Application of rice qGRB1 protein in enhancing plant resistance to rice stem borer

Abstract

This invention discloses the application of rice qGRB1 protein in enhancing rice resistance to the rice stem borer. This invention provides the application of qGRB1 protein, or substances regulating the expression of the gene encoding the qGRB1 protein, or substances regulating the activity or content of the protein, in regulating plant resistance to the rice stem borer. The qGRB1 protein may be a protein with the amino acid sequence of SEQ ID No. 1 or a protein with the amino acid sequence of positions 11-1455 of SEQ ID No. 1. Results show that introducing the qGRB1 gene into rice can significantly increase the expression level of qGRB1 in rice, and significantly improve rice resistance to the rice stem borer.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of rice qGRB1 protein in enhancing plant resistance to rice stem borer. Background Technology

[0002] More than half of the world's population relies on rice (Oryza sativa L.) as their staple food, but herbivorous insects often cause rice yield reductions. The rice stem borer (Chilo suppressalis), belonging to the family Pyralidae in the order Lepidoptera, is also known as the "heart borer" and is one of the most serious rice pests in temperate Asia (Wang, Y., Wilks, JC, Danhorn, T., Ramos, I., Croal, L. and Newman, DK (2011) Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition. J Bacteriol 193, 3606-3617.). Its life cycle includes egg, larval, pupa, and adult stages. It is one of the major pests of rice, causing damage to rice plants mainly through heart rot during the tillering stage and whitehead disease during the heading stage, leading to reduced rice yields and seriously jeopardizing food security. Although many methods have been developed to control the rice stem borer, the use of chemical insecticides remains the most widespread. However, chemical control not only leads to pesticide resistance in pests, but also causes serious environmental pollution and food safety issues (Chagnon, M., Kreutzweiser, D., Mitchell, EA, Mitchell, EA, Morrissey, CA, Noome, DA, Noome, DA, Van der Sluijs, JP and Van der Sluijs, JP (2015) Risks of large-scale use of systemic insecticides to ecosystem functioning and services. Environ Sci Pollut R 22, 119-134. Chagnon et al., 2015).

[0003] With the development of agricultural biotechnology, RNA interference (RNAi) has been increasingly used for pest control. Since RNAi induced by double-stranded RNA (dsRNA) uptake was first reported in *C. elegans* (Timmons, L. and Fire, A. (1998) Specific interference by ingested dsRNA. *Nature* 395, 854-854.), the success of transgenic crops using RNAi technology has greatly broadened the range of target genes for pest control (Huvenne, H. and Smagghe, G. (2010) Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review. *Journal of Insect Physiology* 56, 227-235.). Furthermore, endogenous small RNAs from the rice stem borer itself can also be used for pest control. Studies by He, Jiang, and Zheng et al. have shown that microRNA 14 (He, K., Xiao, HM, Sun, Y., Ding, SM, Situ, GM and Li, F. (2019) Transgenic microRNA-14 rice shows high resistance to rice stem borer. Plant Biotechnol J17, 461-471.) and artificially synthesized microRNA 15 (Jiang, S., Wu, H., Liu, HJ, Zheng, J., Lin, YJ and Chen, H. (2017) The overexpression of insect endogenous small RNAs in transgenic rice inhibits growth and delays pupation of striped stem borer (Chilo suppressalis). Pest Manag Sci 73, 1453-1461.) and microRNAs in rice stem borer can inhibit growth and delays pupation of striped stem borer (Chilo suppressalis). Pest Manag Sci 73, 1453-1461.) and microRNAs can inhibit growth and delays pupation of striped stem borer (Chilo suppressalis). 260(Zheng,XX,Weng,ZJ,Li,H.,Kong,ZC,Zhou,ZH,Li,F.,Ma,WH,Lin,YJand Chen,H.(2021) Transgenic rice overexpressing insect endogenous microRNA csu-novel-260 is resistant to striped stem borer under field conditions. Plant Biotechnol J 19, 421-423.) Overexpression in rice can enhance the resistance of rice to rice stem borer. The mechanism of action is that the miRNA can target the ecdysone signaling pathway in rice stem borer and inhibit the synthesis of ecdysone, thereby affecting the hatching of larvae and the growth and development of adults. (He, K., Xiao, HM, Sun, Y., Ding, SM, Situ, G. and Li, F. (2019) Transgenic microRNA-14 rice shows high resistance to rice stemborer. Plant Biotechnol J 17, 461-471.; Jiang, S., Wu, H., Liu, HJ, Zheng, J., Lin, YJ and Chen, H. (2017) The overexpression of insect Endogenous small RNAs in transgenic rice inhibit growth and delay pupation of striped stem borer (Chilo suppressalis). Pest Manag Sci 73, 1453-1461.; Zheng, XX, Weng, ZJ, Li, H., Kong, ZC, Zhou, ZH, Li, F., Ma, WH, Lin, YJ and Chen, H. (2021) Transgenic rice overexpressing insect endogenous microRNA csu-novel-260 is resistant to striped stem borer under field conditions. Plant Biotechnol J 19, 421-423.). These findings also indicate that utilizing endogenous small RNAs from the rice stem borer as a novel pest control strategy warrants further exploration.

