Rice susceptible brown planthopper related protein OsATL13 as well as coding gene and application thereof

Abstract

The invention discloses a plant insect-resistant related protein as well as a coding gene and application thereof. The protein provided by the invention is a protein as shown in (a) or (b): (a) a protein consisting of a sequence 1 in a sequence table and an amino acid sequence as shown in the sequence 1; and (b) a protein which is formed by substitution and / or deletion and / or addition of one or more amino acid residues of the sequence 1 and the amino acid sequence of the sequence 2, is related to plant insect resistance and is derived from the sequence 1 and the sequence 2. The plant insect-resistant related protein provided by the invention affects the insect resistance of plants. By knocking out the protein coding gene, the brown planthopper resistance of plants can be improved, so that insect-resistant transgenic plants can be cultivated. The protein and the coding gene thereof can be applied to plant genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a rice brown planthopper-associated protein OsATL13, its encoding gene, and its applications. Background Technology

[0002] The brown planthopper, scientifically known as *Nilaparvata lugens* (Stål), belongs to the family Planthopperidae in the order Hemiptera. It is a migratory, piercing-sucking pest that specifically damages rice. Adults and nymphs congregate at the base of rice plants, using their piercing-sucking mouthparts to penetrate the leaf sheaths and stem tissues, feeding on the phloem sap. In addition, the brown planthopper is a major vector for rice straw dwarf virus and serrated leaf dwarf virus, for which there are currently no effective pesticides, and these viral diseases are on the rise in Southeast Asian countries. Furthermore, large-scale brown planthopper outbreaks can cause "planthopper burn," the most typical and severe consequence of such outbreaks. When the planthopper density is extremely high, they feed extensively at the base of the rice plants in a short period, damaging the vascular tissue. This causes the base of entire clumps, patches, or even entire fields of rice plants to turn black, rot, loom, and die around the milk stage, resembling a fire, resulting in a complete crop failure. When brown planthoppers proliferate, chemical control remains a crucial method for managing them. However, the extensive use of chemical pesticides not only increases the burden on farmers and damages the ecological environment but also enhances the planthoppers' resistance, leading to a rapid increase in their numbers and exacerbating the damage. Therefore, breeding resistant varieties and identifying resistance genes are considered more effective and environmentally friendly approaches. In recent years, with the development of molecular markers and the construction of high-density linkage maps for rice, several major genes for brown planthopper resistance in rice have been located on chromosomes 2, 3, 4, 6, 11, and 12, and several brown planthopper resistance genes have been cloned. However, the molecular mechanisms of brown planthopper resistance in rice still require further investigation.

[0003] Ubiquitination is a crucial post-translational modification of proteins in eukaryotes. Through the covalent linkage of ubiquitin molecules to target proteins, it regulates the stability, localization, activity, and interactions of target proteins, participating in core processes such as cellular homeostasis and signal transduction. The ubiquitination process involves three enzymes: ubiquitin activator E1, conjugator E2, and ligase E3. Among them, ubiquitin ligase E3 can specifically recognize substrates, making it a key regulator of the ubiquitination pathway. Recent studies have increasingly revealed the important role of ubiquitin ligases in plant immune regulation. On the one hand, plants can target pathogens through ubiquitination, causing protein degradation, and can also activate immune pathways, enhancing immunity. On the other hand, pathogens can also utilize the ubiquitination pathway to inhibit the host plant's disease resistance response, thereby promoting their own infection. Although many reports have found that ubiquitin ligases can regulate plant immunity to pathogens, there are fewer reports on the involvement of ubiquitination in plant insect resistance, and even fewer studies have investigated whether ubiquitin ligases are involved in rice resistance to brown planthoppers. Summary of the Invention

[0004] To address the aforementioned technical problems in the existing technology, this application provides a rice brown planthopper-associated protein OsATL13, its encoding gene, and its applications.

[0005] The technical solution of this invention is as follows: The first object of the present invention is to provide a protein OsATL13, said protein OsATL13 being selected from (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) A protein derived from SEQ ID NO. 1 with one or more amino acid residues substituted and / or deleted and / or added, and which is associated with plant insect resistance.

[0006] A second objective of this invention is to provide a gene encoding the aforementioned protein.

[0007] Furthermore, the gene is a DNA molecule as described in any one of 1) to 4): 1) The DNA molecule shown in SEQ ID NO.2; 2) The DNA molecule shown in SEQ ID NO.3; 3) DNA molecules that hybridize to the DNA sequence defined in 1) or 2) under stringent conditions and encode the aforementioned protein OsATL13; 4) A DNA molecule that has more than 80% homology with the DNA sequence defined in 1) or 2) and encodes the aforementioned protein OsATL13.

