CN114805512A - Rice OsBBR3 gene and protein coded by same and application of rice OsBBR3 gene - Google Patents

Abstract

The invention discloses a rice OsBBR3 gene and a protein coded by the same and application thereof. The invention specifically discloses that the OsBBR3 protein is any one of the following proteins: (a1) protein with an amino acid sequence of SEQ ID No. 6; (a2) a protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue on the amino acid sequence shown in SEQ ID No.6, has more than 80 percent of identity with the protein shown in a1), and has the same function; (a3) a fusion protein obtained by attaching a tag to the end of the protein defined in any one of (a1) to (a 2). The rice OsBBR3 gene has the function of positively regulating and controlling the rice bacterial blight resistance, and the rice bacterial blight resistance can be obviously improved by over-expressing the coding sequence of the OsBBR3 gene in the rice.

Description

Rice OsBBR3 gene and protein coded by same and application of rice OsBBR3 gene

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a rice OsBBR3 gene, and a protein coded by the gene and application of the gene.

Background

The bacterial leaf blight of rice is an important bacterial disease in the global rice cultivation, and the cultivation of disease-resistant varieties by using resistance genes is the most economic and effective measure for preventing and treating the bacterial leaf blight of rice at present. To date, domesticIn addition, 46 rice bacterial leaf blight resistance genes have been reported: (http://www.shigen.nig.ac.jp/rice/oryzabase/gene/list). However, the disease resistance gene derived from wild rice is difficult to utilize; part of the resistance genes only have adult-stage resistance; the resistance spectrum of most resistance genes is narrow. The rice bacterial blight has complex diversity and high variability, and production practices show that after the disease-resistant variety carrying a single major gene is popularized and planted in a large area, potential toxic microspecies rise to dominant microspecies or new toxic microspecies appear after the germ varies, so that the resistance of the variety is easily lost.

With the development of molecular biology technology, disease-resistant genes can be introduced into susceptible varieties through hybridization or transgenosis, and the disease resistance of improved plants can be improved. Therefore, the identification of the disease-resistant or disease-susceptible gene of the rice bacterial leaf blight has important significance for improving the resistance of the rice.

Disclosure of Invention

The problem to be solved by the invention is how to improve or modify the bacterial blight resistance of plants.

In order to solve the problems in the prior art, the invention provides an application of a substance for expressing a protein or a regulatory gene or regulating the activity or content of the protein in regulating and controlling plant disease resistance, wherein the application can be any one of the following:

p1) protein or the expression substance of the regulatory gene or the substance regulating the activity or content of the protein in regulating the bacterial blight resistance of plants,

p2) protein or substance regulating the expression of the gene or substance regulating the activity or content of the protein in cultivating plants resistant to bacterial blight,

p3) protein or the expression substance of the regulation gene or the application of the substance for regulating the activity or the content of the protein in improving the disease resistance germplasm resources of plants, wherein the disease resistance is the resistance to bacterial blight;

the protein may be any of the following:

(a1) protein with an amino acid sequence of SEQ ID No. 6;

(a2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the amino acid sequence shown in SEQ ID No.6, has more than 80% of identity with the protein shown in a1) and has the function of regulating and controlling the disease resistance of plants, for example, a person skilled in the art can substitute, delete and/or add more than one amino acid according to the amino acid sequence shown in SEQ ID No.6, conservative substitution of the amino acid and other conventional technical means in the field on the premise of not influencing the activity of the protein to obtain an OsBBR3 protein mutant which has more than 80% of identity with the amino acid sequence shown in SEQ ID No.6 and has the function of regulating and controlling the disease resistance of plants;

(a3) a fusion protein obtained by attaching a tag to the end of the protein defined in any one of (a1) to (a 2).

The amino acid sequence of the protein of (a2) above may be SEQ ID No. 3.

The protein of (a1) above was named OsBBR 3. (a2) The protein is OsBBR3 mutant.

In order to facilitate purification or detection of the protein of (a1), a tag protein may be attached to the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence shown by SEQ ID No.6 of the sequence Listing.

Such tag proteins include, but are not limited to: GST (glutathione mercaptotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The nucleotide sequence encoding the protein OsBBR3 of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution or point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of the protein OsBBR3 isolated in the invention are derived from the nucleotide sequence of the invention and are identical with the sequence of the invention as long as the encoded protein OsBBR3 has the function of the protein OsBBR 3.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

Herein, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

Herein, the 80% or greater 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.

Herein, the 90% or greater identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

The protein described in the above application is derived from rice (Oryza sativa L.).

Herein, the substance regulating the activity and/or content of the protein may be a substance regulating the expression of a gene encoding the protein OsBBR 3.