[0004] However, both dsRNA-induced RNAi and the use of endogenous small RNAs in the rice stem borer for control have certain limitations. According to previous reports, the rice stem borer, a lepidopteran insect, seems to have some resistance to dsRNA. In feed-based experiments, all five long dsRNAs caused significant mortality or developmental delay in rice stem borer larvae. However, none of the dsRNAs expressed in transgenic rice plants showed a significant effect on rice stem borer larvae (Jiang, S., Wu, H., Liu, HJ, Zheng, J., Lin, YJ and Chen, H. (2017) The overexpression of insect endogenous small RNAs in transgenic rice inhibits growth and delayspupation of striped stem borer (Chilo suppressalis). Pest Manag Sci 73, 1453-1461.). Furthermore, not all endogenous small RNAs from insects, nor all exogenously synthesized small RNAs, can confer resistance in rice plants. Additionally, it remains unclear whether heterologous expression of genes from the rice stem borer affects rice plant phenotype and yield. Therefore, it is still necessary to start with rice germplasm resources and explore superior insect-resistant genes from natural variations in rice. For example, the planthopper-resistant genes BPH3 and BPH6 have now been discovered. These genes not only enhance rice's resistance to planthoppers but also do not affect rice yield (Guo,JP,Xu,CX,Wu,D.,Zhao,Y.,Qiu,YF,Wang,XX,Ouyang,YD,Cai,BD,Liu,X.,Jing,SL,Shangguan,XX,Wang,HY,Ma,YH,Hu,L.,Wu,Y.,Shi,SJ,Wang,WL,Zhu,LL,Xu,X.,Chen,RZ,Feng,YQ,Du,B.and He,GC(2018)Bph6 encodes an exocyst-localized protein and confers broad resistance to planthoppers in rice.NatGenet 50,297-+.). They have important application value for the breeding of planthopper-resistant rice varieties and the efficient control of pests. However, existing research on rice stem borer resistance genes is scarce. Therefore, mining rice stem borer resistance genes in rice germplasm resources is an effective way to address insect resistance, environmental and food safety issues caused by chemical control, as well as the limitations of RNAi technology. Summary of the Invention

[0005] The purpose of this invention is to provide the application of qGRB1 protein in enhancing plant resistance to rice stem borer.

[0006] To achieve the above objectives, in a first aspect, the present invention provides the application of a protein or a substance that regulates the expression of the protein-encoding gene or a substance that regulates the activity or content of the protein, wherein the application may be any of the following:

[0007] D1) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in regulating plant resistance to rice stem borer.

[0008] D2) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in the preparation of products that regulate plant resistance to rice stem borer.

[0009] D3) The application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in the preparation of products for cultivating rice stem borer resistant plants;

[0010] D4) Application of proteins or substances that regulate the expression of the protein-encoding genes or substances that regulate the activity or content of the proteins in plant breeding.

[0011] The protein may be A1), A2), or A3):

[0012] A1) The amino acid sequence of the protein is SEQ ID No. 1 or the amino acid sequence of the protein is positions 11-1455 of SEQ ID No. 1;

[0013] A2) Proteins obtained by substituting and / or deleting and / or adding one or more amino acid residues to the amino acid sequence shown in A1) have more than 80% identity with the protein shown in A1) and are associated with resistance to rice stem borer in plants.

[0014] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0015] Furthermore, the aforementioned proteins can be derived from rice.

[0016] In this invention, SEQ ID No.1 consists of 1455 amino acid residues.