[0008] A third objective of this invention is to provide a knockout vector for the aforementioned genes.

[0009] Furthermore, the knockout vector is a CRISPR-Cas9 vector, and the target sequence of the CRISPR-Cas9 vector is the DNA molecule shown in SEQ ID NO.2 or SEQ ID NO.3.

[0010] A fourth objective of this invention is to provide the application of the aforementioned protein OsATL13, or the aforementioned gene, or the aforementioned knockout vector in plant breeding.

[0011] Furthermore, downregulating the expression level of the aforementioned protein OsATL13 in rice, or knocking out the aforementioned gene in rice, or transferring the aforementioned knockout vector into rice can result in rice with enhanced resistance to rice planthoppers.

[0012] The beneficial effects of this invention are as follows: The plant insect resistance-related protein OsATL13 of this invention affects the plant's insect resistance response. Knocking out the gene encoding this protein yields transgenic plants with enhanced insect resistance. This protein and its encoding gene can be used for plant genetic improvement. Attached Figure Description

[0013] Figure 1 The state of rice seedlings and the knockout sequence after feeding on the brown planthopper of the OsATL13 knockout family and background parent Ningjing 7.

[0014] Figure 2 Comparison of seedling mortality rates in rice seedlings after feeding on brown planthoppers in OsATL13 knockout families and background parent Ningjing 7. Detailed Implementation

[0015] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0016] Example 1: Discovery of plant insect resistance-related proteins and their encoding genes I. Construction of knockout vectors using the CRISPR / Cas9 genome editing system A pair of primers, Cas9-1, was designed based on the cDNA of OsATL13. PCR amplification was performed using mOsU6 (CN111019946B) as a template. The amplified cDNA was ligated into the pYLCRISPR / Cas9Pubi-H cloning vector (GenBank: KR029109) using the godengate method. The vector was then transformed into DH5α. Single clones were selected and sequenced correctly. The plasmid was extracted, and the OsALT13-Cas9 vector was constructed.

[0017] The PCR primer sequences are as follows: Cas9-1F: GCGGTCTCAGGCGGGCGGCTGGAGCTGCAGCAAGTTTTAGAGCTAGAAATAGCAAG (SEQ ID NO. 4); Cas9-1R: TTGGTCCTAAACGCTCTGGAACGCCGTGTCAACACACAAGCGACAGC (SEQ ID NO. 5).

[0018] Example 2: Obtaining and Identifying Transgenic Plants I. Agrobacterium-mediated transformation Using Agrobacterium strain EHA105 (purchased from Ingenium Biotech, USA) as a mediator, the above-constructed... OsATL13 The gene knockout vector OsALT13-Cas9 was introduced into the disease-susceptible variety Ningjing 7.

[0019] (1) Cultured at 28℃ containing recombinants OsATL13 Agrobacterium was incubated for 16 hours, and the bacterial cells were collected and diluted in N6 liquid medium containing 100 μmol / L to a concentration of OD100. 600 ≈0.5, to obtain bacterial culture; (2) Mix the mature embryonic callus tissue of rice cultured for one month with the above bacterial solution and infect for 30 min. After the bacterial solution is dried with filter paper, transfer it to co-culture medium (N6 solid co-culture medium, purchased from Sigma) and co-culture at 24℃ for 3 days. (3) The above callus was inoculated on N6 solid screening medium containing 150 mg / L hygromycin B (purchased from Sigma) for the first screening for 16 days; (4) Select healthy callus and transfer it to N6 solid selection medium containing 200 mg / L hygromycin B for a second selection. Subculture every 15 days. (5) Select resistant callus and transfer it to a differentiation medium containing 150 mg / L hygromycin B for differentiation; (6) The regenerated rice plants that differentiate into seedlings are the obtained rice plants. OsATL13 Gene knockout plants.

[0020] II. Sequencing comparison of knockout transgenic plants Primers were designed based on the gene sequence using the Primer BLAST tool on the NCBI website. Fragment amplification was performed near the target site of the knockout material. The PCR products were sequenced by the company and compared with the reference sequence to determine the base changes produced after knockout.

