Rice rapid alkalization factor gene ralf37 and application thereof
Patent Information
- Application Number
- CN202311518269.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-14
- Publication Date
- 2026-11-03
- Estimated Expiration
- 2043-11-14
AI Technical Summary
There is currently no research on the role of the RALF gene in the regulation of rice grain shape, making it difficult to effectively improve rice grain shape through genetic engineering, which affects rice yield and quality.
The rice rapid alkalization factor gene RALF37 was cloned and its expression level was reduced by antisense RNA technology. An antisense RNA vector was constructed and introduced into rice cells to regulate rice grain development in order to cultivate small-grained rice.
Genetic improvement of rice grain shape was achieved, with mutant grains becoming shorter and narrower, providing new gene resources and targets, and laying a theoretical foundation for molecular design breeding of rice.
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Figure CN117701583B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a rice rapid alkalization factor gene RALF37 and its application. Background Technology
[0002] Rice is a vital food crop in my country and the world, with over half the global population relying on it as their staple food. Rice yield is a complex agronomic trait controlled by multiple genes, primarily influenced by three factors: effective panicles per unit area, grains per panicle, and thousand-grain weight. Grain shape is a crucial agronomic trait directly related to rice yield and quality. On one hand, grain shape directly determines thousand-grain weight, a key factor in determining rice yield. On the other hand, grain shape is closely related to the appearance, processing quality, and cooking and eating quality of rice, making it a significant factor affecting rice quality. Therefore, discovering and utilizing new grain shape-regulating genes to improve rice grain shape is of great importance for increasing rice yield and improving rice quality.
[0003] High yield and high quality have long been the goals pursued in rice breeding. Rice grain shape is a complex quantitative trait controlled by multiple genes, which has an important impact on both rice yield and quality. It is not only an ideal trait for genetic regulation research, but also an important target for rice domestication and design breeding. Based on the important value of grain shape research in basic and applied research, it has increasingly become a hot topic in rice research.
[0004] Rapid alkalinization factor (RALF) is a plant peptide hormone widely present in plants. It can cause a rapid increase in extracellular pH and participates in the regulation of plant development (Pearce et al. RALF, a 5-kDaubiquitous polypeptide in plants, arrests root growth and development. Proc. Natl. Acad. Sci. USA 2001, 98, 12843-12847). Changes in extracellular pH are an important part of plant regulation of cell growth and responses to external environmental stimuli. In plants, Arabidopsis RALF family members such as RALF1 / 23 and RALF4 / 19 have been shown to function as ligands for FERONIA and BUPS1 / 2, members of the CrRLK1L protein receptor kinase family, respectively, in regulating cell elongation, plant immune response, and double fertilization (Qiang Xiaonan et al. Preliminary analysis of the functional diversity of the Arabidopsis RALF polypeptide family. Biotechnology Bulletin, 2019, 35(1):2-10). However, there are no reports on the role of RALF in regulating rice grain shape. Summary of the Invention
[0005] One objective of this invention is to provide a novel gene, RALF37, for regulating rice grain shape. This gene plays an important role in the regulation of rice grain development; therefore, cloning this gene can not only elucidate the molecular mechanism of rice grain shape regulation but also provide a new gene resource for the genetic improvement of rice grain shape.
[0006] Another object of the present invention is to provide the application of the above-mentioned rice rapid alkalization factor gene RALF37.
[0007] To achieve the above objectives, the present invention provides a rice rapid alkalization factor gene RALF37, wherein the nucleotide sequence of the gene RALF37 is shown in SEQ ID NO: 1.
[0008] On the other hand, the present invention also provides the application of the rice rapid alkalization factor gene RALF37 in regulating rice grain development, wherein the nucleotide sequence of the gene RALF37 is shown in SEQ ID NO: 1.
[0009] Preferably, the gene RALF37 encodes an amino acid sequence as shown in SEQ ID NO: 2.
[0010] Preferably, the regulation of rice grain development is to cultivate small-grained rice grains.
[0011] On the other hand, the present invention also provides the application of the rice rapid alkalization factor gene RALF37 in rice genetic improvement breeding, the nucleotide sequence of the gene RALF37 is shown in SEQ ID NO: 1.
[0012] Preferably, the rice genetic improvement breeding is for cultivating small-grained rice varieties.
