CN118562862A - Protein capable of simultaneously improving rice yield and brown planthopper resistance, nucleotide sequence and application thereof - Google Patents

Abstract

The invention relates to the technical field of gene editing. The invention provides a protein capable of simultaneously improving rice yield and brown planthopper resistance, a nucleotide sequence and application thereof, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 1. In the early stage, the CRISPR-Cas9 gene editing technology is used for editing the rice OsJAZ gene, so that the rice OsJAZ gene frame shift mutation generates a new protein which does not exist in the rice body, and the new protein is named FJ10 (FRAMESHIFT MUTATION OFOSJAZ). Further research shows that FJ10 protein can improve the resistance of rice to brown planthoppers, can promote the field yield of rice, and has important value in field application.

Description

Protein capable of simultaneously improving rice yield and brown planthopper resistance, nucleotide sequence and application thereof

Technical Field

The invention relates to the technical field of gene editing, in particular to a protein capable of simultaneously improving rice yield and brown planthopper resistance, and a nucleotide sequence and application thereof.

Background

Rice (Oryza sativa l.) is one of three world food crops that provides dietary energy to more than half of the world population. Brown planthopper (NILAPARVATA LUGENS)Brownplanthopper, BPH) is one of main pests in paddy fields, and is fed with rice phloem juice, and the rice yield and quality are affected by means of spawning, virus transmission and the like, so that serious losses are caused to agricultural production, and global grain safety is threatened. Chemical pesticides are widely used in farmland ecosystems as one of effective methods for controlling pests, but long-term chemical pesticide use leads to pest resistance and rampant again. Meanwhile, chemical pesticides can have adverse effects on the environment and non-target organisms and humans themselves. Based on the above, the planting of rice having resistance to brown planthoppers is an effective way of ensuring the safety of grains.

Notably, as plants evolve a growth-defense tradeoff mechanism, there is a trade-off between rice yield and insect resistance. Rice breeding maximizes growth, propagation and yield-related traits, which results in loss of insect-resistant related genetic traits; in contrast, high insect-resistant rice tends to be accompanied by unfavorable agronomic traits of dysplasia and reduced yield. In recent years, research has been reported on improving rice traits and improving rice immunity on the premise of ensuring yield, but most of the existing research is focused on improving the resistance of rice to diseases, and only a small amount of research is needed to improve rice yield and insect resistance simultaneously.

Disclosure of Invention

The invention aims to provide a protein capable of improving rice yield and brown planthopper resistance simultaneously, a nucleotide sequence and application thereof, and the rice yield and brown planthopper resistance are improved through a single protein.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a protein for simultaneously improving rice yield and brown planthopper resistance, and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

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

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

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

The invention provides a protein capable of simultaneously improving rice yield and brown planthopper resistance, a nucleotide sequence and application thereof, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 1. In the early stage, the CRISPR-Cas9 gene editing technology is used for editing the rice OsJAZ gene, so that the rice OsJAZ gene frame shift mutation generates a new protein which does not exist in the rice body, and the new protein is named FJ10 (FRAMESHIFT MUTATION OFOSJAZ). Further research shows that FJ10 protein can improve the resistance of rice to brown planthoppers, can promote the field yield of rice, and has important value in field application.

Drawings

FIG. 1 is a diagram showing the genetic mutation of rice OsJAZ to form FJ10 gene;

FIG. 2 is an amino acid sequence alignment of rice self OsJAZ protein and artificially created FJ10 protein;

FIG. 3 is a greenhouse test to evaluate the effect of FJ10 protein on rice growth;

FIG. 4 is a greenhouse test to evaluate the effect of FJ10 protein on resistance to brown planthoppers;

FIG. 5 is a field test to evaluate the effect of FJ10 protein on resistance to brown planthopper in rice;

FIG. 6 is a graph showing the evaluation of the effect of FJ10 protein on rice yield in field trials;

FIG. 7 is a nucleotide sequence alignment of rice self OsJAZ protein and artificially created FJ10 protein.

Detailed Description

The invention provides a protein for simultaneously improving rice yield and brown planthopper resistance, and the amino acid sequence of the protein is shown as SEQ ID NO. 1:

MAMEGKSRRFAVACGVLSQYVRAEQKMAAAAGAAPARAVTTLSLIAWGGGGRRGGGAEGGWGGGGGASDGAGRAADHLLRWEDGRLRGLPRGQGGGGDAHGLLRDGGGAGSAGGRRARGHAHHEEGVAAAVLRQAQGPPRGDHPLRPPVAGGDQGLRAGGEEDAHLMAGPRRLRLRRRAP.

