A protein capable of simultaneously improving rice yield and brown planthopper resistance, its nucleotide sequence and application

Abstract

The present invention relates to the field of gene editing technology. The present invention provides a protein that can simultaneously improve rice yield and brown planthopper resistance, as well as its nucleotide sequence and application, wherein the amino acid sequence of the protein is shown in SEQ ID NO.1. In the early stage of the present invention, the rice OsJAZ10 gene was edited by CRISPR‑Cas9 gene editing technology, resulting in a frameshift mutation of the rice's own OsJAZ10 gene to produce a new protein that does not exist in the rice body, named FJ10 (frameshift mutation of OsJAZ10). Further studies have found that the FJ10 protein can improve rice resistance to brown planthoppers and promote rice field yield, and has important value for field application.

Description

Technical Field

[0001] The present invention relates to the technical field of gene editing, and in particular to a protein capable of simultaneously improving rice yield and brown planthopper resistance, as well as a nucleotide sequence and application thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's three major food crops, providing dietary energy for more than half of the world's population. Brown planthopper (BPH) is one of the main pests in rice fields. It feeds on rice phloem sap and affects rice yield and quality by laying eggs and spreading viruses, causing serious losses to agricultural production and threatening global food security. Chemical pesticides are widely used in farmland ecosystems as one of the effective methods to control pests, but long-term use of chemical pesticides has led to pests developing resistance and becoming rampant again. At the same time, chemical pesticides can have adverse effects on the environment, non-target organisms and humans themselves. Based on this, planting rice that is resistant to brown planthoppers is an effective way to ensure food security.

[0003] It is worth noting that due to the growth-defense trade-off mechanism evolved in plants, there is a trade-off between rice yield and insect resistance. Rice breeding maximizes growth, reproduction and yield-related traits, which leads to the loss of insect resistance-related genetic traits; in contrast, highly insect-resistant rice is often accompanied by unfavorable agronomic traits such as abnormal growth and development and reduced yield. In recent years, studies have reported on the improvement of rice traits to improve rice immunity while ensuring yield, but most of the existing research focuses on improving rice resistance to diseases, and only a small number of studies have simultaneously improved rice yield and insect resistance. Summary of the invention

[0004] The purpose of the present invention is to provide a protein capable of simultaneously improving rice yield and brown planthopper resistance, as well as its nucleotide sequence and application, so as to improve rice yield and brown planthopper resistance through a single protein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a protein capable of simultaneously improving rice yield and brown planthopper resistance. The amino acid sequence of the protein is shown in SEQ ID NO.1.

[0007] The invention also provides application of the protein in simultaneously improving rice yield and brown planthopper resistance.

[0008] The present invention also provides a nucleotide sequence encoding the protein, and the nucleotide sequence is shown as SEQ ID NO.2.

[0009] The invention also provides the application of the nucleotide sequence in preparing transgenic rice.

[0010] The present invention provides a protein capable of simultaneously improving rice yield and brown planthopper resistance, and its nucleotide sequence and application. The amino acid sequence of the protein is shown in SEQ ID NO.1. In the early stage of the present invention, the rice OsJAZ10 gene was edited by CRISPR-Cas9 gene editing technology, resulting in a frameshift mutation of the rice's own OsJAZ10 gene to produce a new protein that does not exist in the rice body, named FJ10 (frameshift mutation of OsJAZ10). Further research found that the FJ10 protein can improve the resistance of rice to brown planthoppers and promote the field yield of rice, and has important value for field application. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The FJ10 gene pattern diagram is formed by the mutation of the rice OsJAZ10 gene;

[0012] Figure 2 Comparison of the amino acid sequences of rice's own OsJAZ10 protein and the artificially created FJ10 protein;

[0013] Figure 3 To evaluate the effect of FJ10 protein on rice growth for greenhouse experiments;

[0014] Figure 4 To evaluate the effect of FJ10 protein on rice resistance to brown planthopper in greenhouse experiments;