[0017] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0018] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0019] Furthermore, in the above applications, the substance regulating the expression of the protein-coding gene or the substance regulating the activity or content of the protein is a biological material, which may be any one of B1) to B9) below:

[0020] B1) Nucleic acid molecules that encode the above proteins;

[0021] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0024] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0025] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0026] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3);

[0027] B8) Nucleic acid molecules that inhibit or reduce the expression of the genes encoding the above proteins or nucleic acid molecules that inhibit or reduce the activity of the above proteins;

[0028] B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8).

[0029] Furthermore, in the above applications, the nucleic acid molecule described in B1) can be any of the following DNA molecules as shown in b1) to b3):

[0030] b1) The coding sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2;

[0031] b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2;

[0032] b3) has 80% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1;

[0033] B8) The nucleic acid molecule may be a DNA molecule expressing a gRNA that targets the protein-coding gene or a gRNA that targets the protein-coding gene.

[0034] In the above applications, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0035] In the above applications, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0036] Furthermore, in the above applications, the target sequence of the gRNA can be the nucleotides shown in positions 227-246 of SEQ ID No. 3 (i.e., 5'-GGACGACCCGTTCGGTCGCG-3').

[0037] In the aforementioned biological materials, the expression cassette described in B2) refers to DNA capable of expressing the aforementioned proteins in host cells. This DNA may include not only promoters that initiate gene transcription but also terminators that terminate gene transcription. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically induced promoters from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by jasmonic acid methyl ester); heat shock promoters (US Patent 5,187,267); tetracycline-inducible promoters (US Patent 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 1)). 0099169.7)), seed storage protein-specific promoters (e.g., promoters of beta-conglycin, napin, oleosin, and soybean beta-conglycin (Beachy et al. (1985) EMBO J.4:3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator (see, e.g., Odell et al. (I 985 Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res. 15:9627.

[0038] In the aforementioned biological materials, the recombinant vector described in B3) may contain the DNA molecule shown in SEQ ID No. 2 for encoding the aforementioned protein.

[0039] Recombinant vectors containing the above-mentioned gene expression cassettes can be constructed using plant expression vectors. These plant expression vectors can be Gateway system vectors or binary Agrobacterium vectors, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1300, pCAMBIA1300-35S, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. When constructing recombinant vectors using coding genes, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0040] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants that encode enzymes or luminescent compounds that produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0041] Among the aforementioned biological materials, the recombinant microorganisms mentioned in B4) can specifically be yeast, bacteria, algae, and fungi.

[0042] Among the aforementioned biological materials, the plant tissues described in B6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0043] Among the aforementioned biological materials, the transgenic plant organs described in B7) can be the roots, stems, leaves, flowers, fruits, and seeds of transgenic plants.

[0044] Among the aforementioned biological materials, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0045] Furthermore, in the above applications, the substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein is any one of the biological materials from B1 to B7, and the regulation of plant resistance to rice stem borer is to improve the plant's resistance to rice stem borer.

[0046] In further applications, the substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein is the biological material of B8 or B9, and the regulation of plant resistance to rice stem borer is to reduce the plant's resistance to rice stem borer.

[0047] Furthermore, in the above applications, the plant can be any one of the following P1)-P5):

[0048] P1) Monocotyledons;

[0049] P2) Plants of the order Poales;

[0050] P3) Gramineae plants;

[0051] P4) Plants of the genus *Oryza*;

[0052] P5) Rice.

[0053] In the above applications, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the gene transcription level; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0054] In the above applications, the regulation of gene expression can be achieved by inhibiting or reducing gene expression, which can be accomplished through gene knockout or gene silencing. Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence. Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing requires no alteration of the DNA sequence to prevent or reduce gene expression. Gene silencing can occur at two levels: transcriptional gene silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA, including antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0055] In the above applications, the substance regulating gene expression can be a reagent that inhibits or reduces the expression of the gene. The reagent that inhibits or reduces the expression of the gene can be a gene knockout reagent, such as a reagent that knocks out the gene through homologous recombination or a reagent that knocks out the gene through CRISPR-Cas9. The reagent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0056] To achieve the above objective, in a second aspect, the present invention provides a method for improving the resistance of plants to the rice stem borer, the method comprising introducing the gene encoding the above-mentioned protein into a recipient plant to obtain a target plant with higher resistance to the rice stem borer than the recipient plant.

[0057] Furthermore, in the above method, the gene encoding the protein is any one of the genes shown in b1)-b3) below:

[0058] b1) The coding sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2;

[0059] b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2;

[0060] b3) has 80% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1.

