Rice RGB1 gene mutant mRGB1 and application thereof

Abstract

The invention discloses a rice RGB1 gene mutant mRGB1 and application thereof, and relates to the technical field of plant genetic engineering, a gene editing technology is utilized, a rice G protein beta subunit coding gene (RGB1, LOCOs03g46650) N-terminal structural domain coding region is targeted, three and six basic groups are deleted from a first exon and a second exon respectively, the mutant is named as mRGB1, the mutation causes plant dwarfing, upright and dense ears, and the rice RGB1 gene mutant mRGB1 has the advantages of high yield and high yield. The seeds become small, and new gene resources can be provided for genetic improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a rice RGB1 gene mutant mRGB1 and application thereof. Background Art

[0002] Among all improvements, increasing yield is undoubtedly the first goal. With the improvement of people's living standards, the quality requirements for rice are getting higher and higher, and more and more attention is paid to environmental protection, which limits the requirements of breeding. It cannot blindly pursue yield, but also needs to focus on quality improvement and resistance screening, nitrogen and phosphorus utilization efficiency, etc. Studies in recent years have shown that the G protein signaling pathway is involved in the regulation of rice yield, resistance, and nitrogen utilization efficiency.

[0003] The G protein heterotrimer consists of one Gα, one Gβ and five Gγ. The Gγ subunit GS3 regulates grain shape, salinity stress and high temperature, the Gγ subunit DEP1 regulates panicle morphology, grain shape and nitrogen use efficiency, and the Gα subunit regulates plant morphology, plant height and grain shape. Studies have shown that Gγ must form a dimer with Gβ to function, and the function of Gγ must rely on Gβ, indicating that Gβ plays an important role in the signal transduction of G proteins.

[0004] CRISPR / Cas9 technology is an accurate and efficient means to study the biological functions of genes. It targets the binding of sgRNA to the gene target site, cuts the target site through the Cas9 protein, and repairs errors after the DNA molecule breaks, resulting in base insertion, deletion and other errors, ultimately changing the function of the gene.

[0005] In the past, rice breeding mainly used backcrossing to introduce some excellent alleles into other varieties. This method has a long cycle, large workload and high cost. CRISPR / Cas9 gene editing technology is used to directly perform site-directed mutations on rice G protein β subunit RGB1 to create mutants and regulate rice plant and grain types. However, the knockout mutant of RGB1 is lethal, and no mutant has been reported yet. Summary of the invention

[0006] Technical problems to be solved: In view of the technical defects of the prior art such as long cycle, heavy workload and high cost, a method for producing a rice RGB1 gene mutant mRGB1 is disclosed, which can use CRISPR / Cas9 technology to quickly obtain dwarf plants, erect, dense-eared and small-grain mutants.

[0007] Technical solution:

[0008] A rice RGB1 gene mutant mRGB1, wherein the nucleotide sequence of the rice RGB1 gene mutant mRGB1 is shown in SEQ ID NO: 1:

[0009]

[0010] The amino acid molecule encoding the rice RGB1 gene mutant mRGB1 has an amino acid sequence as shown in SEQ ID NO: 2:

[0011] MASVAELKEKHAAATASVNSLRERLRQRQMLLDTDVERYSRGRTPVSFNPTDLVCCRTLQGHSGKVYSLDWTPEKNWIVSASQDGRLIVWNALTSQKTHAIKLHCPWVMTCAFAPNGQSVACGGLDSACSIFNLNSQADRDGNIPVSRILTGHKGYVSSCQYVPDQETRLITSSGDQTCVLWDVTTGQR ISIFGGEFPSGHTADVLSLSINSSNSNMFVSGSCDATVRLWDIRIASRAVRTYHGHEGDINSVKFFPDGQRFGTGSDDGTCRLFDVRTGHQLQVYSREPDRNDNELPTVTSIAFSISGRLLFAGYSNGDCYVWDTLLAEVVLNLGNLQNSHEGRISCLGLSSDGSALCTTGSWDKNLKIWAFSGHRKIV.

[0012] Furthermore: the mutant has a 3 bp deletion in the first exon of the LOC_Os03g46650 coding region of the RGB1 gene, and a 6 bp deletion in the second exon.

[0013] The present application also discloses the application of the rice RGB1 gene mutant mRGB1 in regulating rice plant type and seed size.

[0014] A method for obtaining RGB1 mutant rice comprises causing the mutation of the RGB1 gene of the recipient rice as claimed in claim 1, and editing the N-terminal domain coding region of RGB1, so that the plant becomes shorter, the panicles become dense and erect, and the seeds become smaller, thereby obtaining the transgenic rice.