[0013] Preferably, the rice genetic improvement breeding method includes genetic engineering breeding and hybridization breeding.
[0014] Preferably, the genetic engineering breeding involves introducing an antisense RNA vector of the RALF37 gene into plant cells to obtain rice materials with grain shape variations by reducing the transcription level of the RALF37 gene. For example, knocking down the RALF37 gene in rice can breed rice varieties with smaller grains.
[0015] The RALF37 gene knockdown vector can be constructed using existing plant expression vectors; the plant expression vectors include binary Agrobacterium vectors, such as pCubi1390; the targeted knockdown vector containing the RALF37 gene can be introduced into host plant cells or tissues using various physical, chemical and biological methods such as gene gun, protoplast-mediated, pollen tube pathway, and Agrobacterium-mediated; the preferred host plant is rice.
[0016] When constructing a targeted knockdown vector for the RALF37 gene, a suitable antisense RNA target segment is selected on the exon sequence of the RALF37 gene.
[0017] To facilitate the identification and screening of transgenic plant cells or plants, the plant knockout vectors used have incorporated eukaryotic and prokaryotic selection tag genes.
[0018] Preferably, the hybridization breeding includes using rice with grain shape mutations as donor parents to improve rice grain shape through hybridization selection, that is, by hybridizing the obtained rice with grain shape mutations with the rice variety to be improved, and then carrying out targeted improvement of rice grain shape, and selecting rice varieties that achieve the improvement target in the offspring.
[0019] Preferably, the rice variety is Japonica rice Zhonghua 11.
[0020] On the other hand, the present invention also provides an antisense RNA vector, which is an antisense RNA vector of the RALF37 gene.
[0021] On the other hand, the present invention also provides the application of an antisense RNA vector of the rice rapid alkalization factor gene RALF37, a transgenic cell line or recombinant bacteria containing the antisense RNA vector, in regulating rice grain development and / or rice grain shape improvement breeding.
[0022] Reverse genetics is a powerful tool for studying gene function. This invention utilizes antisense RNA knockdown (KD) technology to target and knock down the LOC_Os04g31410 gene in wild-type Zhonghua 11 rice. This gene, located on chromosome 4 of rice and named RALF37, yielded the knockdown mutant material RALF37-KD. This mutant exhibits shorter grain length and narrower grain width. Cytological analysis showed that compared to wild-type Zhonghua 11, the glumes of the RALF37 KD mutant plants were significantly smaller, indicating that RALF37 may affect rice glume cell size by influencing cell growth, ultimately leading to the phenotype of shorter grain length and narrower grain width.
[0023] The present invention has the following advantages and effects compared with the prior art:
[0024] (1) This invention utilizes antisense RNA gene knockdown technology to target and reduce the rice rapid alkalization factor gene RALF37 in wild-type Zhonghua 11, obtaining the mutant material RALF37-KD with reduced RALF37 transcription. Phenotypic analysis shows that the mutant has shorter grain length and narrower grain width, providing new gene resources for the genetic improvement of rice grain shape and new target genes for rice molecular design breeding.
[0025] (2) There is currently no research on the function of RALF37 gene in regulating rice grain shape. The target gene of this invention, RALF37, is a new gene that regulates rice grain shape development. It has important theoretical significance in revealing the mechanism of grain development and provides new examples for broadening the understanding of the existing molecular mechanism of rice grain shape regulation. Attached Figure Description
[0026] Figure 1 The diagram illustrates the chromosomal location of the RALF37 gene, the target region of the knockdown vector antisense RNA, and the phenotypes of the RALF37 gene KD mutant and wild-type (japonica rice, Zhonghua 11) in Example 1. In the diagram, A is a schematic diagram of the RALF37 gene location on the rice chromosome; B is the target region of the RALF37 knockdown vector; C is a schematic diagram of the expression vector used in Example 1; D is the PCR detection results of the hygromycin resistance gene in transgenic plants; E is a phenotypic comparison between wild-type and KD mutant plants; and F is the expression analysis of the RALF37 gene in KD plants. WT represents the wild-type, and KD-1, KD-2, and KD-3 are RALF37 knockdown mutants.