As above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 controls: 1) regulation at the level of transcription of said gene; 2) regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In the above application, the substance increasing, increasing or up-regulating the gene expression and the substance regulating the activity or content of the protein may be biomaterials related to the protein, and the biomaterials may be any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) said nucleic acid molecule, or a recombinant microorganism containing B2) said expression cassette, or a recombinant microorganism containing B3) said recombinant vector;

B5) a transgenic plant cell line containing the nucleic acid molecule according to B1) or a transgenic plant cell line containing the expression cassette according to B2);

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

In the above application, the nucleic acid molecule of B1) may be any one of:

C1) a DNA molecule having the nucleotide sequence of SEQ ID No. 4;

C2) a cDNA molecule with a nucleotide sequence of SEQ ID NO. 5;

C3) the coding sequence is cDNA molecule or DNA molecule of 1-126 site of SEQ ID NO.5 in the sequence table;

C4) a DNA molecule which has 90% or more than 90% identity with the nucleotide sequence defined by C1), C2) or C3), is derived from rice and encodes the protein;

C5) a DNA molecule which hybridizes with the nucleotide sequence limited by C1), C2) or C3) under strict conditions and codes the protein.

The DNA molecule shown in SEQ ID No.5 (OsBBR 3 gene for regulating plant disease resistance) encodes protein OsBBR3 with the amino acid sequence shown in SEQ ID No. 6.

The OsBBR3 gene can be any nucleotide sequence capable of coding protein OsBBR 3. In view of the degeneracy of the codons and the preference of codons for different species, one skilled in the art can use codons suitable for the expression of a particular species as needed.

B1) The nucleic acid molecule also can comprise a nucleic acid molecule obtained by codon preference modification on the basis of the nucleotide sequence shown in SEQ ID No. 5.

The nucleic acid molecule also comprises a nucleic acid molecule which has more than 95 percent of identity with the nucleotide sequence shown in SEQ ID No.5 and is of the same species as the source.

The nucleic acid molecules described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be an RNA, such as a gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

Vectors described herein are well known to those skilled in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. In particular, the vector pGWC or the vector pMDC43 can be used.

The recombinant expression vector containing the OsBBR3 gene can be constructed by using the existing plant expression vector. The plant expression vector includes but is not limited to binary agrobacterium vector, plant microprojectile bombardment vector, etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3 'end of the mRNA precursor, and the untranslated regions transcribed from the 3' end of genes including but not limited to Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopalin synthase Nos), plant genes (e.g., soybean storage protein genes) all have similar functions.

When the OsBBR3 gene is used for constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CAMV)35S promoter and maize ubiquitin promoter (ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.), or chemical agent resistance marker genes (e.g., herbicide resistance gene), etc., which are expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

Any vector capable of guiding the expression of the exogenous gene in the plant is utilized to introduce the OsBBR3 gene or the gene segment provided by the invention into plant cells or receptor plants, so that a transgenic cell line with improved disease resistance and a transgenic plant can be obtained. The expression vector carrying the OsBBR3 gene can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and to culture the transformed plant tissues into plants.

Alternatively, B3) is a plasmid having a DNA molecule represented by SEQ ID No.4 or SEQ ID No.5, such as pMDC43-OsBBR3 prepared in the examples below.

Optionally, according to the above use, the disease resistance is resistance of the plant to bacterial blight.

The regulation of the disease resistance of the plant may be an improvement in the disease resistance of the plant, for example, a reduction in the length of a lesion manifested by rice bacterial blight.

The invention also provides a method for enhancing the bacterial blight resistance of plants, which comprises the step of enhancing the disease resistance of the plants by enhancing and/or increasing the expression level of the coding gene of the protein in the target plants or/and enhancing and/or increasing the activity and/or content of the coding gene of the protein.

The invention also provides a method for cultivating the plant resisting the bacterial blight, which comprises improving and/or increasing the expression of the coding gene of the protein and/or the content and/or the activity of the protein in the original plant, or/and improving and/or increasing the activity and/or the content of the coding gene of the protein to obtain the plant resisting the bacterial blight.

The above-mentioned improvement and/or increase in the expression level of the gene encoding the above-mentioned protein in a target plant, or/and the improvement and/or increase in the activity and/or content of the gene encoding the above-mentioned protein are achieved by introducing the gene encoding the above-mentioned protein into the target plant.

As an embodiment of the present invention, the method for breeding a plant resistant to bacterial blight comprises the steps of:

(1) constructing an expression vector containing an OsBBR3 gene shown as SEQ ID No.4 or an OsBBR3 gene coding sequence shown as SEQ ID No. 5;

(2) introducing the expression vector constructed in the step (1) into a plant;

(3) and screening and identifying to obtain the bacterial blight resistant plant.