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

The invention also provides a nucleotide sequence for encoding the protein, which is shown in SEQ ID NO. 2:

ATGGCGATGGAGGGGAAGAGCAGGAGGTTCGCGGTGGCGTGCGGGGTGCTCAGCCAGTACGTGAGGGCGGAGCAGAAGATGGCGGCGGCGGCGGGGGCGGCACCGGCGAGGGCGGTGACGACGCTGAGCCTGATAGCCTGGGGCGGAGGTGGTCGTCGAGGAGGAGGAGCGGAGGGAGGTTGGGGAGGAGGAGGCGGGGCCAGCGACGGCGCCGGCCGCGCCGCTGACCATCTTCTACGGTGGGAGGATGGTCGTCTTCGAGGACTTCCCCGCGGACAAGGCGGCGGAGGTGATGCGCATGGCCTCCTCCGGGATGGCGGCGGCGCCGGCTCAGCGGGAGGGCGCCGCGCTCGCGGACATGCCCATCATGAGGAAGGCGTCGCTGCAGCGGTTCTTCGCCAAGCGCAAGGACCGCCTCGCGGCGACCACCCCCTACGCCCGCCCGTCGCCGGCGGAGACCAAGGCCTCCGAGCCGGAGGAGAAGAAGACGCCCACCTCATGGCTGGACCTCGCCGCCTCCGCCTCCGCCGCCGCGCGCCGTGA.

the invention also provides application of the nucleotide sequence in preparing transgenic rice.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1 Rice containing FJ10 Gene obtained by Gene editing

In the rice containing the FJ10 gene obtained by the gene editing method, the promoter of the FJ10 gene is the promoter of the rice self OsJAZ gene and has the characteristic of space-time specific expression.

(1) Editing rice OsJAZ gene by CRISPR-Cas9 technology

A OsJAZ gene mutation target (SEQ ID NO.3: CCACCTCCGCCCCAGGCATCAGG) was designed on the CRISPR GE (http:// skl. Scau. Edu. Cn /) online website. The target sequence of OsJAZ gene was inserted into pLYsgRNA-OsU3 vector to obtain guide RNA driven by rice U3 promoter, and then the sgRNA vector was ligated into plant binary expression vector PYLCRISPR/Cas9 Pubi-H. The constructed recombinant plasmid is transformed, monoclonal detected and extracted, and then transferred into agrobacterium EHA105 strain for preservation by an electric shock method. Infection of the callus induced by rice seeds with the agrobacterium tumefaciens EHA105 strain containing the recombinant plasmid, insertion of T-DNA into the genomic DNA of rice, and differentiation of the resistant callus into seedlings to obtain OsJAZ gene-mutated rice seedlings (T0 generation) edited by Cas 9.

(2) Screening of Rice plants containing homozygous FJ10 Gene

Leaves of T0 generation rice seedlings were sampled, and rice genomic DNA was extracted using cetyltrimethylammonium bromide (Cetyltrimethylammonium Bromide, CTAB). Positive plants were identified by Cas9 primer. Subsequently, a specific primer is designed through a CRISPR GE (http:// skl. Scau. Edu. Cn /) online website, PCR amplification and sequencing are carried out on OsJAZ targets, mutation types of OsJAZ genes are analyzed, and homozygous mutation types of OsJAZ genes inserted into 1 base (A) after 134 th base (T) are obtained. Then, the positive rice is picked and transplanted to a phytotron for culture by nutrient solution, planted in the field after three weeks and collected after maturation (T1 generation). And (3) germinating the T1 generation rice seeds, planting the germinated rice seeds in a water culture solution, sampling and extracting DNA after two weeks, and then verifying the mutation type of the mutant through PCR amplification and sequencing to obtain the rice material containing the homozygous FJ10 gene sequence, and carrying out field seed reproduction (T2 generation) again for subsequent experiments.

Example 2 obtaining Rice containing FJ10 Gene by transgenesis

In the rice containing the FJ10 gene obtained by the transgenic method, the promoter of the FJ10 gene is a constitutive promoter, so that the constitutive expression of the FJ10 gene can be maintained.

(1) Cloning of FJ10 Gene

Leaf sampling is carried out after the rice T2 generation material containing the homozygous FJ10 gene is catalyzed to sprout, RNA is extracted and cDNA is obtained by reverse transcription. Specific PCR amplification primers are designed according to the sequence of the FJ10 gene, and the FJ10 gene sequence is subjected to PCR amplification by high-fidelity enzyme. The amplified product is subjected to size and quality detection by agarose gel electrophoresis, the agarose concentration is 1%, the Marker is DL2000, the loading amount is 5 mu L, the voltage is 120V, and the electrophoresis time is 20min. Subsequently, the purified PCR product was recovered using a gel recovery kit. And (3) carrying out double enzyme digestion on the obtained fragment and a pCAMBIA1301 vector by utilizing restriction enzyme, purifying and recovering the enzyme fragments containing the sticky ends and the vector, and then connecting the DNA fragments to the pCAMBIA1301 vector under the action of T4 DNA ligase to obtain the homologous recombinant plasmid.