[0061] Furthermore, in the above method, the recipient plant can be any one of the following P1)-P5):

[0062] P1) Monocotyledons;

[0063] P2) Plants of the order Poales;

[0064] P3) Gramineae plants;

[0065] P4) Plants of the genus *Oryza*;

[0066] P5) Rice.

[0067] Plant expression vectors carrying the protein-coding genes described in this invention can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant cells or tissues can be cultured into plants.

[0068] To achieve the above objectives, in a third aspect, the present invention provides a method for reducing resistance to the rice stem borer, the method comprising reducing resistance to the rice stem borer by knocking out the gene encoding the aforementioned protein in rice.

[0069] Furthermore, in the above method, knocking out the gene encoding the aforementioned protein in rice can be achieved by performing at least one of the following mutations on the rice qGRB1 genome sequence:

[0070] F1) A DNA molecule obtained by inserting nucleotide A between nucleotides 243 and 244 of sequence 3 in the rice genome sequence listing, specifically between nucleotides 130 and 131 of the qGRB1 gene coding sequence shown in sequence 2 of the sequence listing.

[0071] F2) The DNA molecule obtained by deleting 4 nucleotides from position 241-244 of sequence 3 in the sequence listing of the rice genome, that is, deleting nucleotides from position 128-131 of the coding sequence of the qGRB1 gene shown in sequence 2 in the sequence listing;

[0072] F3) The DNA molecule obtained by deleting 13 nucleotides from position 237 to 249 of sequence 3 in the sequence listing of the rice genome, that is, deleting nucleotides from position 124 to 136 of the qGRB1 gene coding sequence shown in sequence 2 in the sequence listing.

[0073] In the method for reducing rice stem borer resistance, the rice can be the japonica rice variety IC 25690::IRGC53973-1 (also called C1054, hereinafter referred to as C1054).

[0074] The beneficial effects achieved by this invention are as follows:

[0075] 1) This invention discloses for the first time the correlation between rice qGRB1 protein and rice stem borer resistance, and provides the application of qGRB1 protein in regulating rice stem borer resistance;

[0076] 2) Compared with the wild type, the rice resistant to rice stem borer obtained by the method of the present invention can significantly reduce the rice dead heart caused by rice stem borer infestation in field experiments, and significantly improve the rice stem borer resistance. Attached Figure Description

[0077] Figure 1 The expression of qGRB1 at the stem base of rice after inoculation with the insect-resistant rice variety G257 and the susceptible rice variety C495 by the rice stem borer was shown in the left figure, which shows the qGRB1 expression level of G257 and the right figure shows the qGRB1 expression level of C495.

[0078] Figure 2 Gene target sequencing analysis for qGRB1-KO transgenic rice.

[0079] Figure 3 This study analyzed the expression level of qGRB1 in the qGRB1-OE transgenic line.

[0080] Figure 4 The insect resistance response of qGRB1-KO transgenic rice to rice stem borer.

[0081] Figure 5 PCR identification of the Hyg marker gene in qGRB1-OE transgenic rice.

[0082] Figure 6 The insect resistance response of qGRB1-OE transgenic rice to rice stem borer.

[0083] Figure 7 This is a chromatogram of the pCBSG032 recombinant vector.

[0084] Figure 8 The spectrum of the recombinant vector 35S::qGRB1.

[0085] Figure 9 The nucleotide sequence is for the pCBSG032 recombinant vector.

[0086] Figure 10 The nucleotide sequence is for the recombinant vector 35S::qGRB1. Detailed Implementation

[0087] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0088] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0089] The rice species WAS 174-B-3-5::C1(G257), MURGI BRINJ::IRGC 73109-1(C495), IC25690::IRGC53973-1(C1054), and OS 4::IRGC 11335-1(G20) are disclosed in the literature “Genomic variation in 3,010 diverse accessions of Asian cultivated rice”, Nature, 2018, 557: 43-49. The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only used for repeating the experiments of this invention and cannot be used for other purposes.

[0090] In Example 1 below, data were processed using EXCEL statistical software. The experimental results are expressed as mean ± standard deviation. The T-test was used, and P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, P < 0.001 (***) indicates a highly significant difference, and "NS" indicates no significant difference.

[0091] In Examples 2 and 3 below, the data were processed and analyzed using R statistical software. The experimental results are expressed as mean ± standard error. Multiple comparisons were performed between the test materials at the significance level of α = 0.01. Different letters indicate extremely significant differences.

