CN116042638A - Novel glufosinate-ammonium-resistant herbicide genetic locus for rice and application thereof - Google Patents

Abstract

The application discloses a novel rice glufosinate-resistant herbicide genetic locus and application thereof, and a method for cultivating glufosinate-resistant plants, which comprises the steps of reducing or knocking out the expression of an SPL8 gene in plants, so that plants with stronger glufosinate resistance compared with wild control plants are cultivated.

Description

Novel glufosinate-ammonium-resistant herbicide genetic locus for rice and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a specific SPL transcription factor family gene OsSPL8 of plants and encoding proteins thereof in aspects of regulating and controlling plant resistance, especially rice resistance to glufosinate herbicide.

Background

Rice (Oryza sativa l.) is one of the most important food crops in the world, with nearly 90% of rice worldwide being produced and consumed in asia, and the yield and quality of rice directly affecting the people's living standard. Therefore, how to promote the yield increase and the quality increase of the rice has important significance for guaranteeing folk life and maintaining the agricultural economy stability. Meanwhile, the rice is used as a typical crop model plant, and has important reference value for researching other crops.

Crops inevitably suffer from various stresses in the life process, wherein weed attack is one of the most common stresses, can compete with crops for various beneficial resources, increases the risk of the crops suffering from pest attack, and seriously affects crop yield and quality. It is estimated that on average, annual crop yield loss due to weed attack is over 13% for global crops, corresponding to one year of ration for 10 million population worldwide. The traditional weed cleaning method needs to consume a large amount of manpower resources, and the use of the herbicide can save a large amount of manpower and material costs. Therefore, cultivation of crops with herbicide resistance has important significance for maintaining high yield of crops and guaranteeing grain quality.

Glufosinate (Glufosinate) is also known as Glufosinate, and is one of the most widely used herbicides at present, and is widely used due to its characteristics of broad spectrum, non-selectivity, low toxicity, environmental friendliness, and the like. At present, the cultivation of glufosinate-resistant crops is mainly realized by using a transgenic technology, however, the application of the transgenic technology in food crops is in great dispute, commercial production of transgenic crops in China is not yet opened, and in addition, crop germplasm resources with glufosinate resistance are not found in China, so that the cultivation of glufosinate-resistant crops has important significance and potential application value for the development of modern agriculture in China.

Disclosure of Invention

The invention aims to find a new gene SPL8 which participates in the resistance of rice to glufosinate-ammonium and is related to the resistance of the rice except GS (glutamine synthetase) target spot resistance, and the gene function deletion can obviously improve the resistance of the rice to glufosinate-ammonium. The invention has important reference value and potential application value for researching how to improve the resistance of plants to glufosinate herbicide. Specifically, the technical problems of the invention are solved by the following technical scheme.

1. A method of growing a glufosinate-resistant plant comprising reducing or knocking out expression of the SPL8 gene in the plant, thereby growing a plant that is more glufosinate-resistant than a wild-type control plant.

2. The method of item 1, wherein the nucleic acid sequence of the transcription factor of SPL8 in a wild-type plant or plant cell is edited, one or more bases are introduced or deleted, an antisense nucleotide technology, or an RNAi gene silencing technology is used to achieve a reduction or knockout of SPL8 gene expression in a plant.

3. The method of any one of clauses 1-2, wherein the plant is a monocot or a dicot.

4. The method of item 3, wherein the plant is rice, maize, wheat, soybean, cotton or canola.

5. The method of any one of clauses 1-2, wherein the protein encoded by the SPL8 gene comprises or consists of the amino acid sequence of:

(1) An amino acid sequence shown in SEQ ID NO. 2;

(2) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 2 by substitution, deletion or addition of one or more amino acids;

(3) An amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more of the amino acid sequence shown in SEQ ID NO. 2,

preferably, the SPL8 gene is rice SPL8 gene and the coding region thereof has a nucleotide sequence shown in SEQ ID NO. 1.

6. The method of any one of items 1-2, wherein in the plant in which the expression of the SPL8 gene is reduced or knocked out, the mutation of cytosine to thymine at position 556 or the insertion mutation of a single nucleotide at position 159 in the nucleotide sequence corresponding to SEQ ID No. 1 in the coding region of the SPL8 gene results in a reduction or loss of the activity of the SPL8 gene expression product.

Use of the SPL8 gene in the selection of glufosinate resistant plants, wherein the expression of the SPL10 gene is reduced or knocked out in glufosinate resistant plants compared to wild type control plants.

Drawings

FIG. 1 is a flow chart of screening of glufosinate-resistant mutants of rice.