[0027] Figure 2 The phenotypes of RALF37 knockdown mutant and wild-type grains are shown. In this diagram, A represents the grain phenotypes of wild-type (WT) and RALF37 knockdown mutant (KD), and B represents the grain phenotypes of wild-type (WT) and RALF37 knockdown mutant (KD). WT represents wild-type, and KD represents the RALF37 knockdown mutant.
[0028] Figure 3 These are electron micrographs of the surface cells of mature grains from wild-type Zhonghua 11 (ZH11) and the KD mutant of RALF37 (KD). In the images, A is a scan of the grain husk surface of Zhonghua 11, B is a scan of the grain husk surface of RALF37-KD, C is a statistical diagram of cell length on the grain husk surface of Zhonghua 11 and RALF37-KD mutants, D is a statistical diagram of cell width on the grain husk surface of Zhonghua 11 and RALF37-KD mutants, and E is a statistical diagram of the number of transverse cells on the grain husk surface of Zhonghua 11 and RALF37-KD mutants. ZH11 is wild-type, and KD is a RALF37 knockdown mutant. Detailed Implementation
[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0030] Unless otherwise specified, the experimental methods described in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.
[0031] In the following examples, the pCubi1390 vector is a commonly used gene expression vector in the art, Escherichia coli DH5α and Agrobacterium EHA105 are commonly used strains, which are preserved in most molecular biology laboratories; the rice variety is wild-type japonica rice Zhonghua 11 (a publicly used rice variety, commercially available).
[0032] The main reagents used in the following examples were: restriction endonucleases, Taq polymerase, T4 ligase, Pyrobest Taq polymerase, KOD, etc., purchased from TAKARA (Dalian), Promega, NEB, ABM, and other biotechnology companies; plasmid miniprep kits and agarose gel extraction kits were purchased from Shanghai Jierui Biotechnology Co., Ltd.; culture media, agar powder, agarose, ampicillin (Amp), kanamycin (Kan), rifampin (Rif), and other antibiotics were purchased from Sigma; other chemical reagents used in the examples were imported or domestically produced analytical grade. Primers used in the examples were synthesized by Riboxing Biotech Co., Ltd., and sequencing was performed by Qingke Biotech Co., Ltd.
[0033] Example 1: Phenotypic and Genetic Effects Analysis of Rice RALF37 Mutant
[0034] This invention utilizes antisense RNA technology to select a target region in the exon region of the rice rapid alkalization factor gene RALF37 (LOC_Os04g31410), and designs and synthesizes specific primers:
[0035] RALF37-BamHI-F: 5′-CGGGATCCATGGCGAGAAACGCCGCAACAG-3′ (SEQ ID NO. 3);
[0036] RALF37-KpnI-R: 5′-GGGGTACCTTAACACGATCGGTTGTGGTAG-3′ (SEQ ID NO. 4)
[0037] Following the product instructions, the target fragment was amplified using reverse-transcribed single-stranded cDNA from rice leaves as a template. After recovery, the target gene fragment was reverse-ligated into the plant expression vector pCubi1390. The ligation product was transformed into *E. coli* DH5α competent cells, plated overnight, and single colonies were selected for sequencing. Plasmids with correct sequencing results were transformed into *Agrobacterium* EHA105 for genetic transformation. Using the *Agrobacterium*-mediated transgenic method, wild-type Zhonghua 11 was transformed to obtain transgenic plants.
[0038] Figure 1 The chromosomal location of the RALF37 gene, the target region of the knockdown vector antisense RNA, and the phenotypes of the RALF37 gene KD mutant and wild type (japonica rice, Zhonghua 11) are shown in Example 1. Figure 1In the diagram, A is a schematic diagram of the location of the RALF37 gene on the rice chromosome. B is the target region of the RALF37 knockdown vector. C is a schematic diagram of the pCubi1390 expression vector used in Example 1. D is the PCR detection result of the hygromycin resistance gene in transgenic plants. E is a comparison of the phenotypic results of wild-type and KD mutant plants. F is the expression analysis result of the RALF37 gene in KD plants, indicating that the RALF37 gene has been knocked down in the mutants. In the figure, WT represents the wild type, and KD-1, KD-2, and KD-3 are all RALF37 knockdown mutants.
