Application of rice gene OsPOK1 to regulation and control of rice grain shape and mutant of rice gene OsPOK1

Abstract

The invention belongs to the technical field of crop breeding, and relates to application of a rice gene OsPOK1 to regulation and control of a rice grain shape and a mutant of the rice gene OsPOK1. The coding sequence of the rice gene OsPOK1 is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein coded by the rice gene OsPOK1 is as shown in SEQ ID NO. 2. According to the invention, the new application of the rice gene OsPOK1 in regulation and control of the rice grain shape is found, a constructed rice modified plant containing the modified rice gene OsPOK1 disclosed by the invention can significantly increase the grain length and yield traits of rice grains, and the improvement of the rice grain shape and yield provides a new gene resource for rice breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of crop breeding, and relates to the use of rice gene OsPOK1 for regulating rice grain shape and a mutant thereof. Background Art

[0002] Rice is one of the most important food crops in the world. It is produced and consumed on a large scale, feeding more than 50% of the world's population. Therefore, it is of great significance to study the molecular mechanism of high and stable rice yield. In 2000, researchers completed the sequencing and annotation of the rice genome sequence, including functional genes regulated by multiple major QTLs, some of which have been cloned and their functions verified. Currently, scientists have discovered many functional genes that regulate rice agronomic traits using map-based cloning, functional genomic analysis and CRISPR / Cas9 gene editing technology.

[0003] Rice yield is mainly determined by three factors: number of grains per ear, number of ears per plant and grain weight, and rice grain shape is an important agronomic trait affecting rice yield. Grain weight (1000-grain weight) is largely determined by grain length, grain width and grain thickness, which synergistically regulate rice grain weight.

[0004] Rice grain shape is the premise for determining rice grain weight and an important basis for rice yield. Properly increasing rice grain shape is conducive to high rice yield. Therefore, the discovery and use of functional genes that regulate rice grain shape are extremely important for rice molecular assisted breeding, cultivating high-yield varieties and enriching rice regulatory networks.

[0005] During the more than 10,000 years of domestication of cultivated rice, many different grain shapes have been formed under different rice genetic backgrounds, providing a large amount of germplasm genetic resources for exploring rice grain shape regulation. Studies have found that several genes that regulate rice grain shape have been applied to modern rice breeding to cultivate new rice varieties with high yield and ideal grain shape.

[0006] However, the current research on rice grain shape has not yet allowed these genes to form a rice grain shape regulatory network. Therefore, discovering and studying more rice grain shape regulating genes will help to further understand how rice regulates grain shape and thus improve rice appearance and yield. Summary of the invention

[0007] The primary purpose of the present invention is to provide the use of the rice gene OsPOK1 for regulating rice grain shape, so that it can be better used for regulating rice grain shape.

[0008] To achieve this object, in a basic embodiment, the present invention provides the use of the rice gene OsPOK1 (LOC_Os07g44400) for regulating rice grain shape, wherein the coding sequence of the rice gene OsPOK1 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2.

[0009] The second object of the present invention is to provide a modified rice gene OsPOK1, which can significantly increase the grain length, thousand-grain weight and yield of rice.

[0010] To achieve this object, in a basic embodiment, the present invention provides a modified rice gene OsPOK1, the coding sequence of the modified rice gene OsPOK1 is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0011] The third object of the present invention is to provide a method for constructing a rice modified plant containing the modified rice gene OsPOK1 as described above, so as to construct a rice modified plant containing the modified rice gene OsPOK1 as described above, and the rice modified plant can significantly increase the grain length, thousand-grain weight and yield of rice.

[0012] To achieve this object, in a basic embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsPOK1 as described above. The construction method is based on the principle of CRISPR / Cas9 gene editing, and an expression vector carrying a gene expressing Cas9 protein and carrying sgRNA is transferred into the original rice plant. After screening and cultivation, the modified rice plant containing the modified rice gene OsPOK1 as described above is obtained.