[0015] Figure 5 To evaluate the effect of FJ10 protein on rice resistance to brown planthopper in field trials;

[0016] Figure 6 To evaluate the effect of FJ10 protein on rice yield in field trials;

[0017] Figure 7 Comparison of the nucleotide sequences of rice's own OsJAZ10 protein and the artificially created FJ10 protein. DETAILED DESCRIPTION

[0018] The present invention provides a protein capable of simultaneously improving rice yield and brown planthopper resistance, wherein the amino acid sequence of the protein is shown in SEQ ID NO.1:

[0019] MAMEGKSRRFAVACGVLSQYVRAEQKMAAAAGAAPARAVTTLSLIAWGGGGRRGGGAEGGWGGGGGASDGAGRAADHLLRWEDGRLRGLPRGQGGGGDAHGLLRDGGGAGSAGGRRARGHAHHEEGVAAAVLRQAQGPPRGDHPLRPPVAGGDQGLRAGGEEDAHLMAGPRRLRLRRRAP.

[0020] The invention also provides application of the protein in simultaneously improving rice yield and brown planthopper resistance.

[0021] The present invention also provides a nucleotide sequence encoding the protein, and the nucleotide sequence is shown in SEQ ID NO.2:

[0022] .

[0023] The invention also provides the application of the nucleotide sequence in preparing transgenic rice.

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1 Obtaining rice containing the FJ10 gene by gene editing

[0026] In the rice containing the FJ10 gene obtained by gene editing, the promoter of the FJ10 gene is the promoter of the rice's own OsJAZ10 gene, which has temporal and spatial specific expression characteristics.

[0027] (1) Editing the rice OsJAZ10 gene using CRISPR-Cas9 technology

[0028] The mutation target of OsJAZ10 gene (SEQ ID NO.3: CCACCTCCGCCCCAGGCATCAGG) was designed on the CRISPR GE (http: / / skl.scau.edu.cn / ) online website. The target sequence of OsJAZ10 gene was inserted into the pLYsgRNA-OsU3 vector to obtain the guide RNA (sgRNA) driven by the rice U3 promoter, and then the sgRNA vector was connected to the plant binary expression vector pYLCRISPR / Cas9Pubi-H. After transformation, single clone detection and plasmid extraction, the constructed recombinant plasmid was transferred into Agrobacterium EHA105 strain by electroporation for preservation. The callus induced from rice seeds was infected with the Agrobacterium tumefaciens EHA105 strain containing the recombinant plasmid, and the T-DNA was inserted into the rice genomic DNA. When the resistant callus differentiated into seedlings, the rice seedlings (T0 generation) with the mutation of OsJAZ10 gene edited by Cas9 were obtained.

[0029] (2) Screening of rice plants containing the homozygous FJ10 gene

[0030] The leaves of T0 generation rice seedlings were sampled, and rice genomic DNA was extracted using Cetyltrimethylammonium Bromide (CTAB). Positive plants were identified using Cas9 primers. Subsequently, specific primers were designed using the CRISPR GE (http: / / skl.scau.edu.cn / ) online website, and the OsJAZ10 target site was PCR amplified and sequenced to analyze the mutation type of the OsJAZ10 gene. A homozygous mutation type of the OsJAZ10 gene with one base (A) inserted after the 134th base (T) was obtained. Subsequently, positive rice was selected and transplanted to an artificial climate chamber for culture with nutrient solution, planted in the field three weeks later, and seeds were collected after maturity (T1 generation). After germination, the T1 generation rice seeds were planted in hydroponic solution. After two weeks, samples were taken and DNA was extracted. The mutation type of the mutant was then verified by PCR amplification and sequencing. Rice materials containing the homozygous FJ10 gene sequence were obtained, and field breeding (T2 generation) was carried out again for subsequent experiments.

