Gene etol3 related to length-width ratio of rice grain and application thereof

Abstract

The application discloses a gene OsETOL3 related to the length-width ratio of rice grains and application thereof, a nucleotide sequence of a coding region of the gene is shown in SEQ ID NO: 3. The gene of the application influences the length-width ratio of rice grains. By using gene editing technology to edit the gene OsETOL3, the editing family can significantly reduce the length-width ratio of rice grains. The OsETOL3 gene can be applied to rice breeding work as a gene influencing the size of rice grains in the future, and has great development value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bioengineering technology, and relates to the gene ETOL3, which is related to the length-to-width ratio of rice grains, and its applications. This invention also relates to the polypeptide sequence encoded by this gene, and a method for reducing the length-to-width ratio of rice grains through gene editing technology. Background Technology

[0002] Rice is an important food crop in my country and a crucial crop for ensuring the country's food security. Because rice grains vary widely in size, the length-to-width ratio directly determines the overall size of the rice grain and affects yield. Therefore, changing the length-to-width ratio of a variety can effectively influence grain size and potentially rice yield.

[0003] The length-to-width ratio of rice grains is a complex quantitative trait. Early researchers started with mutants, using map-based cloning and other methods to clone genes affecting the length-to-width ratio, such as OsGRF1 (Yang et al., 2021) and OsGRX8 (Hao et al., 2021). The discovery of mutants provided more options for rice genetic improvement. However, rice subpopulations are diverse and genetically diverse. Researchers have cloned multiple genes related to grain length-to-width ratio using methods such as parental QTL mapping, such as OsAUX3 (Qiao et al., 2021). With the widespread use of gene editing, researchers have edited genes to change the grain length-to-width ratio, such as GWD1 (Wang et al., 2021). The cloning of these genes provides usable genetic resources for rice breeding. While parental QTL mapping has limited haplotype detection capabilities, utilizing natural variation populations helps analyze different haplotypes of genes. Using the phenotypic differences corresponding to haplotypes can predict gene function, achieving rapid gene localization.

[0004] In other rice traits, such as grain length, haplotype analysis using natural variant populations of japonica rice has shown significant advantages (Si et al., 2016), but research on the length-to-width ratio in natural variant populations of japonica rice is limited. Our understanding of the genes affecting the length-to-width ratio in natural variant populations of japonica rice is still very limited.

[0005] The ETOL family of genes contains SKP1 / BTB / POZ domains and is primarily involved in pathways such as ethylene biosynthesis. Functions of ETOL-like genes have been reported in both Arabidopsis and rice. Rice has three ETOL genes: ETOL1 is associated with drought and flood tolerance; its mutants show better seed setting rate and biomass after drought treatment recovery, but grow slower under flood conditions. ETOL2 and ETOL3 do not have drought-related functions (Du et al., 2014). However, their specific functions are currently unknown, so our understanding of the effects of ETOL-like genes, especially ETOL3, on the length-to-width ratio of rice grains is still very limited. Summary of the Invention

[0006] The purpose of this invention is to provide a rice ETOL3 gene related to the length-to-width ratio of rice grains, its encoded protein, and its application in regulating the length-to-width ratio of rice grains.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] This invention discloses a rice ETOL3 gene. Through mutation detection of the ETOL3 gene and promoter in a population, this invention discovered that the gene is related to the length-to-width ratio of rice grains. Furthermore, gene editing was used to construct transgenic plants, verifying that knocking out the ETOL3 gene can reduce the length-to-width ratio of the grains. This gene has the nucleotide sequence described in (1) or (2) below:

[0009] (1) The coding region nucleotide sequence as shown in SEQ ID NO:3;

[0010] (2) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO:3 and encodes a protein with the same function.

[0011] The gene also has a genomic nucleotide sequence as shown in SEQ ID NO:2.

[0012] The protein encoded by the rice ETOL3 gene described above also falls within the scope of protection of this invention. This protein has the amino acid residue sequence shown in (a) or (b) below:

[0013] (a) The amino acid sequence as shown in SEQ ID NO:1;

[0014] (b) An amino acid sequence with the same function derived from the amino acid sequence shown in SEQ ID NO:1 by substitution and / or deletion and / or addition of one or more amino acid residues.

[0015] Expression cassettes, recombinant vectors, transgenic cell lines or transgenic recombinant bacteria containing the above-mentioned rice ETOL3 gene, or substances used to interfere with, inhibit, silence, target knockout or site-directed mutation of the rice ETOL3 gene, are all within the scope of protection of this invention.

[0016] Preferably, the substance used for interfering with, inhibiting, silencing, targeted knockout, or site-directed mutation of the rice ETOL3 gene includes at least one of the following (1) to (6):

[0017] (1) Interference sequences targeting the rice ETOL3 gene;

[0018] (2) Interference vectors for interfering with the rice ETOL3 gene;

[0019] (3) Transgenic cell lines containing rice ETOL3 gene interference vector;

[0020] (4) Primer sequences for targeted knockout or site-directed mutation of the rice ETOL3 gene based on gene editing technology;

[0021] (5) Vectors for targeted knockout or site-directed mutation of the rice ETOL3 gene based on gene editing technology;

[0022] (6) Cell lines based on gene editing technology that target knockout or site mutation of the rice ETOL3 gene.