[0092] Example 1: Changes in qGRB1 expression levels in rice plants after inoculation with rice stem borer

[0093] 1.1 Inoculation of rice with rice stem borer and extraction of total RNA from rice plants

[0094] Rice seeds of the insect-resistant strain WAS 174-B-3-5::C1 (also known as G257, hereinafter referred to as G257) and the susceptible strain MURGI BRINJ::IRGC 73109-1 (also known as C495, hereinafter referred to as C495) were sown in seedling trays containing sterilized soil and cultured in a greenhouse for 25 days before being transplanted into a net-covered house for single-plant planting. During the peak tillering stage, rice stem borer larvae were inoculated into the leaf sheaths of the rice plants, with three leaf sheaths inoculated per plant. At 0 h and 72 h post-inoculation, the inoculated stems were cut and rapidly frozen in liquid nitrogen, with three biological replicates for each sample. All samples were stored at -70℃ after liquid nitrogen freezing.

[0095] Total RNA was extracted using the TRIzol method. The integrity of the extracted RNA was initially assessed by 1.5% agarose gel electrophoresis. cDNA was synthesized by reverse transcription of the RNA using the Promega A3500 reverse transcription kit according to the manufacturer's instructions.

[0096] 1.2 Detection of qGRB1 gene expression by qRT-PCR

[0097] qGRB1 is a protein derived from rice, and its amino acid sequence is SEQ ID No. 1. The nucleotide sequence of the rice genome for qGRB1 is SEQ ID No. 3. Nucleotides 1-619 of SEQ ID No. 3 are exon 1, nucleotides 706-751 are exon 2, nucleotides 2657-2739 are exon 3, nucleotides 2829-2913 are exon 4, nucleotides 3277-3567 are exon 5, nucleotides 3672-3762 are exon 6, nucleotides 3909-4809 are exon 7, nucleotides 4897-5057 are exon 8, nucleotides 5174-5277 are exon 9, nucleotides 5416-5581 are exon 10, and nucleotides 5671-5814 are exon 11. The nucleotide sequence of the coding sequence (CDS) of qGRB1 is as follows: exon 5955-6061 is exon 12, exon 6201-6533 is exon 13, exon 6644-6850 is exon 14, exon 7012-7095 is exon 15, exon 7355-7488 is exon 16, exon 7647-7874 is exon 17, exon 7990-8161 is exon 18, exon 8657-8911 is exon 19, and exon 9007-9619 is exon 20.

[0098] The sequences of the qGRB1 gene (MSU Locus: LOC_Os11g37700.1; RAP Locus: Os11g0587600) were obtained from the Rice Genome Annotation Project database (http: / / rice.uga.edu / index.shtml / ). Specific primers were designed using Primer5, and the sequences are as follows:

[0099] qGRB1-RT-F: 5′-GCCAACTTCTGGACTTGATGCT-3′

[0100] qGRB1-RT-R: 5′-GGAACACCTGGAACTGCCTCA-3′

[0101] Using cDNA as a template and Actin as an internal reference gene, the quality of cDNA in each sample was detected by PCR, and the cDNA in each sample was homogenized. Take a 0.2 ml micro-PCR tube, prepare a 25 μL reaction system according to Table 1, mix gently, and centrifuge briefly.

[0102] Table 1. Real-time PCR reaction system (25 μL)

[0103]

[0104] The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃ for 10 min.

[0105] Using rice Actin as the reference gene, the primer sequences are as follows:

[0106] Actin-F: 5'-GACTCTGGTGATGGTGTCAGC-3'

[0107] Actin-R: 5'-GGCTGGAAGAGGACCTCAGG-3'

[0108] Real-time PCR was performed according to the instructions for SYBR Premix Ex Taq™ (TaKaRa Code NO. DRR041A). qRT-PCR analysis showed that the melting curves were all single-peaked, indicating good specificity of the amplified products, and the fluorescence curves accurately reflected the amplification results. In the insect-resistant rice variety G257, qGRB1 expression was induced by inoculation with rice stem borer larvae, and the expression level of qGRB1 was significantly upregulated 72 hours after inoculation. However, in the susceptible rice variety C495, the expression level of qGRB1 did not increase significantly 72 hours after inoculation. Figure 1 ).