[0013] The present invention uses a CRISPR / Cas9 gene editing vector to cause a frameshift mutation in the rice gene OsPOK1, thereby stopping the translation of the protein sequence encoded by SEQ ID NO.2 in advance, resulting in the loss of protein function of SEQ ID NO.2.

[0014] Preferably, the construction of the above-mentioned expression vector may include: screening and designing the OsPOK1 gene editing target sequence, amplifying the rice OsU6a promoter, constructing a gene editing expression cassette, and obtaining the CRISPR / Cas9 gene editing vector of the OsPOK1 gene through the steps of fragment and vector enzyme digestion, T4-DNA ligation, transformation of Escherichia coli DH5α competent cells, monoclonal colony PCR identification and vector sequencing.

[0015] Preferably, the acquisition of the above-mentioned rice modified plants may include: using the Agrobacterium-mediated method to directly transform the CRISPR / Cas9 gene editing vector of the OsPOK1 gene constructed above into the callus / cell of japonica rice Zhonghua 11, and obtaining OsPOK1 gene editing vector-positive plants through the steps of hygromycin screening, resistance callus differentiation and rooting culture. Collect leaves from each positive plant, separate the genome of a single rice plant by the CTAB method, and identify whether the obtained OsPOK1 gene-edited positive plants meet the transformation requirements through PCR, agarose gel electrophoresis, recovery of target fragments and sequencing analysis. Transplant the OsPOK1 gene-edited plants that meet the requirements into the field, and analyze and count the rice grain length, thousand-grain weight and yield at the rice maturity stage to determine the changes in the grain length, thousand-grain weight and yield traits of the OsPOK1 gene-edited plants compared with the wild-type japonica rice Zhonghua 11.

[0016] In a preferred embodiment, the present invention provides a method for constructing a rice modified plant containing the modified rice gene OsPOK1 as described above, wherein the target sequence of the sgRNA is shown as SEQ ID NO.5 or SEQ ID NO.6.

[0017] In a preferred embodiment, the present invention provides a method for constructing a rice transformed plant containing the modified rice gene OsPOK1 as described above, wherein the expression vector contains a hygromycin resistance gene and / or a kanamycin resistance gene.

[0018] In a preferred embodiment, the present invention provides a method for constructing a rice modified plant containing the modified rice gene OsPOK1 as described above, wherein the expression vector is transformed into the original rice plant by Agrobacterium-mediated method.

[0019] In a preferred embodiment, the present invention provides a method for constructing a rice modified plant containing the modified rice gene OsPOK1 as described above, wherein the original rice plant is japonica rice Zhonghua 11.

[0020] In a preferred embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsPOK1 as described above, wherein the screening and cultivation include callus induction, Agrobacterium activation and infection, co-cultivation, resistant callus screening and differentiation, rooting culture, positive plant identification, seedling hardening and transplanting to the field.

[0021] The fourth object of the present invention is to provide the use of the modified rice gene OsPOK1 as described above for regulating rice grain shape and / or yield.

[0022] To achieve this object, in a basic embodiment, the present invention provides the use of the modified rice gene OsPOK1 as described above for regulating rice grain shape and / or yield.

[0023] The beneficial effect of the present invention is that the present invention has discovered a new use of the rice gene OsPOK1 for regulating rice grain shape, and the constructed rice modified plants containing the modified rice gene OsPOK1 of the present invention can significantly increase the grain length and yield traits of rice grains. Such improvement of rice grain shape and yield provides a new gene resource for rice breeding.