The invention also provides a method for preparing the plant for improving the bacterial blight resistance, which can replace LOC _ Os05g26630 in plant genome DNA by DNA with a sequence shown as SEQ ID NO.4 or SEQ ID NO.5 ^Hap1 DNA shown in SEQ ID No.1 or SEQ ID No.2 in the coding gene is used for obtaining the plant with improved bacterial leaf blight resistance.

The plant may be E1) or E2) or E3) or E4) or E5): E1) monocotyledonous or dicotyledonous plants, E2) plants of the order gramineae, E3) plants of the family gramineae, E4) plants of the genus oryza, E5) rice.

The OsBBR3 protein or the biological material also belongs to the protection scope of the invention.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the tag is a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

Herein, the introduction may be to transform the vector carrying the DNA molecule of the present invention into a host bacterium by any known transformation method such as chemical transformation or electroporation. The introduced DNA molecule may be in single or multiple copies. The introduction may be the integration of the foreign gene into the host chromosome or the extrachromosomal expression from a plasmid.

The OsBBR3 gene is cloned in the embodiment of the invention, and the resistance of rice to bacterial leaf blight is obviously improved by transgenic over-expression of the gene.

The invention discovers that the rice OsBBR3 gene has the function of positively regulating and controlling the bacterial blight resistance of rice, and the resistance of the rice to the bacterial blight can be obviously improved by over-expressing the coding sequence of the OsBBR3 gene in the rice. The OsBBR3 gene can be used for improving the resistance of rice to bacterial blight and has important significance for cultivating new varieties of bacterial blight resistant rice.

Drawings

FIG. 1 is a map of an OsBBR3 gene expression vector constructed in example 2 of the present invention.

FIG. 2 shows the PCR identification result of the transgenic plant with OsBBR3 gene over-expression in example 4 of the present invention; wherein M is DNA molecular weight standard (DL2000 DNA marker), V is positive control, CK is negative control, OE-1 and OE-2 are OsBBR3 gene overexpression transgenic plants.

FIG. 3 shows the results of analyzing the relative expression level of OsBBR3 gene over-expressing transgenic plants in example 4 of the present invention.

FIG. 4 shows the lesion length of bacterial blight strain GD1358 inoculated with transgenic lines overexpressing genes of Nipponbare (Nip) and OsBBR3 in example 5 of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative experiments in the following examples, unless otherwise specified, were set up in triplicate.

In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

The vector pGWC: from Biovector NTCC type culture Collection.

Vector pMDC 43: from Biovector NTCC type culture Collection.

Agrobacterium tumefaciens EHA 105: from Biovector NTCC type culture Collection.

Bacterial leaf blight strain GD1358, described in the article "Fangzhong, Ching, Chong, Yin Shang Zhi, Wushang Zhong, Xuanming, Qi, research of pathogenic type of bacterial leaf blight of Chinese rice, report of plant pathology, 1990,20(2): 81-88", publicly available from the institute of crop science of Chinese academy of agricultural sciences.

The biological material is publicly available to the applicant for rice variety Nipponbare, IRIS _ 313-.

LOC _ Os05g26630 mainly comprises 2 single coding proteins with different lengths in 3K germplasm resourcesDoublet type, LOC _ Os05g26630 respectively ^Hap1 And LOC _ Os05g26630 ^Hap2 。LOC_Os05g26630 ^Hap1 The sequence (genome sequence) is shown as SEQ ID No.1, the 1 st to 271 th sites are 5 'UTR sequence, the 272 and 523 th sites are exon sequence, the 524 and 759 th sites are 3' UTR sequence, the coding sequence is shown as SEQ ID No.2, and the coded protein sequence is shown as SEQ ID No. 3. LOC _ Os05g26630 ^Hap2 The sequence is shown as SEQ ID No.4, the 1 st to 271 th sites are 5 'UTR sequence, the 272 and 523 th sites are exon sequence, the 524 and 759 th sites are 3' UTR sequence, the coding sequence is shown as the 1 st to 126 th sites of SEQ ID No.5, and the coded protein sequence is shown as SEQ ID No. 6. LOC _ Os05g26630 ^Hap2 Is named OsBBR 3.

Example 1 cloning of Rice LOC _ Os05g26630 Gene

1. Acquisition of genome sequence of rice LOC _ Os05g26630 gene

Extracting the DNA of the leaves of the rice variety Nipponbare and IRIS _313-11058, taking the DNA as a template, and using a primer OsBBR 3-F: 5'-ACCAAACTCCCTCGAGTCTCTCC-3' and OsBBR 3-R: 5'-CCACAATGATGGTTCGATCAAG-3', PCR amplification was performed using PrimeSTAR GXL DNA Polymerase (Code: R050A, Takara) to obtain an amplification product (i.e., the genomic sequence of LOC _ Os05g26630 gene). The LOC _ Os05g26630 gene represents a nucleotide sequence shown as SEQ ID NO.1 of a genome sequence of Nipponbare of a variety in Hap 1; the nucleotide sequence shown as SEQ ID NO.4 of the genome sequence in the Hap2 representative variety IRIS _ 313-11058.