[0109] Example 2: Construction of qGRB1 gene editing knockout vector and acquisition and identification of transgenic plants

[0110] 2.1 qGRB1 gene editing knockout target primer design and CRISPR / Cas9 vector construction

[0111] Based on the principles of gene editing, a specific 20bp region in the full-length qGRB1 cDNA sequence was selected as the target region for gene editing knockout through online BLAST homology analysis. The target sequence is the DNA shown at positions 227-246 of sequence 3 in the sequence listing, i.e., 5'-GGACGACCCGTTCGGTCGCG-3'. Annealing primers were designed based on the target sequence to synthesize DNA molecules containing the target sequence, which were then constructed into pCBSG032. The pCBSG032 recombinant vector was obtained, expressing Cas9 protein and sgRNA targeting the aforementioned target sequence. The pCBSG032 recombinant vector map is shown below. Figure 7 As shown, the nucleotide sequence of the recombinant vector pCBSG032 is as follows: Figure 9 As shown.

[0112] 2.2 Obtaining qGRB1-KO transgenic rice

[0113] The pCBSG032 recombinant vector was transformed into Agrobacterium EHA105 using the freeze-thaw method (Holsters et al., Molecular & General Genetics, 1978, 163(2):181-7). The japonica rice variety IC25690::IRGC 53973-1 (also called C1054, hereinafter referred to as C1054) was transformed using Agrobacterium-mediated transformation. Mature C1054 seeds were mechanically dehulled, and plump, smooth, and spotless seeds were selected, sterilized, and then inoculated onto induction medium for induction culture. Rice callus tissue with good appearance and growth vigor was selected as recipient material. The pCBSG032 recombinant vector was transformed into the rice callus tissue using Agrobacterium-mediated transformation. Transformation was performed using a culture medium containing 100 μM acetylsylcholine and Agrobacterium with an OD value of 0.3-0.5. The callus tissue soaked in the transformation medium was then placed on a co-culture medium and co-cultured at 28℃ in the dark for 50-55 h. Callus tissue without obvious Agrobacterium on the surface was selected and transferred to N6 antibacterial medium containing 2.0 mg / L 2,4-D and 500 mg / L cephalosporin, and cultured in the dark at 28℃ for 3-4 days. The callus tissue was then transferred to a selection medium and cultured for 30 days, with subculture every 10 days. Take fresh hygromycin-resistant callus, inoculate it in pre-medium, and culture it in the dark at 28°C for 7 days. Then place it in a light culture room (12h light / 12h dark) and continue to culture for 7 days. After that, transfer it to regeneration medium (250mL tissue culture bottle) and continue to culture under light until regenerated plants grow.

[0114] The culture medium formulation involved in this embodiment is as follows:

[0115] Table 2. Culture media and their formulations used for genetic transformation

[0116]

[0117] 2.3 Identification of qGRB1-KO transgenic positive lines

[0118] To obtain T1 generation qGRB1-KO positive transgenic lines, all transformant lines were amplified by PCR and sequenced using gene editing target information.

[0119] Gene editing target-specific primers are:

[0120] KGRB1-F: 5′-TCGATCTCGCAGTCGTTCC-3′,

[0121] KGRB1-R: 5′-ATATACCTGGAGGACGTTGGT-3′

[0122] Sequencing results showed that three homozygous mutants of the target sequence were obtained in the transformant lines: qgrb1-1 with a 1bp target insertion, qgrb1-2 with a 4bp target deletion, and qgrb1-3 with a 13bp target deletion. Figure 2 This indicates that the qGRB1 gene has been knocked out in the C1054 background.

[0123] Compared with wild-type rice C1054, the homozygous mutant qGRB1-1 has the following changes in the qGRB1 genome on two homologous chromosomes: In the rice C1054 genome, nucleotide A is inserted between nucleotides 243 and 244 of sequence 3 in the sequence listing, that is, a single base N is inserted between nucleotides 130 and 131 of the qGRB1 gene coding sequence shown in sequence 2 in the sequence listing, resulting in a frameshift mutation, thereby knocking out the qGRB1 gene;

[0124] Compared with wild-type rice C1054, the homozygous mutant qGRB1-2 has the following changes in the qGRB1 genome on two homologous chromosomes: In the rice C1054 genome, a total of 4 nucleotides are deleted from positions 241-244 of sequence 3 in the sequence listing, that is, nucleotides 128-131 of the qGRB1 gene coding sequence shown in sequence 2 in the sequence listing are deleted, resulting in a frameshift mutation, thereby knocking out the qGRB1 gene;

[0125] Compared with wild-type rice C1054, the homozygous mutant qGRB1-3 exhibits the following changes in the qGRB1 genome on two homologous chromosomes: In the rice C1054 genome, a total of 13 nucleotides are deleted from positions 237-249 of sequence 3 in the sequence listing, which is the deletion of nucleotides 124-136 of the qGRB1 gene coding sequence shown in sequence 2 in the sequence listing, resulting in a frameshift mutation that knocks out the qGRB1 gene.

