A gene suitable for breeding ideal rice plant types and its application

Abstract

The present invention discloses a gene suitable for breeding ideal rice plant types and its application. The nucleotide sequence of the gene suitable for breeding ideal rice plant types is shown in SEQ ID NO.1, bases 134 to 738, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2. The present invention first determines that knocking out the LOC_Os01g68500 gene can reduce rice plant height. Therefore, the present invention provides technical support for regulating rice plant height and breeding ideal rice plant types.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a gene suitable for breeding ideal rice plant types and its application. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important staple crops, serving as a staple food for over 60% of my country's population. Plant height is a key trait of particular interest in current rice breeding for optimal plant architecture. Breeding rice varieties with moderate plant height not only optimizes plant architecture and increases harvest index, but also improves fertilizer tolerance and lodging resistance. Identifying genes that regulate rice plant height will facilitate molecular breeding for optimal rice plant architecture. Summary of the Invention

[0003] The first object of the present invention is to provide a functional protein for regulating semi-dwarfism in rice.

[0004] The functional protein for regulating semi-dwarf rice of the present invention has an amino acid sequence as shown in SEQ ID NO. 2, specifically: MASSSSWWVVMLLMVVAAAGWGGVAAATAAEAAHEVLRAHGLPRGLLPAGIADFRHD EGSGRFEAALGESCTAQFEVGLRYNATVAGVISYGRIASLSGVSAQDLFLWFPVRGIRVDV PSSGVIYFDVGVVFKHFPLAVFEAPPPCTPDPLLLLTQVCEDGSVAGGGAASQ*.

[0005] The second object of the present invention is to provide a gene encoding the above functional protein for regulating semi-dwarfism in rice.

[0006] Preferably, the nucleotide sequence of the gene regulating semi-dwarf rice is as shown in the sequence from base 134 to base 738 of SEQ ID NO. 1 (containing an 89 bp intron), specifically:

[0007] ATTCCATTTCTTTTCTTTTCTTTCTCCCCCTCAATTATTGCCGCTTCTGTCCATCCCCTCC

[0008] CCCTCCAATTCCATTCCATCTTCATCTCCATATCTTCTCCTCCTCGCGTCGGCAGTTATCT

[0009] CCTCGTTGATAATGGCGTCGTCGTCGTCGTGGTGGGTGGTGATGTTGTTGATGGTGGTG

[0010] GCGGCGGCGGGGTGGGGCGGGGTGGCGGCGGCGACGGCGGCGGAGGCGGCGCACG

[0011] AGGTGCTGCGGGCGCACGGGCTGCCGCGGGGGCTCCTGCCGGCGGGGATCGCGGATT

[0012] TCAGGCACGACGAAGGGAGCGGGAGGTTCGAGGCGGCGCTCGGGGAGTCGTGCACG

[0013] GCGCAGTTCGAGGTCGGGCTGCGGTACAACGCGACGGTGGCCGGGGTCATCAGCTAC

[0014] GGCCGGATCGCGTCGCTGTCGGGCGTCTCCGCGCAGGACCTCTTCCTCTGGTTCCCCG

[0015] TCCGCGGCATCCGCGTCGACGTCCCTTCCTCCGGCGTCATCTACTTCGACGTCGGCGTC

[0016] GTGTTCAAGCACTTCCCCCTCGCCGTCTTCGAGGCCCCGCCGCCCTGCACCCCCGACC

[0017] CCCTCCTCCTCCTCACGCAGGT AATCTAATCCTTCTTCCTCGAACCAATCACGAAGTGA AATCTCCACCCACCTAGTTCATCTCTGAATTGGGCGCTCACGCGGGGCAGGT(This 89bp is an intron)

[0018] GTGTGAGGACGGATCGGTCGCCGGCGGCGGCGCGGCGTCGCAATGAAGGGGACGGA

[0019] CTCGATGGTTGAAATCGGCGAGGCGGAAACCAAAAGAATGCGCAGTTTTGAGGCATG

[0020] GGAGTGGTCGGCCGGTCCGTCGCCGGCGGGCCGGCATCTGTGCAGATAGATAGAATG

[0021] AACATTCCTTTCGTTTTCTACTACTAGTTAGTAACCTTTCATTCCTCATCACTCTGTTCTT

[0022] CTTGCCGTTCTTGTTGTCCAGTGCGATCAAAGCCGGCCGACGGTGGCCGGTAGAGGA

[0023] GGCTGCCGGCCTCCGGTCGCCGCCACGGCGATAATTTTTCGTGGTTATGATGGTTCGGGGATTACAACGTGTTGAA.

[0024] It should be understood that, taking into account the degeneracy of codons, modifications to the nucleotide sequence of the above-mentioned coding gene without changing the amino acid sequence also fall within the scope of protection of the present invention.

[0025] The third object of the present invention is to provide the use of the above-mentioned rice semi-dwarf regulating functional protein or its encoding gene in regulating rice plant height. Preferably, the rice plant height is reduced by interfering, silencing or knocking out the rice semi-dwarf regulating gene through transgenic technology.

[0026] The fourth object of the present invention is to provide the use of the functional protein for regulating semi-dwarf rice or its encoding gene in increasing rice yield.