2. Obtaining of coding sequence of rice LOC _ Os05g26630 gene

Extracting total RNA of leaves of a rice variety Nipponbare and IRIS _313-11058, synthesizing cDNA by adopting FastKing gDNA Dispelling RT SuperMix (Code: KR118, TIANGEN), taking the cDNA as a template, and utilizing a primer OsBBR 3-CDS-F: 5'-ATGAGGGAGAGGGTGGCTGC-3' and OsBBR 3-CDS-R: 5'-TCACCCAAGGCTCGTC-3', PCR amplification was performed using PrimeSTAR GXL DNA Polymerase (Code: R050A, Takara) to obtain an amplification product (i.e., the coding region of LOC _ Os05g26630 gene). The LOC _ Os05g26630 gene represents a nucleotide sequence shown as SEQ ID NO.2 of a Nipponbare coding sequence of a variety in Hap1, and an amino acid sequence of a coding protein is shown as SEQ ID NO. 3; the coding sequence in the Hap 2-representing variety IRIS _313-11058 is the nucleotide sequence shown in the 1 st to 126 th sites of SEQ ID NO.5, and the amino acid sequence of the coded protein is shown in SEQ ID NO. 6.

Example 2 construction of expression vector of representative variety of Rice disease-resistant Gene OsBBR3

The CDS sequence (the nucleotide sequence shown in the 1 st-126 th position of SEQ ID NO. 5) of the OsBBR3 gene in IRIS _313-11058 is transferred to an expression vector pMDC43 from an entry vector pGWC of a target gene OsBBR3 by a Gateway system to obtain an expression vector pMDC43-OsBBR3 containing the OsBBR3 gene. The operation steps are as follows:

(1) extracting total RNA of rice varieties IRIS _313-11058, carrying out reverse transcription to obtain cDNA, taking the cDNA as a template, and utilizing a forward primer OsBBR 3-CDS-F: 5'-ATGAGGGAGAGGGTGGCTGC-3' (SEQ ID No.9) and a reverse primer OsBBR 3-CDS-R: 5'-TCACCCAAGGCTCGTC-3', performing PCR amplification to obtain amplification product (SEQ ID NO.5, containing coding sequence (CDS) of OsBBR 3), and cutting and recovering.

(2) A treatment is carried out on the cut rubber recovery product obtained in the step (1), and the specific steps are as follows: mu.L of the recovered product was mixed with 20. mu.L of PCR Supermix (Code: AS111-11, TRANSGEN BIOTECH) to carry out PCR reaction. Reaction procedure: storing at 95 deg.C for 5min, 72 deg.C for 20min, and 4 deg.C. The PCR product was then purified and recovered using a common DNA product purification kit (Code: DP204-02, TIANGEN).

(3) And (3) carrying out TA cloning connection on the recovered product obtained in the step (2) and a vector framework obtained by carrying out Eam1105 enzyme digestion on an entry vector pGWC to obtain a recombinant vector containing a DNA fragment shown by SEQ ID NO.5 and with a correct sequence, and naming the recombinant vector as a positive entry cloning plasmid pGWC-OsBBR 3.

(4) And (3) carrying out LR reaction on the positive phylum-entering clone plasmid pGWC-OsBBR3 obtained in the step (3) and a target vector pMDC43 to obtain a recombinant vector named pMDC43-OsBBR3 with a correct sequence and containing the DNA fragment shown in SEQ ID NO.5, wherein the map of the recombinant vector is shown in figure 1. The nucleotide sequence shown in the 1 st to 126 th positions of SEQ ID NO.5 is the CDS sequence of the OsBBR3 gene.

LR reaction system: pGWC-OsBBR 31 uL (50-100ng), vector pMDC 431 uL (50-100ng), LR enzymemix 0.5 uL.

LR reaction conditions: and (3) incubating for 6h at 25 ℃, transforming the Escherichia coli DH5 alpha in the reaction system, and screening positive clones to obtain a correct expression vector pMDC43-OsBBR3 containing the OsBBR3 gene. pMDC43-OsBBR3 is a recombinant expression vector obtained by replacing the fragment between attR1 site and attR2 site of pMDC43 vector with attL1-OsBBR3-attL2, and keeping the other nucleotides of pMDC43 unchanged. pMDC43-OsBBR3 contains OsBBR3 gene CDS sequence shown in SEQ ID NO.5 in the sequence table and 35S promoter, can express OsBBR3 protein shown in SEQ ID NO.6 and GFP fusion protein, and the expression of the protein is driven by 35S promoter.