[0031] Example 2 Obtaining rice containing FJ10 gene by genetic modification

[0032] In the rice containing the FJ10 gene obtained by transgenic methods, the promoter of the FJ10 gene is a constitutive promoter, which can maintain the constitutive expression of the FJ10 gene.

[0033] (1) Cloning of FJ10 gene

[0034] After the T2 generation of rice materials containing the homozygous FJ10 gene were accelerated, the leaves were sampled, RNA was extracted and reverse transcribed to obtain cDNA. According to the sequence of the FJ10 gene, specific PCR amplification primers were designed, and the FJ10 gene sequence was PCR amplified by a high-fidelity enzyme. The amplified product was size and quality detected by agarose gel electrophoresis, with an agarose concentration of 1%, a marker of DL2000, a sample volume of 5 μL, a voltage of 120 V, and an electrophoresis time of 20 min. Subsequently, the PCR product was recovered and purified using a gel recovery kit. The obtained fragment was double-digested with the pCAMBIA1301 vector using restriction endonucleases. After the digested fragments containing sticky ends and the vector were recovered by DNA purification, the DNA fragments were ligated to the pCAMBIA1301 vector under the action of T4 DNA ligase to obtain homologous recombinant plasmids.

[0035] (2) FJ10 gene transformed rice plants

[0036] After transformation, single clone detection, and plasmid extraction, the constructed recombinant plasmid was transferred into the Agrobacterium EHA105 strain by electroporation for preservation. The callus induced from rice seeds was infected with the Agrobacterium tumefaciens EHA105 strain containing the FJ10 gene, and the T-DNA was inserted into the rice genomic DNA. After the resistant callus differentiated into seedlings, it was planted in hydroponic solution to obtain rice materials containing the FJ10 gene (T0 generation).

[0037] (3) GUS staining to identify homozygous rice plants containing FJ10 protein

[0038] The positive transgenic plants containing FJ10 protein were screened by GUS staining. 5mm tender root tips of T0 generation rice plants differentiated from callus were cut and immediately placed in PCR tubes. 15μL dye solution was added to immerse the root tips. The root tips were stained in a 37℃ constant temperature incubator for 2h. The positive plants with blue root tips were selected for field breeding. The seeds (T1 generation) produced by self-pollination of positive T0 generation rice were germinated. At least 40 seedlings were tested for each line. The root tips of rice seedlings were cut and stained in a 37℃ constant temperature incubator. The positive plants with a line segregation ratio of 3:1 were selected for breeding. The seeds (T2 generation) produced by self-pollination of T1 generation were verified by the same method. The homozygous lines were all positive for GUS staining.

[0039] (4) Western immunoblotting assay to detect rice FJ10 protein content

[0040] The leaves of T2 homozygous rice plants were sampled and the total plant protein was extracted. The leaves were hybridized with monoclonal antibodies and alkaline phosphatase-labeled sheep anti-mouse IgG, and chemiluminescence signals were detected. The lines with signals were positive plants, and appropriate rice plants were selected for subsequent experiments based on the content of FJ10 protein.

[0041] Example 3 Greenhouse test to evaluate the growth status and brown planthopper resistance of rice containing FJ10 protein

[0042] (1) Growth status assessment

[0043] The rice seeds containing FJ10 protein and the ordinary rice seeds (not containing FJ10 protein) were placed in a culture dish, soaked in water for 24 hours, placed in a light incubator (temperature 28±1°C, photoperiod 14h L:10h D) to germinate the seeds and cleaned regularly to keep the surface of the seeds moist. After 7 days, the healthy seedlings were transferred to a greenhouse (temperature 28±2°C, photoperiod 14h L:10h D, humidity 45%), cultured with nutrient solution and regularly replaced with new nutrient solution. During this period, the growth status of the rice was photographed, and the plant height and leaf sheath height of the rice plants containing FJ10 protein and ordinary rice were recorded respectively.