[0023] The recombinant vector containing the rice ETOL3 gene can be a recombinant cloning vector or a recombinant expression vector, and the recombinant expression vector of the gene can be constructed using existing plant expression vectors.

[0024] The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microbombardment. These vectors may also contain the 3' untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist in the untranslated regions transcribed at the 3' end of Agrobacterium crown leptocyst-inducing (Ti) plasmid genes (such as the lipase N's gene) and plant genes (such as the soybean storage protein gene).

[0025] When constructing recombinant plant expression vectors using the aforementioned genes, any type of enhancing promoter or constitutive promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter or the maize ubiquitin promoter, can be added before the transcription initiation nucleotide. 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.

[0026] 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, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0027] The above-mentioned rice ETOL3 gene, the above-mentioned protein, or the above-mentioned expression cassette, recombinant vector, transgenic cell line or transgenic recombinant bacteria are used in regulating the length-to-width ratio of rice grains or in cultivating rice with low grain length-to-width ratio.

[0028] The aforementioned substances used to interfere with, suppress, silence, target knockout, or perform site-directed mutations of the rice ETOL3 gene are applied in reducing the length-to-width ratio of rice grains or in breeding rice with low length-to-width ratios. For example, the application of site-directed mutation vectors of the rice ETOL3 gene in reducing the length-to-width ratio of rice grains or in breeding rice with low length-to-width ratios.

[0029] The above-mentioned application specifically involves reducing the length-to-width ratio of rice grains by gene editing, interference, suppression, or silencing the expression of the rice ETOL3 gene, thereby cultivating rice with a low grain length-to-width ratio.

[0030] A method for breeding rice with low grain length-to-width ratio involves reducing the grain length-to-width ratio of rice by gene editing, interference, suppression, or silencing the rice ETOL3 gene.

[0031] The results of this invention show that introducing a gene-editing vector into recipient rice results in the editing of the rice ETOL3 gene, yielding transgenic rice. Compared to the recipient rice, the transgenic rice grains have a lower length-to-width ratio. The recipient rice is the japonica rice variety Zhonghua 11, but it is not limited to this variety.

[0032] The beneficial effects of this invention are:

[0033] The ETOL3 gene, which affects the length-to-width ratio of rice grains, is a key component of this invention. Knocking out this gene reduces the length-to-width ratio of rice grains, thus enabling the breeding of transgenic rice with low grain length-to-width ratios. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description

[0034] Figure 1 The variation of the 5UTR region of the ETOL3 gene in different rice varieties.

[0035] Figure 2 Statistical analysis of the length-to-width ratio of grains from different haplotype varieties.

[0036] Figure 3 Sequencing analysis of ETOL3 gene-edited families showed that CR1 and CR2 were homozygous knockout families.

[0037] Figure 4 Protein translation analysis of CR1 and CR2 families after ETOL3 gene editing.

[0038] Figure 5 Statistical analysis of grain length-width ratio in recipient rice, CR1 and CR2 families. Detailed Implementation

[0039] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0040] Example 1

[0041] Evolutionary analysis of a large number of rice varieties revealed that the gene encoding the Ethylene overproducer1-like protein ETOL3 (LOC_Os11g37520) exhibits different haplotypes in different varieties (Figure 1). Sequence analysis showed that LOC_Os11g375200 has a 16bp deletion variant in its 5UTR (physical location 22150227), as shown in Figure 1. Figure 1 As shown.

[0042] This invention, through UTR variation and phenotypic analysis of ETOL3 in a population, discovered that the gene is associated with the length-to-width ratio of rice grains. The gene has a coding region sequence as shown in SEQ ID NO:3, and the genome sequence of the gene is shown in SEQ ID NO:2.

[0043] Example 2: Investigation and Analysis of the Length-to-Width Ratio of Different Varieties

[0044] Seeds of different varieties were harvested after maturity and planted in Nanjing in May, with transplanting in June. After normal water and fertilizer management in the field, the length-to-width ratio of the grains of different haplotype varieties was observed. The results showed that the length-to-width ratio of the grains varied among different haplotype varieties, such as... Figure 2 As shown.

[0045] Example 3: Obtaining the ETOL3 gene-edited transgenic line and determining its length-to-width ratio.