FIG. 2 shows that LOC_Os04g56170 (OsSPL 8) is verified by sequencing the candidate mutant gene generation in the glufosinate herbicide resistant mutant gar-3.

FIG. 3 OSSL 8 mutant phenotype in different settings before and after spraying. A and B are respectively the phenotype before and after spraying the medicine of the rice Longjing 31 type background wild type and gar18-3 mutant; c and D are the rice Tp309 background wild type and osspl8 mutant pre-and post-spray phenotypes, respectively, with a white scale of 10cm.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

Definition of the definition

"homology" or "identity" or "similarity" refers to the degree of sequence similarity between two peptide chain molecules or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence, and can be compared by alignment. When there are identical bases or amino acids at positions in the sequences being compared, the molecules at that position are homologous. Homology between sequences is a function of the number of paired or homologous sites shared by the sequences. The "unrelated" or "non-homologous" sequences have less than 40% homology, but preferably less than 25% homology, to one of the sequences of the present application.

A polynucleotide or polynucleotide region (or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that when aligned, the percentage of bases (or amino acids) is the same when the two sequences are compared. Such alignments and percent homology or sequence identity may be determined using software programs known in the art, such as those described by Ausubel et al (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment.

The SPL8 (squamosa protomer-binding-like 8) Gene is a Gene found in plants, for example, the SPL8 Gene in rice (Oryza sativa l.) has a Gene ID number 4337258 (also referred to as loc_os04g 56170), the SPL8 Gene in arabidopsis thaliana (Arabidopsis thaliana) has a Gene ID number 839275, and the SPL8 Gene in peanut (Arachis hypogaea) has a Gene ID number 112735625. The SPL8 Gene in sunflower (Helianthus annuus) has a Gene ID number of 110923138. The SPL8 Gene in turnip (Brassica rapa) has Gene ID number 103844692. In the context of the present application, the SPL8 gene includes the rice SPL8 gene (Os SPL 8) and SPL8 genes in other plants, which are homologous to the rice SPL8 gene and have the same or similar functions in plants.

Example 1 obtaining and Gene location of Rice glufosinate-resistant mutant gar-3

Wild seeds (provided by Pan national Jun of the institute of agricultural sciences of Heilongjiang province) of the rice Longjing 31 (LG 31) variety background are mutagenized by ethyl methylsulfonate (Ethyl methyl sulfonate, EMS for short) (CAS: 62-50-0), and an M2-generation rice mutagenesis mutant library is created by selfing. The glufosinate-ammonium herbicide resistant mutant is screened by taking glufosinate with the spraying concentration of 2g/L (the purity of the glufosinate is 95% -96% of that of the glufosinate-ammonium of Shijia Ruiki chemical Co., ltd.) as screening conditions. The specific operation is as follows: the M2 generation seeds are soaked in single steam water to accelerate germination for 2-3 days, and then are uniformly spread in plant nutrient soil to normally germinate and grow for about two weeks until the rice grows to about three leaf stage; the glufosinate herbicide with the concentration of 2g/L is prepared and uniformly sprayed on the overground parts of seedlings by a spray can, the leaves of the seedlings without glufosinate resistance are observed to turn green and yellow after 3-5 days, the seedlings which are still green on the overground parts and can normally grow are selected as primary screening glufosinate herbicide-resistant positive seedlings (see figure 1) after 7-10 days, and the seedlings are moved to the field to normally grow until flowering and seed setting. After the seeds are mature, M3 generation mutant seeds are harvested and subjected to glufosinate-resistant rescreening by the same method. The above mutants were further determined by M3 generation screening for their glufosinate herbicide resistance phenotype, designated glufosinateammonium resistance18-3(gar18-3)。

Seeds of the M3 generation of the gar-3 mutant are germinated in the field and grown to a flowering period, the gar-18-3 mutant (serving as a male parent) and LG31 (serving as a female parent) are hybridized, and the BC1F1 generation hybrid seeds are harvested. Transplanting the BC1F1 generation hybrid seeds again and performing selfing on the seeds in the field, harvesting the BC1F2 generation seeds, and constructing BC1F2 character segregation populations.