(2) FJ10 gene transformed rice plant

The constructed recombinant plasmid is transformed, monoclonal detected and extracted, and then transferred into agrobacterium EHA105 strain for preservation by an electric shock method. Infection of callus induced by rice seeds with Agrobacterium tumefaciens EHA105 strain containing FJ10 gene, insertion of T-DNA into rice genomic DNA, differentiation of the resistant callus into seedlings, and planting in water culture to obtain rice material (T0 generation) containing FJ10 gene.

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

Positive transgenic plants containing FJ10 protein were screened using GUS staining. Cutting young root tips of rice plants with the T0 generation differentiated from the callus at the length of 5mm, immediately placing the young root tips into a PCR tube, adding 15 mu L of dye liquor to immerse the root tips, dyeing the rice plants in a constant temperature incubator at 37 ℃ for 2 hours, and selecting positive plants with the blue root tips for field seed reproduction. And (3) accelerating germination of seeds (T1 generation) generated by the selfing of the positive T0 generation rice, detecting at least 40 seedlings of each strain, cutting root tips of the seedlings of the rice, dyeing in a constant-temperature incubator at 37 ℃, and selecting positive plants with the strain separation ratio of 3:1 for seed reproduction. The seeds (T2 generation) generated by T1 generation selfing are verified by the same method, and the homozygous strain is obtained after GUS staining is positive.

(4) Western immunoblotting experiment for detecting rice FJ10 protein content

Sampling the T2 generation homozygous rice plant leaves, extracting plant total proteins, respectively hybridizing with monoclonal antibodies and alkaline phosphatase-labeled goat anti-mouse IgG, detecting chemiluminescence signals, obtaining a signal-appearing strain which is a positive plant, and selecting a proper rice plant according to the content of FJ10 protein for subsequent experiments.

Example 3 greenhouse assay to assess the growth status of rice containing FJ10 protein and brown planthopper resistance

(1) Growth status assessment

The rice seeds containing FJ10 protein and the common rice seeds (without FJ10 protein) are respectively placed in a culture dish, fully soaked in water for 24 hours, placed in an illumination incubator (the temperature is 28+/-1 ℃ C., the photoperiod is 14h L:10h D) to germinate and regularly cleaned, and the surface of the seeds is kept moist. After 7 days, healthy seedlings were transferred to a greenhouse (temperature 28.+ -. 2 ℃ C., photoperiod 14h L:10h D, humidity 45%), cultured with nutrient solution and periodically replaced with new nutrient solution. During this period, the rice growth state was photographed, and the plant height and leaf sheath height of rice plants containing FJ10 protein and ordinary rice were recorded, respectively.

(2) Evaluation of brown planthopper resistance

In the experiment, the egg hatching rate of the brown planthopper female adults on rice plants is taken as a measurement index, and the egg laying performance of the brown planthopper on rice without FJ10 protein and rice with FJ10 protein is estimated. Selecting rice plants with consistent growth vigor and good growth state in different rice, transplanting the rice plants into a plastic cup, and recovering and culturing the rice plants for 4 days by using a nutrient solution. 10 brown planthoppers are inoculated to the rice leaf sheath part to carry egg female adults, all female adults are removed after 24 hours, and honeydew is wiped off. The rice was observed daily, and when the nymphs were hatched at the spawning sites, newly hatched nymphs were counted every 24 hours until no newly hatched nymphs were present for 3 consecutive days. The rice stem base of brown planthoppers spawning is cut, the unhatched egg quantity is dissected and counted under a stereoscopic microscope (Nikon), and the hatchability is calculated.

Example 4 field evaluation of rice yield and brown planthopper resistance containing FJ10 protein

(1) Evaluation of brown planthopper resistance

The field test was carried out in Xingxing county, city, long, and county, an Zhen, xinfeng village, zhejiang university, xingxing agricultural science and technology garden (30 DEG 53 '48' N,119 DEG 39 '8' E). And setting 30 blocks in a field district, wherein 10 blocks are planted with rice plants without FJ10 protein, and the other 20 blocks are planted with rice plants with FJ10 protein. 9 rice seedlings were planted on average in each block. To evaluate brown planthopper performance of different rice, brown planthoppers were collected into plastic trays by gently patting the plants. The number of brown planthoppers was counted from a random block and investigated at regular time intervals.

(2) Yield assessment

To evaluate rice yield, fully mature plants were harvested 110 days after planting and the rice was subjected to indoor copy. The term "seed copy" refers to a study of properties such as grains of a mature limited rice random sample, and the main index is the yield of a single plant.