Example 3 obtaining of transgenic Rice

Transgenic rice was prepared using pMDC43-OsBBR3 of example 2. The Nipponbare is used as a starting plant to prepare transgenic rice, wherein the Nipponbare of a rice variety shows moderate disease resistance to a bacterial blight strain GD1358 in China. The method comprises the following specific steps:

(1) taking out the mature seeds of the plants, shelling, selecting the full, smooth and clean seeds without bacterial plaque, and sterilizing.

(2) Inoculating the sterilized seeds to an induction culture medium, culturing in the dark at 28 ℃ for about 14 days, and selecting the callus with good appearance and good growth power.

(3) The recombinant vector pMDC43-OsBBR3 constructed in example 2 is introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant strain, which is named as EHA105/pMDC43-OsBBR 3.

(4) And (4) taking the recombinant bacteria obtained in the step (3), and suspending the bacteria by using an infection culture medium to obtain an EHA105/pMDC43-OsBBR3 bacterial suspension.

(5) And (3) soaking the Nipponbare callus obtained in the step (2) in the EHA105/pMDC43-OsBBR3 bacterial suspension prepared in the step (4) for 20 min. Pouring off the bacterial suspension after infection, taking the callus, sucking water by using sterile filter paper, then placing on a co-culture medium added with acetosyringone and glucose, and culturing for 50-55h in dark at 28 ℃.

(6) After the step (5) is completed, selecting the callus without obvious agrobacterium on the surface, transferring the callus into an antibacterial culture medium added with the cefamycin, and carrying out dark culture for 3-4 days at 28 ℃.

(7) Transferring the cultured callus to a screening culture medium added with hygromycin and cefamycin, and culturing the callus in dark at 28 ℃ for 30 days, wherein subculture is carried out once every 10 days.

(8) And (3) after the step (7) is finished, taking the fresh hygromycin-resistant callus, inoculating the callus into a pre-regeneration culture medium, performing dark culture at 28 ℃ for 7 days, then placing the callus into a light culture room (12h light/12 h dark), continuing to perform culture for 7 days, transferring the callus onto a regeneration culture medium, and continuing to perform light culture until a regeneration plant grows out to obtain a transgenic plant.

The transgenic plant obtained by using the recombinant vector pMDC43-OsBBR3 is marked as OsBBR3 transgenic plant.

The culture medium and the formula used for genetic transformation are as follows:

induction medium: CaCl ₂ ·2H ₂ O 440mg，KH ₂ PO ₄ 170mg，MgSO ₄ ·7H ₂ O 370mg，NH ₄ NO ₃ 1650mg，KNO ₃ 1900mg，KI 0.83mg，CoCl ₂ ·6H ₂ O 0.025mg，H ₃ BO ₃ 6.2mg，Na ₂ MoO ₄ ·2H ₂ O 0.25mg，MnSO ₄ ·4H ₂ O 22.3mg，CuSO ₄ ·5H ₂ O 0.025mg，ZnSO ₄ ·7H ₂ O 8.6mg，Na ₂ -EDTA·2H ₂ O 37.3mg，FeSO ₄ ·7H ₂ 27.8mg of O, 10.1mg of VB10.5mg of VB60.5mg, 0.5mg of nicotinic acid, 100mg of inositol, 2mg of glycine, 2, 4-D2 mg, 2g of hydrolyzed casein, 30g of maltose, 3g of agar and deionized water to 1L.

Infection culture medium: see references for formulation methods: hiei Y, Ohta S, Komari T, et al, efficient transformation of rice (Oryza sativa, L.) mediated by Agrobacterium, and sequence analysis of the nucleic acids of the T-DNA [ J ] Plant Journal,1994,6(2):271-282. The concentration of acetosyringone in the reference was replaced with 200 μ M.

Co-culture medium: adding acetosyringone and glucose into the induction culture medium to make the final concentration of acetosyringone in the culture medium 200 μ M and the final concentration of glucose in the culture medium 10 g/L.

And (3) an antibacterial culture medium: the cefamycin is added into the induction culture medium, so that the final concentration of the cefamycin in the culture medium is 500 mg/L.

Screening a culture medium: hygromycin and cefamycin are added into an induction culture medium, so that the final concentration of the hygromycin in the culture medium is 65mg/L, and the final concentration of the cefamycin in the culture medium is 500 mg/L.