[0044] (2) Evaluation of brown planthopper resistance

[0045] In this experiment, the egg hatching rate of female adults of brown planthoppers on rice plants was used as a measurement index to evaluate the egg-laying performance of brown planthoppers on rice without FJ10 protein and rice containing FJ10 protein. Three-week-old rice plants with consistent growth and good growth status among different rice varieties were selected and transplanted into plastic cups for 4 days of recovery culture with nutrient solution. Ten female adults of brown planthoppers with eggs were inoculated in the rice sheath. After 24 hours, all female adults were removed and the honeydew was wiped off. The rice was observed every day. When nymphs hatched at the oviposition site, the newly hatched nymphs were counted every 24 hours until no new nymphs hatched for 3 consecutive days. The base of the rice stem where the brown planthoppers laid eggs was cut, and the number of unhatched eggs was dissected and counted under a stereo microscope (Nikon) to calculate the hatching rate.

[0046] Example 4 Field evaluation of rice yield and brown planthopper resistance containing FJ10 protein

[0047] (1) Evaluation of brown planthopper resistance

[0048] The field experiment was conducted at the Changxing Agricultural Science and Technology Park of Zhejiang University, Xinfeng Village, Si'an Town, Changxing County, Huzhou City, Zhejiang Province (30°53′48″N, 119°39′8″E). Thirty blocks were set up in the field plot, of which 10 blocks were planted with rice plants without FJ10 protein, and the other 20 blocks were planted with rice plants containing FJ10 protein. An average of 9 rice seedlings were planted in each block. In order to evaluate the performance of brown planthoppers on different rice varieties, brown planthoppers were collected into plastic trays by gently tapping the plants. The number of brown planthoppers was counted from random blocks and surveyed at the specified time.

[0049] (2) Yield Assessment

[0050] To assess rice yield, fully mature plants were harvested 110 days after planting and the rice was subjected to indoor seed-copying. The so-called seed-copying is to investigate the grain and other traits of a limited random sample of mature rice, with the main survey indicator being the yield per plant.

[0051] Example 5 Data Analysis

[0052] The data obtained from the experiment were statistically analyzed using SPSS software. The comparison between two samples was performed using the Student's t-test, and the comparison between multiple samples was performed using the Turkey test in one-way analysis of variance. The asterisks in the figure indicate significant differences between the groups (*, p<0.05; **, p<0.01; ***, p<0.001; Student's ttest); different letters in the figure represent significant differences between the groups (p<0.05).

[0053] Experimental Results

[0054] 1. Obtaining the FJ10 gene through CRISPR-Cas9 gene editing

[0055] Through gene editing technology, rice plants with mutations in the OsJAZ10 gene were obtained. After DNA was extracted, the samples were amplified by PCR and sequenced. The results showed that a base (A) was inserted after the 134th base (T) of the rice OsJAZ10 gene, resulting in a frameshift mutation in the OsJAZ10 gene and the formation of a new gene FJ10. Compared with the OsJAZ10 gene, the FJ10 gene terminated prematurely at the 540th base ( Figure 1 ).

[0056] 2. Amino acid sequence comparison between rice's own OsJAZ10 protein and artificially created FJ10 protein

[0057] Since the mutation site occurs after the 134th base of the OsJAZ10 gene, the first 44 amino acids of the FJ10 protein are actually completely consistent with the amino acid sequence of the OsJAZ10 protein. However, due to the frameshift mutation, the 45-180 amino acid sequence of the FJ10 protein is completely different from the 45-187 amino acid sequence of the OsJAZ10 protein. This shows that the FJ10 protein is a completely new protein different from the OsJAZ10 protein and does not exist in rice itself ( Figure 2 ).

