CN116042637B

CN116042637B - Rice glufosinate-ammonium-resistant herbicide genetic locus and application thereof

Info

Publication number: CN116042637B
Application number: CN202111260833.8A
Authority: CN
Inventors: 向成斌; 夏金球
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2021-10-28
Filing date: 2021-10-28
Publication date: 2025-09-09
Anticipated expiration: 2041-10-28
Also published as: CN116042637A

Abstract

A genetic locus of glufosinate-ammonium resistant herbicide for paddy rice and its application are disclosed. The application discloses a method for cultivating glufosinate-resistant plants, which comprises the steps of reducing or knocking out the expression of SPL10 genes in plants, so as to cultivate plants with stronger glufosinate resistance compared with wild type control plants.

Description

Rice glufosinate-ammonium-resistant herbicide genetic locus and application thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to an SPL transcription factor family gene OsSPL special for plants and application of coded 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), also known as glufosinate, 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, etc. 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 SPL10 which participates in the resistance of rice to glufosinate-ammonium and is related to the resistance of the rice except GS (glutamine synthetase) target point 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 the expression of the SPL10 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 SPL10 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 SPL10 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 SPL10 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 SPL10 gene is rice SPL10 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 SPL10 gene is reduced or knocked out, the mutation of cytosine to thymine at position 685 or the insertion mutation of a single nucleotide at position 50 in the nucleotide sequence corresponding to SEQ ID No. 1 in the coding region of the SPL10 gene results in a reduction or loss of the activity of the SPL10 gene expression product.

7. Use of the SPL10 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_Os06g44860 is verified by sequencing candidate mutant genes in glufosinate herbicide resistant mutant gar-2 (OsSPL 10).

FIG. 3 shows the osSPL mutant phenotypes in different settings before and after spraying. A and B are respectively the wild type of the background of the Longjing type 31 and the phenotype before and after spraying the drug of the gar-2 mutant, C and D are respectively the wild type of the background of the Tp309 and the phenotype before and after spraying the drug of the osSPL10 mutant, and the white scale is 10 cm.

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 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 SPL10 (squamosa protomer-binding-like 10) Gene is a Gene found in plants, for example, the SPL10 Gene (also called Os SPL10 or loc_os06g 44860) in rice (Oryza sativa l.) has a Gene ID number of 4341729, the SPL10 Gene in arabidopsis (Arabidopsis thaliana) has a Gene ID number of 839626, and the SPL10 Gene in grape (VITIS VINIFERA) has a Gene ID number of 100246757. In the context of the present application, the SPL10 gene includes the rice SPL10 gene (Os SPL 10) and SPL10 genes in other plants, where the SPL10 gene is homologous to the rice SPL10 gene and has the same or similar function in plants.

Example 1 obtaining and Gene location of Rice glufosinate-resistant mutant gar-2

A background wild type seed of the Oryza sativa Longjing 31 (LG 31) variety (provided by Pan monarch, national institute of agricultural sciences, heilongjiang province) was mutagenized by ethyl methylsulfonate (ETHYL METHYL sulfonate, EMS for short) (CAS: 62-50-0), and an M2 generation rice mutagenesis mutant library was created by selfing. The glufosinate-ammonium herbicide resistant mutant is screened by taking glufosinate with the spraying concentration of 2 g/L (the purity of the glufosinate is 95% -96% of that of the glufosinate of Shijia Ruiki chemical Co., ltd.) as screening conditions. The method comprises the steps of immersing M2 generation seeds in single-distilled water to accelerate germination for 2-3 days, then uniformly spreading the seeds in plant nutrient soil for normal germination and growth for about two weeks until rice grows to about three-leaf period, preparing glufosinate herbicide with the concentration of 2 g/L, uniformly spraying the herbicide on the overground parts of seedlings by using a watering can, and after 3-5 days, observing that leaves of the seedlings without glufosinate resistance lose green and yellow, and after 7-10 days, picking out seedlings which are still green in the overground parts and can grow normally as primary screening glufosinate herbicide-resistant positive seedlings (see figure 1), and moving the seedlings to the field for normal growth 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 and were designated glufosinate ammonium resistance 6-2 (gar 6-2).