Pre-regeneration medium: CaCl ₂ ·2H ₂ O 440mg，KH ₂ PO ₄ 170mg，MgSO ₄ ·7H ₂ O 370mg，NH ₄ NO ₃ 1650mg，KNO ₃ 1900mg，KI 0.83mg，CoCl ₂ ·6H ₂ O 0.025mg，H ₃ BO ₃ 6.2mg，Na ₂ MoO ₄ ·2H ₂ O 0.25mg，MnSO ₄ ·4H ₂ O 22.3mg，CuSO ₄ ·5H ₂ O 0.025mg，ZnSO ₄ ·7H ₂ O 8.6mg，Na ₂ -EDTA·2H ₂ O 37.3mg，FeSO ₄ ·7H ₂ 27.8mg of O, 10.1mg of VB10.5 mg of VB60.5 mg of nicotinic acid, 100mg of inositol, 2mg of glycine, 2g of hydrolyzed casein, 30g of maltose, 3g of agar, 2mg of kinetin, 1mg of naphthylacetic acid and 1L of deionized water; hygromycin was added to a concentration of 50mg/L before being poured onto the plate.

Regeneration culture medium: CaCl ₂ ·2H ₂ O 440mg，KH ₂ PO ₄ 170mg，MgSO ₄ ·7H ₂ O 370mg，NH ₄ NO ₃ 1650mg，KNO ₃ 1900mg，KI 0.83mg，CoCl ₂ ·6H ₂ O 0.025mg，H ₃ BO ₃ 6.2mg，Na ₂ MoO ₄ ·2H ₂ O 0.25mg，MnSO ₄ ·4H ₂ O 22.3mg，CuSO ₄ ·5H ₂ O 0.025mg，ZnSO ₄ ·7H ₂ O 8.6mg，Na ₂ -EDTA·2H ₂ O 37.3mg，FeSO ₄ ·7H ₂ 27.8mg of O, 10.1mg of VB10.5 mg of VB60.5 mg of nicotinic acid, 100mg of inositol, 2mg of glycine, 2g of hydrolyzed casein, 30g of maltose, 6g of agar, 2mg of kinetin, 1mg of naphthylacetic acid and 1L of deionized water; hygromycin was added to a concentration of 50mg/L before being poured onto the plate.

Example 4 identification of transgenic Rice

1. Screening and identification of positive OsBBR3 transgenic plant

And (3) the plant to be detected: japanese nitrile (CK) and the OsBBR3 transgenic plants obtained in example 3.

Extracting the genome DNA of a plant to be detected, taking the genome DNA as a template, carrying out PCR amplification by using a primer pair consisting of pMDC43-TF (the sequence is 5'-GGAGACACCCTCGTCAACAG-3', which corresponds to the 1151-1170 bit fragment of pMDC43-OsBBR 3) and OsBBR3-CDS-R (the sequence is 5'-TCACCCAAGGCTCGTC-3', which corresponds to the 237-252 bit sequence of the sequence 5), using pMDC43-OsBBR3 plasmid as a positive control (V), and using receptor Nipponbare as a negative Control (CK).

The PCR amplification product is subjected to 1% agarose gel electrophoresis, positive control (V) and positive OsBBR3 overexpression transgenic plants show a 743bp band, and negative Control (CK) can not amplify any band. The electrophoretogram of a portion of the sample is shown in FIG. 2. Two positive OsBBR3 transgenic plants are selected and marked as OE-1 and OE-2 respectively.

2. Identification of positive OsBBR3 transgenic plant on RNA level

Extracting total RNA of positive OsBBR3 transgenic plants OE-1 and OE-2 and wild Nipponbare, carrying out reverse transcription, detecting the expression condition of the OsBBR3 gene on the RNA level by adopting qRT-PCR, wherein the used primers OsBBR 3-qF: 5'-AATCCTTCCTTACCTCGTGTTT-3' and OsBBR 3-R: 5'-CCACAATGATGGTTCGATCAAG-3' are provided. The primer UBQ-F is adopted: 5'-ACCTACTGCCCACCAAACTCCC-3' and primer UBQ-R: 5'-GCCCTCTAGCCACCAGCGAAA-3' detection of an internal reference gene (UBQ).

As a result, as shown in FIG. 3, the expression level of OsBBR3 gene in OE-1 and OE-2 was significantly increased compared to that of wild type Nippon (Nip), and the expression level was 3 times or more higher than that of wild type.

Positive OsBBR3 overexpression transgenic plants OE-1 and OE-2 are transplanted to a greenhouse for cultivation, and the plants are harvested individually to obtain T1 generation transgenic seeds, and then homozygous T3 generation seeds are obtained by propagation to obtain positive OsBBR3 overexpression transgenic plants OE-1 and OE-2 respectively, and the following experiments are carried out.

Example 5 identification of transgenic lines against bacterial blight

The plants to be tested are: nipponbare (Nip), positive OsBBR3 transgenic lines OE-1 and OE-2.

1. And culturing each plant line to be tested in a greenhouse for about 25 days, transplanting the plant lines to a net room for planting, planting the plant lines individually, and planting 20 plant lines in each plant line.

