant of the rice aminoacyl-tRNA synthetase encoding gene, characterized in that, The mutant sequence of the rice aminoacyl-tRNA synthetase encoding gene is shown in SEQ ID NO.

3. The mutant rice aminoacyl-tRNA synthetase encoding gene is a mutation of C to T at position 956 of the wild-type rice aminoacyl-tRNA synthetase encoding gene shown in SEQ ID NO.

1.

3. Knockout lines of rice aminoacyl-tRNA synthetase encoding genes, characterized in that, The knockout target points are: T1 as shown in SEQ ID NO.5: GCTGTTCGACACGAGGACGA and T2 as shown in SEQ ID NO.6: TCGCCTTCGACGTCCTCTTC.

4. The knockout carrier according to claim 3, characterized in that, The knockout primers were: hgz2 gRT1: CTGTTCGACACGAGGACGAgttttagagctagaaat (SEQ ID NO.7), hgz2 U6aT1: TCGTCCTCGTGTCGAACAGCggcagccaagccagca (SEQ ID NO.8), hgz2 gRT2: TCGCCTTCGACGTCCTCTTCgttttagagctagaaat (SEQ ID NO.9), and hgz2 U6bT2: GAAGAGGACGTCGAAGGCGACaacacaagcggcagc (SEQ ID NO.10).

5. The application of the rice aminoacyl-tRNA synthetase as described in claim 1, or the rice aminoacyl-tRNA synthetase encoding gene as described in claim 2, or the rice aminoacyl-tRNA synthetase mutant as described in claim 1, or the rice aminoacyl-tRNA synthetase encoding gene mutant as described in claim 2, or the knockout vector as described in claim 3 in increasing the zinc content in rice grains and / or maintaining the rice's ability to be passaged.

6. The application according to claim 5, characterized in that, Mutate the rice aminoacyl-tRNA synthetase as described in claim 1, or knock out the rice aminoacyl-tRNA synthetase encoding gene as described in claim 2, or transfer the knockout vector as described in claim 3 into rice to increase the zinc content in rice grains.

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