[0015] Principle explanation: The method of targeted knockout of RGB1 gene uses CRISPR / Cas9 technology to select the target site sequence of rice G protein β subunit RGB1 to construct an editing vector, and transforms the receptor ZH11 through Agrobacterium-mediated transformation to obtain mutants of RGB1 gene and corresponding transgenic rice plants, complete the editing of G protein gene RGB1, and perform functional verification.

[0016] Beneficial effects:

[0017] 1. The present invention creates a method for creating a non-lethal mutant of the RGB1 gene. It is found that the mutation can control the seed size and plant type of rice, reduce plant height, and form upright dense panicles. It can provide a new RGB1 allele and creation method for genetic improvement of rice.

[0018] 2. Rice plant type and seed size play an important role in yield. G protein signaling pathway is an important element for yield and resistance regulation. Regulation of it can provide genetic resources for high-yield and high-resistance rice breeding. RGB1 is a core element of G protein signal transduction. Complete loss of its function is lethal. The mining of its weak alleles will provide genetic resources for genetic improvement. The mRGB1 mutant created by the present invention is characterized by dwarfed plants, upright and dense panicles, and smaller seeds, which can provide genetic resources for the future improvement of rice in terms of plant height, panicle type, and seed size. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A diagram comparing the protein structures of the RGB1 protein and mRGB1 of the present application;

[0020] Figure 2 This is a verification diagram of the interaction between the N-terminal and C-terminal domains of the RGB1 protein and the Gγ subunit in this application;

[0021] Figure 3 The agronomic traits comparison diagram of the mRGB1 mutant of the present application and the control parent ZH11, wherein A is a comparison diagram of the plant type of mRGB1 and ZH11, B is a comparison diagram of the panicle type of mRGB1 and ZH11, C is a comparison diagram of the seed size of mRGB1 and ZH11, and D is a comparative analysis diagram of plant height, panicle length, number of grains per panicle, grain length, grain width and 1000-grain weight between mRGB1 and ZH11; *** represents extremely significant difference, t test P<0.01;

[0022] Figure 4 These are the co-segregation and complementation verification diagrams of the mRGB1 mutants of the present application, wherein A is the co-segregation detection diagram of mRGB1, in which W represents the wild type, H represents the heterozygous genotype, and M represents the mutant genotype; B is the phenotypic diagram of RGB1 restoring the plant height of the mRGB1 mutant, C is the statistical analysis diagram of RGB1 restoring the plant height of the mRGB1 mutant, D is the phenotypic diagram of RGB1 restoring the ear length of the mRGB1 mutant, E is the statistical analysis diagram of RGB1 restoring the ear length of the mRGB1 mutant, F is the phenotypic diagram of RGB1 restoring the grain length of the mRGB1 mutant, and G is the statistical analysis diagram of RGB1 restoring the grain length of the mRGB1 mutant. Different letters in the diagram represent extremely significant differences in the one-way analysis of variance, P<0.01. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] Example 1

[0027] A method for obtaining a rice RGB1 gene mutant, specifically:

[0028] like Figure 1 As shown, the protein structure of RGB1 was structurally analyzed using the website http: / / swissmodel.expasy.org / . The protein is mainly composed of an α-helical domain at the N-terminus and seven WD repeat domains at the C-terminus;

[0029] like Figure 2 As shown, yeast two-hybrid experiments were used to prove that the N-terminal domain of RGB1 is responsible for the interaction with the Gγ subunit; therefore, an editing target was designed in the N-terminal domain coding region, and the target nucleotide sequence O1 is shown in SEQ ID NO:3: GGGAGCGGCTCCGTCAGAGGcgg; the target nucleotide sequence O2 is shown in SEQ ID NO:4: GGTACTCGAGGACGCAGGGGcgg, and the last three bases are PAM sites, which are located in the first and second exons of the coding region, respectively.