Example 5 data analysis

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

Experimental results

1. FJ10 gene obtained by CRISPR-Cas9 gene editing

Through gene editing technology, osJAZ gene mutated rice plants are obtained. After DNA extraction, the sample was subjected to PCR amplification and sequencing, and the result showed that the 134 th base (T) of rice OsJAZ gene was inserted with 1 base (A) to cause frame shift mutation of OsJAZ gene, forming 1 new gene FJ10. In contrast to OsJAZ gene, the FJ10 gene was terminated early at base 540 (FIG. 1).

2. Amino acid sequence alignment of rice self OsJAZ protein and artificially created FJ10 protein

Since the mutation site occurs after the 134 th base of OsJAZ gene, the first 44 amino acids of the FJ10 protein are virtually identical to the amino acid sequence of OsJAZ protein. However, the 45 th to 180 th amino acid sequence of FJ10 protein is completely different from the 45 th to 187 th amino acid sequence of OsJAZ th protein due to frame shift mutation. This suggests that FJ10 protein is an entirely new protein, distinct from OsJAZ protein, and that rice itself is not present (FIG. 2).

3. Greenhouse test to evaluate the effect of FJ10 protein on rice growth

The rice containing FJ10 protein and the rice not containing FJ10 protein are planted in a greenhouse at the same time, and after the plant height is measured, the plant height and leaf sheath height of the rice containing FJ10 protein are obviously higher than those of common rice not containing FJ10 protein. Wherein, the rice plants containing FJ10 protein obtained by gene editing have plant heights increased by 34.2% and 25.7% respectively compared with the control, and leaf sheath heights increased by 111.4% and 58.7% respectively; and the heights of different rice strains containing FJ10 protein obtained through transgenosis are respectively improved by 32.1 percent and 46.0 percent compared with common rice, and the heights of leaf sheaths are respectively improved by 91.2 percent and 104.5 percent, which shows that the FJ10 protein can accelerate the growth of the rice (figure 3).

4. Greenhouse test to evaluate the effect of FJ10 protein on resistance to brown planthopper in rice

The effect of FJ10 protein on brown planthopper resistance in rice was assessed by bioassay experiments. The result shows that the hatching rate of brown planthoppers on the rice lines containing FJ10 protein obtained by gene editing is obviously lower than that of common rice without FJ10 protein, and the hatching rate of brown planthoppers is respectively reduced by 22.7 percent and 23.7 percent on lines 1 and 2; whereas the hatchability of brown planthoppers on rice lines containing FJ10 protein obtained by the transgenesis was significantly lower than that of ordinary rice without FJ10 protein, the hatchability of brown planthoppers was reduced by 11.0% and 14.6% on lines 1 and 2, respectively, indicating that FJ10 protein can increase resistance of rice to brown planthoppers (fig. 4).

5. Evaluation of FJ10 protein Effect on resistance to brown planthopper in Rice by field experiments

The rice containing FJ10 protein and the common rice are planted in the field, and the number of brown planthoppers on rice plants is counted on days 70, 77 and 85, so that the result shows that the number of brown planthoppers on the rice containing FJ10 protein is obviously lower than that of the common rice. In three brown planthopper quantity surveys, the brown planthopper quantity on the rice containing FJ10 protein obtained through gene editing is respectively reduced by 54.0%, 24.3% and 17.2% compared with that of common rice; the quantity of brown planthoppers on the rice containing the FJ10 protein obtained through the transgenesis is reduced by 52.3 percent, 25.9 percent and 34.9 percent respectively compared with that of common rice, which shows that the FJ10 protein can improve the resistance of the brown planthoppers in the field (figure 5).

6. Field test to evaluate the effect of FJ10 protein on rice yield

The rice containing FJ10 protein and common rice are planted in the field, the rice is harvested after the rice is fully mature (110 days), and the result shows that the single plant yield of the rice strain containing FJ10 protein is obviously higher than that of the common rice. Compared with common rice, the single plant yield of the rice containing FJ10 protein obtained by two methods of gene editing and transgenosis is respectively improved by 17.6 percent and 22.7 percent, which shows that the FJ10 protein can improve the field yield of the rice (figure 6).

As can be seen from the above examples, the present invention provides a protein for simultaneously improving rice yield and brown planthopper resistance, and a nucleotide sequence and application thereof, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 1. In the early stage, the CRISPR-Cas9 gene editing technology is used for editing the rice OsJAZ gene, so that the rice OsJAZ gene frame shift mutation generates a new protein which does not exist in the rice body, and the new protein is named FJ10 (FRAMESHIFT MUTATION OFOSJAZ). Further research shows that FJ10 protein can improve the resistance of rice to brown planthoppers, can promote the field yield of rice, and has important value in field application.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (4)

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

2. Use of the protein of claim 1 for increasing both rice yield and brown planthopper resistance.

3. A nucleotide sequence encoding the protein of claim 1, wherein said nucleotide sequence is set forth in SEQ ID No. 2.

4. Use of the nucleotide sequence according to claim 3 for the preparation of transgenic rice.