[0126] Rice plants containing homozygous mutants qgrb1-1, qgrb1-2, and qgrb1-3 were transplanted into a greenhouse for cultivation. Individual plants were harvested to obtain T1 generation transgenic seeds, which were then propagated to obtain homozygous T2 generation seeds, resulting in three T2 generation qGRB1-KO transgenic rice plants (qgrb1-1, qgrb1-2, and qgrb1-3).

[0127] 2.4 Identification of rice stem borer resistance in qGRB1-KO transgenic lines

[0128] Wild-type C1054 and three T2 generation qGRB1-KO transgenic rice varieties (qgrb1-1, qgrb1-2, and qgrb1-3) were sown in seedling trays filled with nutrient soil treated with soil fungicide. After being cultured in a greenhouse for approximately 25 days, they were transplanted to a greenhouse at the Zhejiang Jinhua Academy of Agricultural Sciences for planting. Each material was planted in one row with 10 plants per row, with a total of three replicates. During the peak tillering period, the plants were artificially inoculated with larvae of the rice stem borer. To eliminate marginal effects, 5 to 6 individual plants from the middle of each material were selected for inoculation. One third-instar larva was inoculated into each tiller of each individual plant. Four weeks after inoculation, the number of tillers with insect bites at the base of the stem and the total number of tillers were investigated in the inoculated individual plants, and the dead heart rate of each individual plant was calculated (dead heart rate of individual plant = number of tillers bitten by insects / total number of tillers of individual plant × 100%).

[0129] Phenotypic results showed that the qGRB1-KO transgenic lines qgrb1-1, qgrb1-2, and qgrb1-3 were more susceptible to the rice stem borer than the wild-type C1054. After inoculation with the rice stem borer, the average single-plant dead heart rate of the three replicates (16 plants) of wild-type C1054 was 23.9%, that of the three replicates (16 plants) of qgrb1-1 was 72.9%, that of the three replicates (15 plants) of qgrb1-2 was 57.9%, and that of the three replicates (15 plants) of qgrb1-3 was 58.8%. The dead heart rate of all three qGRB1-KO transgenic lines was significantly higher than that of the wild-type C1054. Figure 4 ).

[0130] Example 3: Construction of qGRB1 gene overexpression vector and acquisition and identification of transgenic plants

[0131] 3.1 qGRB1 gene overexpression vector

[0132] The DNA molecule shown in Sequence 2 of the sequence listing was constructed into plasmid BGV002 to obtain the recombinant vector 35S::qGRB1. Recombinant vector 35S::qGRB1 contains the qGRB1 coding sequence shown in Sequence 2 of the sequence listing and can express the qGRB1 protein. The map of recombinant vector 35S::qGRB1 is shown below. Figure 8 As shown, the nucleotide sequence of the recombinant vector 35S::qGRB1 is as follows: Figure 10 As shown; among them, the nucleotide sequence of the recombinant vector 35S::qGRB1 nucleotide sequence from position 1172 to 5539 is the qGRB1 gene shown in Sequence 2 of the table.

[0133] 3.2 Obtaining transgenic plants overexpressing the qGRB1 gene

[0134] The overexpression vector 35S::qGRB1 obtained above was transformed into Agrobacterium EHA105 using the freeze-thaw method (Holsterset al., Molecular & General Genetics, 1978, 163(2):181-7). The japonica rice variety OS4::IRGC 11335-1 (also called G20, hereinafter referred to as G20) was transformed using Agrobacterium-mediated transformation to obtain T0 generation qGRB1 overexpression transgenic rice (abbreviated as T0 generation qGRB1-OE).

[0135] The specific procedures for rice genetic transformation are as shown in step 2.2 of Example 2.