[0030] The gene editing method used in this laboratory is the CRISPR / Cas9 system provided by Liu Yaoguang's research group at South China Agricultural University. The knockout vector KO-RGB1 of RGB1 was constructed by referring to the method provided in the paper (Yuming Lu; et al. Genome-wide targeted mutagenesis in rice using CRISPR / Cas9 system, Molecular Plant, 2017, 10, 1242-1245). KO-RGB1 was transformed into callus induced by immature embryos of wild-type Zhonghua 11 by Agrobacterium-mediated method, and positive transgenic plants were obtained by hygromycin screening. Leaf DNA was extracted from the obtained positive transgenic plants, and the target segment was amplified with primers mRGB1F as shown in SEQ ID NO: 5: GCTGATTGTTTTGGGGTGTGG and mRGB1R as shown in SEQ ID NO: 6: TTCCGCTGTGGCCTTGAAG and first-generation sequencing was performed to analyze the editing form of the mutant, and finally a mutant with 3 bases missing in the first exon of the coding region and 6 bases missing in the second exon was obtained. Protein structure prediction of the mutant revealed that the mutation resulted in damage to the N-terminal α-helical domain, such as Figure 1 Yeast two-hybrid and luciferase complementation assays demonstrated that the mutation resulted in a weakened interaction with Gγ.

[0031] A rice RGB1 gene mutant mRGB1, wherein the nucleotide sequence of the rice RGB1 gene mutant mRGB1 is shown in SEQ ID NO: 1:

[0032]

[0033] An amino acid molecule encoding a rice RGB1 gene mutant mRGB1, the amino acid sequence of which is shown in SEQ ID NO: 2:

[0034] MASVAELKEKHAAATASVNSLRERLRQRQMLLDTDVERYSRGRTPVSFNPTDLVCCRTLQGHSGKVYSLDWTPEKNWIVSASQDGRLIVWNALTSQKTHAIKLHCPWVMTCAFAPNGQSVACGGLDSACSIFNLNSQADRDGNIPVSRILTGHKGYVSSCQYVPDQETRLITSSGDQTCVLWDVTTGQR ISIFGGEFPSGHTADVLSLSINSSNSNMFVSGSCDATVRLWDIRIASRAVRTYHGHEGDINSVKFFPDGQRFGTGSDDGTCRLFDVRTGHQLQVYSREPDRNDNELPTVTSIAFSISGRLLFAGYSNGDCYVWDTLLAEVVLNLGNLQNSHEGRISCLGLSSDGSALCTTGSWDKNLKIWAFSGHRKIV.

[0035] The mutant consists of a 3 bp deletion in the first exon of the coding region of the RGB1 gene LOC_Os03g46650 and a 6 bp deletion in the second exon;

[0036] Application of the rice RGB1 gene mutant mRGB1 in regulating rice plant type and seed size;

[0037] The method for obtaining RGB1 mutant rice comprises causing the mutation of the RGB1 gene of the recipient rice as claimed in claim 1, and editing the N-terminal domain coding region of RGB1, so that the plant becomes shorter, the panicles become dense and erect, and the seeds become smaller, thereby obtaining the transgenic rice.

[0038] Example 2

[0039] Phenotypic and agronomic trait analysis of mRGB1:

[0040] The mutant obtained was named mRGB1, and its performance was dwarfing of plants, erect dense panicles, and smaller seeds. In order to further conduct phenotypic analysis, the applicant sowed the seeds of mRGB1 harvested at T0 in the nursery field after soaking and germination, and transplanted them to the field 20 days later to obtain T1 transgenic plants. The planting density was 15 cm × 24 cm, and the planting location was the experimental field of Yangzhou University in Yangzhou City, Jiangsu Province. Field management was carried out according to conventional rice planting methods under the conditions of safety protection facilities. The T1 plants of the mRGB1 mutant were compared with the wild-type Zhonghua 11. The plant height of the mutant plants was significantly reduced, the panicle length was shortened, and the seeds were smaller. This shows that RGB1 is a gene that positively regulates seed size, and its mutation will reduce the seeds, plant height and panicle length. The phenotypic changes and data of the relevant plants of the T1 generation, including plant height, panicle length, number of grains per panicle, grain length and grain width, were investigated and recorded, and statistical analysis was performed separately. The results are shown in the table. Figure 3 shown.

[0041] Example 3

[0042] Co-segregation detection and complementation verification of mRGB1

[0043] In order to exclude that the phenotypic changes of mRGB1 were not caused by transgenic effects and off-target effects, co-segregation and complementation verification were further performed. The mRGB1 mutant plants were crossed with the ZH11 wild type to obtain F1 plants. The harvested F1 seeds were used to obtain the F2 population. Individual plants were randomly selected for genotyping and grain length was examined. The results showed that the seed length of the mRGB1 homozygous mutant plants was significantly shorter than that of the heterozygous and wild type plants, such as Figure 4 As shown in A. At the same time, the mRGB1 plants were crossed with the RGB1 overexpression plants, and the phenotypes of the F2 population were examined and it was found that the overexpression of RGB1 restored the phenotype of mRGB1, as shown in Figure 4 As shown in B. In summary, it is proved that the phenotypic changes of mRGB1 are caused by the mutation of RGB1.