The gar-2 mutant M3 generation seeds are germinated in the field and grow to the flowering period, the gar-2 mutant (serving as a male parent) is hybridized with LG31 (serving as a female parent), 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 those with glufosinate resistance were able to survive normally, and the BC1F2 segregating population of gar-2 mutants exhibited survival: death ≡1:3 (survival: death=79:234; χ ²= 0.001< χ² _(0.05) =3.84), thus indicating that the gene responsible for the gar-2 mutant with glufosinate resistance was a single-gene recessive genetic mutation inherited by the nucleus. Then, 30 seedlings with glufosinate resistance are selected from BC1F2 segregating groups of gar6-2, 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, thereby obtaining candidate genes involved in regulating and controlling glufosinate resistance, 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_Os06g44860/OsSPL10 gene coding region (LOC_Os06 g44860/OsSPL gene coding region) in gar-2 mutant genome is shown as SEQ ID NO:1, the coded amino acid sequence is shown as SEQ ID NO: 2), mutation is carried out at 685 th cytosine (C685T), the amino acid of the gene coding protein is caused to have missense mutation, tyrosine (H311V) is further verified by first generation sequencing, and the nucleotide sequence of the gene coding region of LOC_Os06g44860/OsSPL shown as SEQ ID NO:2, and the amino acid sequence of the gene coding region of LOC_Os06g44860/OsSPL gene coding region is shown as SEQ ID NO:2, and the amino acid sequence of the gene coding region coding for the gene coding for the glufosinate resistance of the gene coding region is shown as SEQ ID NO: 2-62, respectively, so that the herbicide coding region coding the gene coding to obtain the glufosinate resistance is shown as SEQ ID 6.

Example 2 verification of the anti-glufosinate phenotype of Rice osspl Gene knockout mutants

To further verify that the gar6-2 mutant obtained by screening was generated by a OsSPL10 gene loss of function, we obtained a rice Tp309 background wild-type seed and a Tp309 background seed of OsSPL gene knockout mutant osspl10 (provided by the university of south-Beijing M.Brian Traw professor task group) which had inserted a base thymine at position 50 in the OsSPL10 gene coding region, resulting in premature termination of translation of the gene.

Then, the LG31, gar6-2, tp309 and osspl gene editing mutant seeds are respectively planted in soil to germinate and grow for about 2 weeks to three leaf periods, and a glufosinate solution with the concentration of 2 g/L is sprayed uniformly together, so that gar-2 mutant on the background of LG31 can be observed to show stronger glufosinate resistance than that of LG31 on the background of LG31 after 3-5 days, as shown in fig. 3A-B, and similar to that, osspl gene editing mutant on the background of Tp309 shows stronger glufosinate resistance than that of Tp309 on the background of Tp309, as shown in fig. 3C-D. The experimental result again shows that OsSPL gene function deletion can obviously enhance the resistance of 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

1. A method of growing a glufosinate-resistant plant comprising reducing or knocking out expression of the SPL10 gene in the plant, thereby growing a plant that is more glufosinate-resistant than a wild-type control plant,

Wherein the plant is rice, and the protein encoded by the SPL10 gene consists of an amino acid sequence shown in SEQ ID NO. 2.

2. The method of claim 1, wherein the nucleic acid sequence of the transcription factor of SPL10 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 SPL10 gene expression in a plant.

3. The method of any one of claims 1-2, wherein the SPL10 gene is a rice SPL10 gene and the coding region thereof is the nucleotide sequence set forth in SEQ ID No. 1.

4. The method of any one of claims 1-2, wherein in the plant in which the expression of the SPL10 gene is reduced or knocked out, the mutation of cytosine to thymine at position 685 or the insertion mutation of a single nucleotide at position 50 in the nucleotide sequence of SEQ ID No. 1 in the coding region of the SPL10 gene results in a reduction or loss of the activity of the SPL10 gene expression product.

Use of the SPL10 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, wherein the plants are rice, wherein the protein encoded by the SPL10 gene consists of the amino acid sequence shown in SEQ ID No. 2.