2. In the full tillering stage of the rice plant in the step 1, inoculating the rice plant with the bacterial blight strain GD1358, artificially inoculating the rice plant by a leaf-cutting method, and inoculating 5 leaves (the concentration of bacterial liquid is 1 multiplied by 10) to each plant ⁹ cfu/mL), the inoculum size was equal for each leaf, 40. mu.L.

3. The lesion length of the leaves of each plant was measured about 14 days after inoculation, each leaf had a lesion along the veins, and the lesion length of 3 inoculated leaves was measured for each plant, and the average value was calculated.

The statistical results are shown in fig. 4. The average lesion length of Nipponbare (Nip) is 3.9cm, the average lesion length of the OsBBR3 overexpression transgenic lines OE-1 and OE-2 is 1.3cm and 2.7cm respectively, and the average lesion length is obviously shorter than that of Nipponbare, so that the OsBBR3 gene can improve the disease resistance of rice to bacterial leaf blight. The results show that the OsBBR3 gene can positively regulate and control the resistance of rice to bacterial blight.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

<120> rice OsBBR3 gene and protein coded by same and application

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 759

<212> DNA

<213> Rice (Oryza sativa)

<400> 1

accaaactcc ctcgagtctc tccctcctcc ctcggtccct ctgattccct cctctttcta 60

tctctctccc actcttctcc ctcctccctt ctctctccac cgtcgccagt tgttgtcgcc 120

tccaacaccg ctggttgcag tcgcctccac tgctgcccag taccgctgtc acatcggcca 180

acggggaggc ctaggcctcc agatctagcg accccatcct tcccgctagt ggaactagct 240

agatagagcg ggccgagtac aagcgacgac gatgagggag agggtggctg cgcggggaag 300

gggccggcgc ggcgcaggga aggaggtggt ggcaacagca ccgggaaggg attggcggtg 360

gctcacgacg gtagaggtgc gggagagggt gcggcgagga ggggatccac tgatgccggg 420

cctcgggagc ggtgctggga aggggcagca gggactctcg gggttagcgc cacgggggag 480

gggacgacag ggaacggatc cggtgccgac gagccttggg tgagtggatc tggcgccacc 540

gagcctcagg agcagagcgg ggaagggggt gacggcggca cttggcggca gcgacagagg 600

gagcaaatct ggcaccgacc attcacaccc ttcctcggga gcacaaatcc ttccttacct 660

cgtgtttcgt gatgtgatgt gaattttttg tgcttgatct gtgatgtgga atttatgctc 720

tgtgatatga atttgatctt gatcgaacca tcattgtgg 759

<210> 2

<211> 252

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgagggaga gggtggctgc gcggggaagg ggccggcgcg gcgcagggaa ggaggtggtg 60

gcaacagcac cgggaaggga ttggcggtgg ctcacgacgg tagaggtgcg ggagagggtg 120

cggcgaggag gggatccact gatgccgggc ctcgggagcg gtgctgggaa ggggcagcag 180

ggactctcgg ggttagcgcc acgggggagg ggacgacagg gaacggatcc ggtgccgacg 240

agccttgggt ga 252

<210> 3

<211> 83

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Arg Glu Arg Val Ala Ala Arg Gly Arg Gly Arg Arg Gly Ala Gly Lys

1 5 10 15

Glu Val Val Ala Thr Ala Pro Gly Arg Asp Trp Arg Trp Leu Thr Thr Val

20 25 30

Glu Val Arg Glu Arg Val Arg Arg Gly Gly Asp Pro Leu Met Pro Gly Leu

35 40 45 50

Gly Ser Gly Ala Gly Lys Gly Gln Gln Gly Leu Ser Gly Leu Ala Pro Arg

55 60 65

Gly Arg Gly Arg Gln Gly Thr Asp Pro Val Pro Thr Ser Leu Gly

70 75 80

<210> 4

<211> 759

<212> DNA

<213> Rice (Oryza sativa)

<400> 4

accaaactcc ctcgagtctc tccctcctcc ctcggtccct ctgcctccct cctctttcta 60

tctctctccc actcttctcc ctcctccctt ctctctccac cgtcgccagt tgttgtcgcc 120

tccaacaccg ctggttgcag tcgcctccac tgctgcccag taccgctgtc acatcggcca 180

acggggaggc ctaggcctcc agatctagcg accccatcct tcctgctaat ggaactagct 240

agatagagcg ggccgagtac aagcgacgac gatgagggag agggtggctg cgcggggaag 300

gggccggcgc ggcgcaggga aggaggtggt ggcaacagca ccgggaaggg attggcggtg 360

gctcacgacg gtagaggtgc gggagagggt gcggtgagga ggggatccac tgatgccggg 420

cctcgggagc ggtgctggga aggggcagca gggactctcg gggttagcgg cacgggggag 480

gggacgacag ggaatggatc cggtgctgac gagccttggg tgagtggatc cggtgccacc 540

gagcctcagg agcagagcgg ggaagggggt gacggcggca cttggcgaca gcggcagagg 600

gagcaaatct ggcaccgacc attcacaccc ttcctcggga gcacaaatcc ttccttacct 660

cgtgcttcgt gatgtgatgt gaattttttg tgcttgatct gtgatgtgga atttatgctc 720

tgtgatatga atttgatctt gatcgaacca tcattgtgg 759

<210> 5

<211> 252

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgagggaga gggtggctgc gcggggaagg ggccggcgcg gcgcagggaa ggaggtggtg 60