Claims

1. A rice RGB1 gene mutant mRGB1, It is characterized in that The nucleotide sequence of the rice RGB1 gene mutant mRGB1 is shown in SEQ ID NO:1: ATGGCGTCCGTGGCGGAGCTCAAGGAGAAGCACGGCGGCCACGGTCGGTGAACTCCCCTGCGGGAGCGCTCCGTCAGCGGCAGATGCTGCTCGACACCGACGTGGAGAGGTACTCGAGGGGCGGACGCCGGTGAGC TTCAACCCGACGGATCTGGTGTGCTGCCGCACGCTTCAAGGCCACAGCGGAAAGGTATATTCTCTGGATTGGACCCCCTGAAAAGAATTGGATAGTCAGTGCCTCACAAGATGGAAGGCTAATTGTATGGAATGCATTAACAA GTCAAAAAACACATGCCATAAAGTTACATTGCCATGGGTGATGACATGTGCATTTGCACCCAATGGCCAATCTGTTGCCTGTGGTGGTCTTGACAGCGCATGCTCTATCTTCAATCTTAACTCAAGCAGAGAGATGG GAATATACCAGTATCAAGAATACTTACCGGACACAAAAGGCTATGTTTCATCCTGTCAGTATGTCCCCAGATCAGGAAACCCGCCTAATTACTAGCTCTGGTGATCAAACATGTGTCCTGTGGATGTTACTACTGGCCAAAGG ATATCAATATTTGGCGGTGAATTCCCCATCAGGGCATACGGCAGATGTTTTGAGCTTGTCCATAAACTCATCAAATTCGAATATGTTTGTTTCGGGTTCATGTGATGCAACTGTAAGGCTGTGGGATATCAGAATTGCAAGCC GGGCAGTTAGAACATATCATGGTCATGAGGGTGACATTAACAGTGTCAAGTTTTTCCCTGATGGCCAGAGGTTTGGTACTGGTTCAGATGATGGAACGTGTAGATTTTGACGTGAGAACAGGGCACCAACTTCAAGTATA CAGTCGGGAACCTGATAGAAATGAAATGAACTCCCCAACTGTTACATCTATTGCATTTTCGATATCAGGAAGGCTTCTTTTTGCTGGATACTCCCAATGGTGACTGTTATGTGGGACACTTCTCGCTGAGGTGGGACACTTCTCGCTGAGGTGGTACTT AATTTGGGAAACCTCCCAAAACTCTCATGAGGGGCGTATAAGCTGCCTTGGTCTTCTTCTGATGGGAGTGCATTGTGTACAGGAAGTTGGGACAAGAATTTGAAGATTTGGGCCTTCAGCGGACACCGGAAAATAGTTTGA.

2. An amino acid molecule encoding the rice RGB1 gene mutant mRGB1 of claim 1, It is characterized in that Its amino acid sequence is shown in SEQ ID NO:2: MASVAELKEKHAAATASVNSLRERLRQRQMLLDTDVERYSRGRTPVSFNPTDLVCCRTLQGHSGKVYSLDWTPEKNWIVSASQDGRLIVWNALTSQKTHAIKLHCPWVMTCAFAPNGQSVACGGLDSACSIFNLNSQADRDGNIPVSRILTGHKGYVSSCQYVPDQETRLITSSGDQTCVLWDVTTGQR ISIFGGEFPSGHTADVLSLSINSSNSNMFVSGSCDATVRLWDIRIASRAVRTYHGHEGDINSVKFFPDGQRFGTGSDDGTCRLFDVRTGHQLQVYSREPDRNDNELPTVTSIAFSISGRLLFAGYSNGDCYVWDTLLAEVVLNLGNLQNSHEGRISCLGLSSDGSALCTGSWDKNLKIWAFSGHRKIV.

3. The rice RGB1 gene mutant mRGB1 according to claim 1, It is characterized in that The mutant was missing 3 bp from the first exon of the RGB1 gene LOC_Os03g46650 coding region, and 6 bp missing from the second exome.

4. The use of rice RGB1 gene mutant mRGB1 as claimed in claim 1 in regulating rice plant type and seed size.

5. A method for obtaining RGB1 mutant rice, It is characterized in that The RGB1 gene of the receptor rice is included to cause the mutation of the RGB1 gene as claimed in claim 1 and edit the N-terminal domain coding region of RGB1, so that the plants become shorter, dense ears stand upright, and the seeds become smaller, and the transgenic rice is obtained.

Images