[0027] A fifth objective of the present invention is to provide the use of the aforementioned rice semi-dwarfism regulating protein or its encoding gene in rice breeding for ideal plant types. Preferably, the rice semi-dwarfism regulating gene is disrupted, silenced, or knocked out through transgenic technology to reduce plant height, thereby improving the harvest index and lodging resistance of rice.

[0028] The sixth object of the present invention is to provide a substance for inhibiting the expression of the aforementioned gene regulating semi-dwarf rice for use in reducing rice plant height and breeding ideal rice plant types.

[0029] A seventh object of the present invention is to provide a method for reducing rice plant height, comprising the step of deleting the function of the protein regulating semi-dwarfism in rice. Specifically, the method involves interfering with, silencing, or knocking out the gene regulating semi-dwarfism in rice through transgenic technology, thereby deleting the protein function.

[0030] This study demonstrates for the first time that knocking out the LOC_Os01g68500 gene can reduce rice plant height. The present invention utilizes Crispr / Cas9 vectors to construct a knockout transformation vector for the LOC_Os01g68500 gene. Transformation of rice with this vector knocks out the LOC_Os01g68500 gene, resulting in significantly lower plant height than wild-type control plants. Therefore, the function of the LOC_Os01g68500 gene can be applied to breeding ideal rice plant types, improving harvest index, fertilizer tolerance, and lodging resistance, thereby ensuring food security. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the Crispr / Cas9 vector map.

[0032] Figure 2 The results of sequencing identification of mutant strains KO-1 and KO-2 are shown.

[0033] Figure 3 Knocking out the LOC_Os01g68500 gene significantly reduced rice plant height, where NIP represents the wild-type Nipponbare, and KO-1 and KO-2 are knockout transgenic plants. DETAILED DESCRIPTION

[0034] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0035] Example 1

[0036] 1. Construction of LOC_Os01g68500 gene knockout vector

[0037] Using CRISPR-GE software developed by Professor Liu Yaoguang's team, we selected the specific target site of LOC_Os01g68500, 5'-CGGATTTCAGGCACGACGAA-3' (with a 3' PAM sequence of GGG), and designed knockout primers: OsU6aT1F: gccgCGGATTTCAGGCACGACGAA; OsU6aT1R: aaacTTCGTCGTGCCTGAAATCCG. The single guide RNA (sgRNA) sequence for LOC_Os01g68500 was cloned using the pYLgRNA-OsU3 vector provided by Professor Liu Yaoguang's team (Ma et al., 2015, Molecular Plant, 8:1274-1284). The target site sequence of LOC_Os01g68500 is 5'-CGGATTTCAGGCACGACGAA-3', with a 3' PAM sequence of GGG. Using Crispr / Cas9 ( Figure 1) vector was constructed to obtain the Crispr / Cas9 knockout vector of LOC_Os01g68500.

[0038] 2. Genetic Transformation of LOC_Os01g68500 Knockout Vector

[0039] The Crispr / Cas9 knockout vector for the LOC_Os01g68500 gene was introduced into the normal japonica rice variety Nipponbare using Agrobacterium tumefaciens EHA105-mediated genetic transformation method. DNA was extracted from the leaves of T0 and T1 transgenic plants, and primers (F: 5'-CCCCCTCAATTATTGCCGCT-3'; R: 5'-AGTAGAGACTTAACCCGCGA-3') were designed to PCR amplify the gene sequence of LOC_Os01g68500. Through third-generation sequencing, homozygous mutant strains KO-1 and KO-2 with the LOC_Os01g68500 gene knocked out were screened. The sequencing identification results of the mutant strains KO-1 and KO-2 are shown in Figure 2. Figure 2 shown.

[0040] 3. Statistics of plant height of LOC_Os01g68500 gene knockout transgenic plants

[0041] Seeds from the two homozygous knockout transgenic T2 lines (KO-1 and KO-2) and wild-type Nipponbare plants were dried at 49°C for 72 hours, soaked in water at 30°C for 24 hours, and germinated at 30°C for 36 hours before being sown in a transgenic field. Seedlings with consistent growth were selected and planted in the transgenic field. Normal cultivation and management were maintained.

[0042] After rice panicles matured, the plant heights of the LOC_Os01g68500 gene knockout lines KO-1, KO-2, and the wild-type Nipponbare were measured and counted. Figure 3 .Depend on Figure 3 It can be seen that the plant height of the transformed plants KO-1 and KO-2 with the LOC_Os01g68500 gene knocked out was significantly lower than that of the wild-type Nipponbare control plants.

[0043] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rice semi-dwarf knockout protein or its encoding gene for reducing rice plant height, wherein the amino acid sequence of the functional protein is shown in SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown in the sequence from base 134 to base 738 of SEQ ID NO.

1.

2. A method for reducing rice plant height, characterized in that: The method comprises the step of causing the function of a protein regulating semi-dwarf rice to be lost; the amino acid sequence of the functional protein regulating semi-dwarf rice is shown in SEQ ID NO.

2.

3. The method according to claim 2, characterized in that The protein function is lost by knocking out the gene regulating semi-dwarf rice; the nucleotide sequence of the gene regulating semi-dwarf rice is shown in the 134th to 738th base sequence of SEQ ID NO.1.

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