gcaacagcac cgggaaggga ttggcggtgg ctcacgacgg tagaggtgcg ggagagggtg 120

cggtgaggag gggatccact gatgccgggc ctcgggagcg gtgctgggaa ggggcagcag 180

ggactctcgg ggttagcggc acgggggagg ggacgacagg gaatggatcc ggtgctgacg 240

agccttgggt ga 252

<210> 6

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Arg Glu Arg Val Ala Ala Arg Gly Arg Gly Arg Arg Gly Ala Gly Lys

1 5 10 15

Glu Val Val Ala Thr Ala Pro Gly Arg Asp Trp Arg Trp Leu Thr Thr Val

20 25 30

Glu Val Arg Glu Arg Val Arg

35 40

Claims (10)

1. The application is characterized in that: the application is the application of protein or an expression substance of a regulatory gene or a substance for regulating the activity or the content of the protein in regulating and controlling the bacterial blight resistance of plants, and the application is any one of the following applications:

p1) protein or the expression substance of the regulatory gene or the substance regulating the activity or content of the protein in regulating the bacterial blight resistance of plants,

p2) protein or substance regulating the expression of the gene or substance regulating the activity or content of the protein in cultivating plants resistant to bacterial blight,

p3) protein or the expression substance of the regulation gene or the application of the substance for regulating the activity or the content of the protein in improving the disease resistance germplasm resources of plants, wherein the disease resistance is the resistance to bacterial blight;

the protein is any one of the following proteins:

(a1) protein with an amino acid sequence of SEQ ID No.6,

(a2) a protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in SEQ ID No.6, has more than 80 percent of identity with the protein shown in a1) and has the function of regulating and controlling the bacterial blight resistance of plants,

(a3) a fusion protein obtained by attaching a tag to the end of the protein defined in any one of (a1) to (a 2).

2. Use according to claim 1, characterized in that: (a2) the amino acid sequence of the protein is SEQ ID No. 3.

3. Use according to claim 1 or 2, characterized in that: the protein is derived from rice.

4. Use according to claim 1 or 2, characterized in that: the substance regulating gene expression is a substance which increases or up-regulates the gene expression.

5. Use according to any one of claims 1 to 4, characterized in that: the substance regulating gene expression and the substance regulating the activity or content of the protein are biomaterials related to the protein, and the biomaterials are any one of the following B1) to B7):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) said nucleic acid molecule, or a recombinant microorganism containing B2) said expression cassette, or a recombinant microorganism containing B3) said recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

6. Use according to claim 5, characterized in that: the nucleic acid molecule is a DNA molecule shown in any one of the following items:

C1) a DNA molecule having the nucleotide sequence of SEQ ID No. 4;

C2) a cDNA molecule with a nucleotide sequence of SEQ ID NO. 5;

C3) the coding sequence is cDNA molecule or DNA molecule of 1-126 site of SEQ ID NO.5 in the sequence table;

C4) a DNA molecule having 90% or more 90% identity to a nucleotide sequence defined by C1), C2) or C3), derived from rice and encoding the protein of claim 1;

C5) a DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined by C1), C2) or C3) and encodes a protein as claimed in claim 1.

7. A method of enhancing resistance of a plant to bacterial blight, characterized by: the method comprises the steps of increasing and/or increasing the expression quantity of a gene coding for the protein in claim 1 in a target plant, or/and increasing and/or increasing the activity and/or content of the gene coding for the protein in claim 1, so as to enhance the disease resistance of the plant.

8. A method for producing a plant resistant to bacterial blight, which comprises increasing and/or increasing the expression level of a gene encoding the protein of claim 1 in a target plant, or increasing and/or increasing the activity and/or content of a gene encoding the protein of claim 1, to obtain a plant resistant to bacterial blight.

9. Use according to any one of claims 1 to 6, or a method according to claims 7 and 8, wherein: the plant is any one of the following plants:

E1) a monocotyledonous plant or a dicotyledonous plant,

E2) a plant of the order of the gramineae,

E3) a plant belonging to the family of the family Poaceae,

E4) a plant of the genus Oryza,

E5) a rice plant.

10. A protein as claimed in claim 1, 2 or 3, or a biomaterial as claimed in claim 5 or 6.

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