[0021] The sequencing primer sequences are as follows: sq34550-F: 5'-GTTGTTCTTCCCTCGCTTGCA-3' (SEQ ID NO. 6); sq34550-R: 5'-AGTCGAACGGGTCCTTCCTG-3' (SEQ ID NO.7); The results showed that: Knockout transgenic plants OsATL13 The genes each contain 10 bases ( osatl13-1 ) and 21 bases ( osatl13-2 The absence of ) (see Figure 1 The message indicates that the removal was successful.

[0022] III. Identification of Insect Resistance in Transgenic Plants II. Transgenic material with OsATL13 knockout confirmed by sequencing ( osatll3-l , osatll3-2 Seeds of both the parent variety (Ningjing 7) and the recipient variety (NJ7) were soaked and germinated separately, then sown in round plastic pots (5.8 cm in diameter and 6.0 cm high) filled with nutrient soil (each pot had a small hole at the bottom for water absorption). These pots were placed in plastic storage boxes (65 cm × 44 cm × 14 cm) with a water level of approximately 2 cm. 30 sprouting seeds were sown per pot. Four days before inoculation, seedlings were thinned, discarding diseased and weak seedlings, leaving 20 uniform, healthy seedlings per pot for inoculation with brown planthopper larvae. The inoculation was repeated twice. When the seedlings reached the two-leaf stage, inoculation was conducted. Ten 2-3 instar brown planthoppers were inoculated per seedling. Inoculated seedlings were placed in greenhouse storage boxes or transplanted to the field, with careful water and fertilizer management.

[0023] Seven days after inoculation with brown planthoppers, the resistance of the saltl13 knockout transgenic family was significantly higher than that of the wild-type Ningjing 7 (see...). Figure 1 When the mortality rate of Ningjing 7 was approximately 100%, the mortality rates of knockout families were approximately 38% and 30%, respectively (see...). Figure 2 The results showed that knocking out OsATL13 in rice effectively improved resistance to brown planthoppers.

[0024] SEQ ID NO.1 MSHYTMHAHINYTALPPTSPLQLPLPYLPPPPPPPQPPLLQLQPPPPPSSPVSFDTAFQSRISPSILLIILILAVIFFVSGLLHLLVRFILRPAPRDAGDADSGDANVTAFQGQLQQLFHLHDAG VDQSFIDTLPVFLYGAVVGAGRKDPFDCAVCLCEFADDDRLRLLPKCSHAFHVDCIDTWLLSHSTCPLCRRSLLADFSPYGGGCSPLLFVLESGSEGSISDRLDAASSAHLSLVMEQEEEAEPEQ KHAEAAEKADEVVVSVKLGKFRSQATEVAAGGGCGGSSSANPEQDVRRCYSMGTYEYVMDERSLLRVAVKPPAKKRPTTQMPGHRVAMSECDCHSKRESFRGLDALPGGKLAQAQPPKPPAKVGK KESFSFSKIWMRGAPRIRKDGASSRRASSFRLSSVLQRAASDVGATAAPKPLRPDVVSPVTESEYNVSAWDKSEKSASSGSVADWDLESAAATAAVPGAGAGNGLSSRADEAPSFARRTLLWIRGHL SEQ ID NO.2 SEQ ID NO.3

Claims

1. Protein OsATL13, characterized in that, The protein OsATL13 is selected from either (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) Proteins derived from SEQ ID NO.1 with one or more amino acid residues substituted and / or deleted and / or added, and which are associated with plant insect resistance.

2. The gene encoding the protein of claim 1.

3. The gene as described in claim 2, characterized in that, The gene is any one of the DNA molecules described in 1) to 4) below: 1) The DNA molecule shown in SEQ ID NO.2; 2) The DNA molecule shown in SEQ ID NO.3; 3) A DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and encodes the protein OsATL13 of claim 1; 4) A DNA molecule that has more than 80% homology with the DNA sequence defined in 1) or 2) and encodes the protein OsATL13 of claim 1.

4. The gene knockout vector of claim 2 or 3.

5. The knockout carrier according to claim 4, characterized in that, The knockout vector is a CRISPR-Cas9 vector, and the target sequence of the CRISPR-Cas9 vector is the DNA molecule shown in SEQ ID NO.2 or SEQ ID NO.

3.

6. The application of the protein OsATL13 of claim 1, or the gene of claim 2 or 3, or the knockout vector of claim 4 or 5 in plant breeding.

7. The application according to claim 9, characterized in that, Downregulating the expression level of the protein OsATL13 of claim 1 in rice, or knocking out the gene of claim 2 or 3 in rice, or transferring the knockout vector of claim 4 or 5 into rice can produce rice with enhanced resistance to rice planthoppers.

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