[0058] 3. Greenhouse experiment to evaluate the effect of FJ10 protein on rice growth

[0059] Rice containing FJ10 protein and rice without FJ10 protein were planted in a greenhouse at the same time. After measuring the plant height, it was found that the plant height and leaf sheath height of rice containing FJ10 protein were significantly higher than those of ordinary rice without FJ10 protein. Among them, the plant height of rice plants containing FJ10 protein obtained by gene editing increased by 34.2% and 25.7% respectively compared with the control, and the leaf sheath height increased by 111.4% and 58.7% respectively; and the plant height of different rice strains containing FJ10 protein obtained by genetic modification increased by 32.1% and 46.0% respectively compared with ordinary rice, and the leaf sheath height increased by 91.2% and 104.5% respectively, indicating that FJ10 protein can accelerate the growth of rice ( Figure 3 ).

[0060] 4. Greenhouse experiment to evaluate the effect of FJ10 protein on rice resistance to brown planthopper

[0061] The effect of FJ10 protein on rice resistance to brown planthoppers was evaluated through bioassay tests. The results showed that the hatching rate of brown planthoppers on rice lines containing FJ10 protein obtained through gene editing was significantly lower than that of ordinary rice without FJ10 protein, and the hatching rate of brown planthoppers on lines 1 and 2 was reduced by 22.7% and 23.7%, respectively; while the hatching rate of brown planthoppers on rice lines containing FJ10 protein obtained through genetic modification was significantly lower than that of ordinary rice without FJ10 protein, and the hatching rate of brown planthoppers on lines 1 and 2 was reduced by 11.0% and 14.6%, respectively, indicating that FJ10 protein can improve rice resistance to brown planthoppers ( Figure 4 ).

[0062] 5. Field trials to evaluate the effect of FJ10 protein on rice resistance to brown planthoppers

[0063] Rice containing FJ10 protein and ordinary rice were planted in the field, and the number of brown planthoppers on the rice plants was counted on the 70th, 77th and 85th days. The results showed that the number of brown planthoppers on rice containing FJ10 protein was significantly lower than that on ordinary rice. In the three brown planthopper population surveys, the number of brown planthoppers on rice containing FJ10 protein obtained through gene editing was 54.0%, 24.3% and 17.2% lower than that on ordinary rice, respectively; while the number of brown planthoppers on rice containing FJ10 protein obtained through genetic modification was 52.3%, 25.9% and 34.9% lower than that on ordinary rice, respectively, indicating that FJ10 protein can improve rice's resistance to brown planthoppers in the field ( Figure 5 ).

[0064] 6. Field trials to evaluate the effect of FJ10 protein on rice yield

[0065] Rice containing FJ10 protein and ordinary rice were planted in the field, and the rice was harvested after it was fully mature (110 days). The yield per plant was counted, and the results showed that the yield per plant of rice lines containing FJ10 protein was significantly higher than that of ordinary rice. Compared with ordinary rice, the yield per plant of rice containing FJ10 protein obtained by gene editing and transgenic methods increased by 17.6% and 22.7%, respectively, indicating that FJ10 protein can increase the field yield of rice ( Figure 6 ).

[0066] As can be seen from the above embodiments, the present invention provides a protein that can simultaneously improve rice yield and brown planthopper resistance, as well as its nucleotide sequence and application, and the amino acid sequence of the protein is shown in SEQ ID NO.1. In the early stage of the present invention, the rice OsJAZ10 gene was edited by CRISPR-Cas9 gene editing technology, resulting in a frameshift mutation of the rice's own OsJAZ10 gene to produce a new protein that does not exist in rice, named FJ10 (frameshift mutation of OsJAZ10). Further studies have found that the FJ10 protein can improve rice resistance to brown planthoppers and promote rice field yield, and has important value for field application.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A protein for simultaneously improving rice yield and brown planthopper resistance, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. Use of the protein according to claim 1 in simultaneously improving rice yield and brown planthopper resistance.

3. A nucleotide sequence encoding the protein according to claim 1, characterized in that: The nucleotide sequence is shown as SEQ ID NO.

2.

4. Use of the nucleotide sequence according to claim 3 in preparing transgenic rice.

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