[0136] 3.3 Molecular identification of transgenic plants overexpressing the qGRB1 gene

[0137] The marker genes hpt / hyg were used to detect the T0 generation qGRB1-OE plants by PCR. The results were analyzed by 1.0% agarose gel electrophoresis and observation under UV light. The marker gene-specific primers were:

[0138] Hyg-F: 5′-CTATTTCTTTGCCCTCGGAC-3′,

[0139] Hyg-R: 5′-CCTGACCTATTGCATCTCCC-3′

[0140] PCR results showed that 16 T0 generation transformed plants under the G20 background exhibited obvious positive bands, indicating they were T0 generation qGRB1-OE positive plants. Other plants showed specific bands consistent with the hygromycin gene in the plasmid vector, demonstrating that the T-DNA carrying the hpt / hyg marker gene in the transgenic plants had been inserted and integrated into the recipient genome. Figure 5 T0 generation qGRB1-OE positive plants were transplanted to a greenhouse for cultivation, and individual plants were harvested to obtain T1 generation transgenic seeds. These were then propagated to obtain homozygous T2 generation seeds. In subsequent experiments, T2 generation qGRB1-OE positive lines numbered GRO-1, GRO-2, and GRO-3 were selected as rice materials.

[0141] T2 generation qGRB1-OE positive lines numbered GRO-1, GRO-2, and GRO-3 were selected. The changes in qGRB1 expression levels in each line were analyzed using qRT-PCR to identify the overexpression efficiency of the transgenic lines. Results are as follows: Figure 3 , Figure 3 The expression levels of qGRB1 in GRO-1, GRO-2, and GRO-3 were significantly upregulated compared to wild-type G20, with qGRB1 expression in GRO-3 being upregulated by about 9-fold.

[0142] 3.4 Identification of rice stem borer resistance in transgenic lines overexpressing the qGRB1 gene

[0143] Seeds of wild-type G20 and T2 generation qGRB1-OE positive lines numbered GRO-1, GRO-2, and GRO-3 were sown in sterilized nutrient soil seedling trays and cultured in a greenhouse for approximately 25 days before being transplanted to a greenhouse at the Zhejiang Jinhua Academy of Agricultural Sciences. Each material was planted in one row with 10 plants per row, with three replicates. During the peak tillering period, *Taxodium difficile* larvae were artificially inoculated. To eliminate marginal effects, 5 to 6 individual plants from the middle of each material were selected for inoculation. One third-instar larva was inoculated onto each tiller of each individual plant. The dead heart rate of individual plants was measured four weeks after inoculation.

[0144] The method for determining the dead heart rate is the same as in Example 2, section 2.4.

[0145] Phenotypic results showed that after inoculation with the rice stem borer, the average dead heart rate per plant was 63.8% for wild-type G20, 41.1% for GRO-1, 40.5% for GRO-2, and 42.3% for GRO-3. The dead heart rate of transgenic lines overexpressing the qGRB1 gene was significantly lower than that of wild-type G20. Figure 6 The above results indicate that the qGRB1-OE transgenic lines GRO-1, GRO-2, and GRO-3 exhibit enhanced resistance to rice stem borer compared to the wild-type G20.

[0146] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of biomaterials for regulating the expression of protein-coding genes, characterized by: The application is any one of the following: D1) Application of biomaterials that regulate the expression of the protein-coding genes in improving resistance to rice stem borer; D2) Application of biomaterials that regulate the expression of the protein-coding genes in the preparation of reagents to improve resistance to rice stem borer; D3) Application of biological materials that regulate the expression of the protein-coding genes in the preparation of reagents for cultivating rice resistant to rice stem borer; D4) The application of biological materials that regulate the expression of the protein-coding genes in rice breeding, wherein the purpose of rice breeding is to cultivate rice resistant to rice stem borer; The protein is either A1) or A2): A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1); The biomaterial is any one of B1) to B7) below: B1) The nucleic acid molecule that encodes the protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), transgenic plant organs containing the expression cassette described in B2), or transgenic plant organs containing the recombinant vector described in B3).

2. The application according to claim 1, characterized in that: B1) The nucleic acid molecule is any one of the DNA molecules shown in b1) to b3) below: b1) The coding sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2; b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 3; b3) has 80% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1.

3. A method for improving rice resistance to the rice stem borer, comprising introducing the gene encoding the protein of claim 1 into recipient rice to obtain target rice with higher resistance to the rice stem borer than the recipient rice.

4. The method as described in claim 3, characterized in that: The gene encoding the protein is a DNA molecule shown in any one of the following b1)-b3): b1) The coding sequence of the coding strand is the DNA molecule shown in SEQ ID No. 2; b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No. 3; b3) has 80% or more identity with the nucleotide sequence defined by b1) or b2) and is a DNA molecule encoding the protein of claim 1.

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