The BC1F2 population was again screened for glufosinate herbicide tolerance and survival in the BC1F2 population was counted 7-10 days later. All seedlings without glufosinate resistance died, and the glufosinate resistant seedlings were able to survive normally, and the BC1F2 isolate of the gar-3 mutant appeared to survive: death is approximately 1:3 (survival: death=60:198; χ) ² ＝0.42<χ ² _(0.05) =3.84), thus indicating that the gene responsible for the glufosinate resistance of the gar18-3 mutant is a single-gene recessive genetic mutation inherited by the nucleus. Then, 30 seedlings with glufosinate resistance are selected from the BC1F2 segregating population of gar18-3, genomic DNA is extracted respectively, genomic DNA in the 30 samples is mixed in equal quantity, genome re-sequencing (Nanjing Ovison Biotechnology Co., ltd.) is carried out by using a MutMap method, so that candidate genes involved in regulating and controlling glufosinate resistance are obtained, then candidate gene mutation sites are further verified by first generation sequencing, gene association analysis is carried out by first generation sequencing, analysis results show that LOC_Os04g56170/OsSPL8 gene coding region (LOC_Os04 g56170/OsSPL8 gene coding region nucleotide sequence is shown as SEQ ID NO:1, amino acid sequence coded by the LOC_Os04g56170/OsSPL8 gene coding region is shown as SEQ ID NO: 2), the 556 th cytosine mutation is thymine (C556T) so that nonsense mutation occurs to the 186 th amino acid of the gene coding protein, peptide chain synthesis is terminated in advance, as shown in FIG. 2 (the nucleotide sequence of the gene mutated by the C556T mutation and the amino acid sequence of the polypeptide are shown as SEQ ID NO:1, and the amino acid sequence coded by the LOC_Os 04/OsSPL 8 gene coding region is shown as SEQ ID NO:1, so that the amino acid sequence coded by the amino acid sequence is shown as SEQ ID NO:2, and the amino acid sequence coded by SEQ ID 4 is shown in SEQ ID NO. 2, so that the amino acid encoding the coding sequence is shown respectively, and the coding gene has the coding activity is encoding position encoding coding gene.

Example 2 Rice osspl8 Gene knockout mutant anti-glufosinate phenotype validation

To further verify that the glufosinate resistant phenotype of the screening-obtained gar-3 mutant was produced by loss of OsSPL8 gene function, we obtained rice Tp309 background wild-type seed and Tp309 background seed of OsSPL8 gene knockout mutant OsSPL8 (provided by the university of Nanjing M.Brian Traw professor task group), which had an adenine and a base thymine inserted at position 159 of the coding region of OsSPL8 gene, respectively, resulting in premature termination of translation of the gene.

Then, respectively planting LG31, gar18-3, tp309 and osspl8 gene editing mutant seeds in soil to germinate and grow for about 2 weeks to a trefoil period, uniformly spraying glufosinate solution with the concentration of 2g/L together, and observing that gar18-3 mutant bodies in the LG31 background show stronger glufosinate resistance compared with the LG31 wild type after 3-5 days, as shown in fig. 3A-B; similarly, the osspl8 gene editing mutant in the context of Tp309 exhibited more glufosinate resistance than Tp309 wild type, as shown in figures 3C-D. The experimental result again shows that the OsSPL8 gene function deficiency can obviously enhance the resistance of the rice to glufosinate herbicide.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (7)

1. A method of growing a glufosinate-resistant plant comprising reducing or knocking out expression of the SPL8 gene in the plant, thereby growing a plant that is more glufosinate-resistant than a wild-type control plant.

2. The method of claim 1, wherein the nucleic acid sequence of the transcription factor of SPL8 in a wild-type plant or plant cell is edited, one or more bases introduced or deleted, an antisense nucleotide technique, or an RNAi gene silencing technique by a gene editing technique to achieve a reduction or knock-out of SPL8 gene expression in a plant.

3. The method of any one of claims 1-2, wherein the plant is a monocot or dicot.

4. The method of claim 3, wherein the plant is rice, maize, wheat, soybean, cotton or canola.

5. The method of any one of claims 1-2, wherein the protein encoded by the SPL8 gene comprises or consists of the amino acid sequence:

(1) An amino acid sequence shown in SEQ ID NO. 2;

(2) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO. 2 by substitution, deletion or addition of one or more amino acids;

(3) An amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more of the amino acid sequence shown in SEQ ID NO. 2,

preferably, the SPL8 gene is rice SPL8 gene and the coding region thereof has a nucleotide sequence shown in SEQ ID NO. 1.

6. The method of any one of claims 1-2, wherein in a plant in which expression of the SPL8 gene is reduced or knocked out, the mutation of cytosine to thymine at position 556 or the insertion mutation of a single nucleotide at position 159 in the nucleotide sequence corresponding to SEQ ID No. 1 in the coding region of the SPL8 gene results in a reduction or loss of activity of the SPL8 gene expression product.

Use of the SPL8 gene in the selection of glufosinate resistant plants, wherein the expression of the SPL10 gene is reduced or knocked out in glufosinate resistant plants compared to wild type control plants.

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