[0046] First, a suitable spacer sequence (as shown in nucleotides 77-99 of SEQ ID NO:3) was found using the CRISR-P website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), and two complementary primers with adapters were synthesized (front primer: GGCAGGAGAGCTTCCCTTTCTCGA, back primer: AAACTCGAGAAAGGGAAGCTCTCC). After slow annealing in a PCR instrument, double-stranded oligos were obtained, and the double-stranded oligos were ligated into the BGK03 vector digested with BsaI. The constructed vector was transformed into recipient rice callus using Agrobacterium EHA105 to obtain T0 generation gene-edited transgenic plants. First-generation sequencing identified 10 knockout plants with the ETOL3 gene edited, among which CR1 and CR2 were homozygous mutants. Figure 3 In CR1, the ETOL3 gene has a four-base deletion (AAAG) from 85-88 bp after the ATG, resulting in a frameshift mutation at amino acid 29 and premature termination of translation (844 amino acids shorter than the wild type). In CR2, the ETOL3 gene has a 17-base deletion (CTTCCCCTCGAGAAAGG) from 73-89 bp after the ATG, resulting in a frameshift mutation at amino acid 25 and premature termination of translation (821 amino acids shorter than the wild type). Figure 4 An investigation of the grain length-to-width ratio in the above transgenic knockout families revealed that the grain length-to-width ratio of the knockout plants CR1 and CR2 was significantly lower than that of the recipient rice. Figure 5 ).

[0047] The above results indicate that ETOL3 is a gene associated with the length-to-width ratio of rice grains. Rice with a low grain length-to-width ratio can be obtained through gene editing.

[0048] SEQ ID NO:1

[0049] MRKLFFSELTSCKETKLHSAPHSWLPLEKGKLSKFAGHSTSSIESLMKMPEPVVLPHFKPADYVDILAQIHEELESCPPDEKSCLYLLQFQVFRGLGEVKLSRRSLQSAWEKASTIHEKLIFGAWLKYEKKGEEPISDLLSSCGKCSQEFKLLDFVSQISTGSHEISYDDESDVFWGSPVVHFRIRDDMIACDRRKLAALSTPLYAMLNGGFRESHLEVIDMSRNGISSIGMRAISKFSLTGRLPYLSADAILEMLDFANKFCCNGLKDACERKLASFICSRQDAIDFMECALELGCSILAAACLQVLLNELPECLNDEQVVRIFSCASKQQRSTMAGNASFSLYCLLSEVSMSINATSDVTVTFLEKLVDSASDSRQKQLALHQLACTRLLRKDYAEAERLFNAAFTAGHLYSVVGLARLASMRGNKHFSLKLLDSVMSSRWPLGWMYQERALYLEGDNKLENLNKATELDPTLTYPYMFRAASLMKRQSVEAALMEINRILGFKLVLECLELRFCCYLALEDHRAALCDVQAILTLAPDYRMIGGRVSAKQLRMLVMENVEQWTTADCWMQLYDRWSSVDDIGSLSVIYQMLESDAAKGVLYFRQSLLLLRLNCPEAAMRSLQLAREHAASQHEQLVYEGWILYDTGHCEEGLQKAEASIAIQRSFEAFFLKAYALADSSLDPSTSATVVSLLEDALRCPSDRLRKGQALNNLGSVYVDCGKLDLAAECYINALKIGHTRAHQGLARVHFLRNSRTGAYEEMTKLIEKARSNASAYEKRSEYCDRELTKSDLQMVTKLDPLRVYPYRYRAAVLMDNHKEKEAIAELTKAIAFKADLNLLHLRAAFHEHVGDISSALRDCRAALSVDPNHQEMLELHHRVNSQEP

[0050] SEQ ID NO:2

[0051]

[0052] SEQ ID NO:3

[0053]

Claims

1. Application of interfering with, inhibiting, silencing or knocking out the rice OsETOL3 gene with nucleotide sequences as shown in SEQ ID NO:3 in reducing the length-to-width ratio of rice grains or in breeding rice with low grain length-to-width ratio.

2. Application of substances that interfere with, inhibit, silence, or target knockout the rice OsETOL3 gene with nucleotide sequences such as SEQ ID NO:3 in reducing the length-to-width ratio of rice grains or in breeding rice with low grain length-to-width ratio; The substance used to interfere with, suppress, silence or target knockout the rice OsETOL3 gene is at least one of the following (1) to (6): (1) Interference sequence targeting the rice OsETOL3 gene; (2) Interference vector for interfering with the rice OsETOL3 gene; (3) Transgenic cell lines containing rice OsETOL3 gene interference vector; (4) Primer sequences for targeted knockout of the rice OsETOL3 gene based on gene editing technology; (5) Vectors for targeted knockout of the rice OsETOL3 gene based on gene editing technology; (6) Cell lines that target and knock out the rice OsETOL3 gene based on gene editing technology.

3. The application according to claim 1 or 2, characterized in that, By interfering with, inhibiting, silencing, or targeting and knocking out the expression of the rice OsETOL3 gene, the length-to-width ratio of rice grains is reduced, thereby cultivating rice with a low grain length-to-width ratio.

4. A method for cultivating rice with low grain length-to-width ratio, characterized in that, By interfering with, inhibiting, silencing, or targeting and knocking out the rice OsETOL3 gene with nucleotide sequences such as SEQ ID NO:3, the length-to-width ratio of rice grains can be reduced, thus cultivating rice with a low grain length-to-width ratio.

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