[0024] The modified rice gene OsPOK1 and the modified rice plants containing the same of the present invention are of great significance for studying rice grain shape and yield regulation pathways, and provide a new breeding scheme for rationally and appropriately utilizing the OsPOK1 gene to cultivate large-grain and high-yield rice varieties, which has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of pOsU6a and pCRISPR / Cas9 related plasmid vector map. Figure 1 In A, U6apromoter represents the U6a promoter sequence of rice, Insert site is represented by a black box, which is the insertion position of the target sequence, sgRNA represents the scaffold sequence of the editing vector, AMP R Represents the ampicillin resistance gene sequence, AMP R promoter represents the promoter used to express the ampicillin resistance gene sequence, Ori represents the plasmid replication origin, and BsaI and BsmBI are restriction sites used to construct the vector; Figure 1 In B, CaMV 35S promoter is the 35S promoter sequence of cauliflower virus, Hyg R is the hygromycin resistance gene sequence, CaMV poly(A)signal represents the CaMV transcription termination sequence, KanR represents the kanamycin resistance gene sequence, LB T-DNA repeat and RB T-DNA repeat represent the left and right border sequences, respectively, Ubi-promoter represents the Ubiquitin promoter sequence of the maize ubiquitin protein gene, Cas9 represents the nuclease Cas9 gene sequence, NOSterminator represents the NOS transcription terminator sequence, and ccdB represents a gene sequence toxic to Escherichia coli, which is used to improve the screening of positive clones; Figure 1 C and 1D are schematic diagrams of the two target sequences of OsPOK1 inserted into the pOsU6a vector respectively; Figure 1E and 1F are schematic diagrams of the two target sequence expression cassettes of OsPOK1 inserted into the pCRISPR / Cas9 vector respectively.

[0026] Figure 2 The target sequence of OsPOK1 gene editing and the schematic diagram of the edited sequence of OsPOK1 gene editing positive plants. In the figure, black boxes represent exons, black lines represent introns, ATG represents the start codon, TGA represents the stop codon, bold fonts and green fills represent the missing bases at that location, and red fonts represent the PAM sequence.

[0027] Figure 3 The results of the grain phenotype, grain shape and yield-related traits of OsPOK1 gene-edited plants are shown in Figure 1. Figure 3 A is the observation result of grain length phenotype of mature plants, scale bar is 10 mm; Figure 3 B is the statistical result of grain length of mature plants; Figure 3 C is the statistical result of thousand-grain weight of mature plants; Figure 3 D is the statistical result of single plant yield of mature plants; Figure 3 E is the statistical result of plant height. Figure 3 In B, 3C, 3D, and 3E, *: significant difference at p<0.05. WT: japonica rice Zhonghua 11 (wild type); M1-1: plant obtained after gene editing of OsPOK1 target sequence 1; M2-1: plant obtained after gene editing of OsPOK1 target sequence 2. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are further described below by way of examples. The experimental methods involved in the examples are all conventional experimental methods unless otherwise specified, and the primer sequence synthesis and DNA fragment sequencing analysis were all completed by Shanghai Sangon Biotechnology Co., Ltd.

[0029] Example 1: Construction of OsPOK1 gene editing vector and its rice genetic transformation

[0030] The coding sequence of rice OsPOK1 gene was obtained from NCBI database (https: / / www.ncbi.nlm.nih.gov) as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2. The target sequence for gene editing was screened using online software CRISPR-GE (skl.scau.edu.cn / home / ), and finally two suitable sequences SEQ ID NO.5 and SEQ ID NO.6 were selected, named target 1 (Target-1) and target 2 (Target-2), respectively. According to the restriction site of OsU6a vector, the relevant primer sequence was designed for target 1, the forward primer Target1-F sequence was shown in SEQ ID NO.7, and the reverse primer Target1-R sequence was shown in SEQ ID NO.8; the relevant primer sequence was designed for target 2, the forward primer Target2-F sequence was shown in SEQ ID NO.9, and the reverse primer Target2-R sequence was shown in SEQ ID NO.10.