[0039] During the grain-filling stage of wild-type Zhonghua 11, photographs were taken of wild-type plants and KD mutants of RALF37 (parallel experimental plants KD-1, KD-2, and KD-3). The phenotypes are as follows: Figure 1 As shown in Figure E (from left to right: wild type (WT), KD mutants KD-1, KD-2, and KD-3), the results indicate that the plant height and growth period of the KD mutants were not significantly different from those of the wild type.
[0040] Example 2: RALF37 mutant and wild-type husk phenotypes
[0041] Ten grains of wild type and RALF37 knockdown mutant were randomly selected for comparison. The phenotypes of the grains and the phenotypes of the rice grains after hulling were observed, including the length and width of the grains.
[0042] Figure 2 The results show the phenotypic results of the RALF37 knockdown mutant and wild-type grains. In the figures, A represents the grain phenotypes of wild-type (WT) and the RALF37 knockdown mutant (KD), and B represents the grain phenotypes of wild-type (WT) and the RALF37 knockdown mutant (KD). WT represents the wild-type, and KD represents the RALF37 knockdown mutant.
[0043] Observations revealed that, compared to the wild type, the grains and rice grains of the RALF37 homozygous mutant material were significantly shorter and narrower, making it suitable for breeding small-grained rice.
[0044] Example 3: Scanning electron micrographs of RALF37-KD and wild-type rice husks
[0045] Electron microscopy was used to scan grain sections of mature RALF37-KD and wild-type rice to observe the cell size on the surface of the grain husk. Results are as follows: Figure 3 As shown. Figure 3These are electron micrographs of the surface cells of mature grains from wild-type Zhonghua 11 (ZH11) and the KD mutant of RALF37 (KD). In the images, A is a scan of the grain husk surface of Zhonghua 11, B is a scan of the grain husk surface of RALF37-KD, C is a statistical diagram of cell length on the grain husk surface of Zhonghua 11 and RALF37-KD mutants, D is a statistical diagram of cell width on the grain husk surface of Zhonghua 11 and RALF37-KD mutants, and E is a statistical diagram of the number of transverse cells on the grain husk surface of Zhonghua 11 and RALF37-KD mutants. ZH11 is wild-type, and KD is a RALF37 knockdown mutant.
[0046] As shown in the figure, compared with the wild-type Zhonghua 11, the length of the surface cells of the husk of the mutant RALF37-KD is significantly shorter. Figure 3 (A to C) Cell width significantly narrowed ( Figure 3 (A, B, and D), while the number of transverse cells showed no significant change ( Figure 3 (A, B, and E). This indicates that the grain size difference between the mutant RALF37-KD and the wild-type Zhonghua 11 is mainly due to differences in cell size.
[0047] In summary, the RALF37 gene of this invention can regulate rice grain shape; mutations in this gene can shorten the grain length and narrow the grain width. Reducing RALF37 gene expression using antisense RNA technology can genetically improve rice grain shape.
[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. Knock down the rice rapid alkalization factor gene RALF37 Its application in regulating the shortening of rice grain length and the narrowing of grain width is characterized by: The gene RALF37 The nucleotide sequence is shown in SEQ ID NO:
1.
2. The application according to claim 1, characterized in that: The gene RALF37 The amino acid sequence is encoded as shown in SEQ ID NO:
2.
3. The application according to claim 1, characterized in that: The adjustment of rice grain length to be shorter and grain width to be narrower is for the purpose of cultivating small-grained rice.
4. The application according to claim 3, characterized in that: Rice is cultivated through genetic improvement breeding, including genetic engineering breeding and hybridization breeding.
5. The application according to claim 4, characterized in that: The genetic engineering breeding includes... RALF37 The antisense RNA vector of the gene is introduced into plant cells, by reducing RALF37 Rice materials with grain shape variations obtained at the gene transcription level.
6. The application according to claim 4, characterized in that: The hybridization breeding includes using rice with grain shape mutations as donor parents and improving rice grain shape through hybridization selection.
7. The application according to any one of claims 1 to 6, characterized in that: The rice variety in question is Japonica rice Zhonghua 11.
8. Rice rapid alkalization factor gene RALF37 Antisense RNA vectors, transgenic cell lines, or recombinant bacteria used for knockdown RALF37 The application of genes in making rice grains shorter and narrower, the genes mentioned RALF37 The nucleotide sequence is shown in SEQ ID NO: 1.