[0031] pOsU6a uses pUC57 as the vector backbone. First, the BsmBI and BsaI restriction sites contained in pUC57 are mutated to eliminate these two restriction sites. Then, the vector is linearized using the restriction endonuclease EcoRV, and the U6a-sgRNA expression cassette sequence is inserted into the pUC57 linearized sequence to form the Cas9 expression cassette intermediate vector. The vector structure is as follows: Figure 1 As shown in A.

[0032] pCRISPR / Cas9 is based on pCABMBIA1300. First, the Bsa1 restriction site contained in pCABMBIA1300 is mutated to eliminate this restriction site. Then, the vector is linearized using restriction endonucleases KpnI and HindIII, and the Ubiquitin promoter sequence, Cas9 protein sequence, and ccdB sequence are inserted into the pCABMBIA1300 linear sequence to form the pCRISPR / Cas9 gene editing vector. The vector structure is as follows: Figure 1 As shown in B.

[0033] First, the primer pairs of target 1 and target 2 were dissolved in sterile ultrapure water to prepare a 100 μM mother solution; then, the target adapter was prepared according to the reaction system in Table 1 (the reaction was carried out in a 1.5 mL centrifuge tube; incubated at 95°C for 30 s, placed at room temperature for 5 min, and the adapter was prepared); then, the target adapter was incubated with the pOsU6a plasmid vector according to the reaction system in Table 2 (37°C for 5 min, 10°C for 1 min, 20°C for 5 min, and reacted for 5 cycles). Finally, the reaction product was transformed into DH5α Escherichia coli competent cells, placed on ice for 30 min, heat-shocked at 42°C for 30 s, then placed on ice for 2 min, 500 μL of LB liquid culture medium was added, and incubated at 37°C and 220 rpm for 30 min. Finally, the product was evenly spread on an LB solid culture dish containing AMP (50 mg / L). Colony PCR was performed using specific primers (SEQ ID NO.11 and SEQ ID NO.8; or SEQ ID NO.11 and SEQ ID NO.10), and positive single clones were selected for sequencing analysis. The vectors with correct sequencing were named pOsU6a-Target1 and pOsU6a-Target2, and their vector structures were as follows: Figure 1 C and Figure 1 D.

[0034] Table 1 Target linker reaction system

[0035]

[0036] Table 2 Target linker and pOsU6a plasmid vector reaction system

[0037]

[0038] Use Bsa1 to digest pOsU6a-Target1 and pOsU6a-Target2, and recover the gene editing expression cassette fragments (about 500 bp) by agarose gel electrophoresis. The recovered fragments were incubated with the pCRISPR / Cas9 vector according to the reaction system in Table 3 (37°C for 5 min, 10°C for 1 min, 20°C for 5 min, and 10 cycles). Finally, the reaction products were transformed into DH5α Escherichia coli competent cells. Colony PCR was performed using specific primers (SEQ ID NO.12 and SEQ ID NO.8; or SEQ ID NO.12 and SEQ ID NO.10), and positive single clones were picked for shaking. The plasmids were extracted and sequenced correctly, which were OsPOK1 gene editing vectors, named pCRISPR / Cas9-OsPOK1-Target1 and pCRISPR / Cas9-OsPOK1-Target2, respectively. Their vector structures are as shown below. Figure 1 E and Figure 1F.

[0039] Table 3 Reaction system of recovered fragments and pCRISPR / Cas9 vector

[0040]

[0041] Based on the Agrobacterium-mediated method, japonica rice Zhonghua 11 (wild type WT) was transformed. After callus induction, Agrobacterium activation and infection, co-cultivation, resistant callus screening and differentiation, rooting culture, positive plant identification, seedling hardening and field transplanting, 10 genetically transformed T0 rice positive plants were obtained. The specific operations refer to the corresponding part of Example 1 of the specification of Chinese patent application CN118086362A (paragraphs 0043-0119).

[0042] Example 2: Molecular Identification of OsPOK1 Genome Sequence Modification

[0043] According to the target sequence information of the OsPOK1 gene, two pairs of specific primers were designed about 100-200bp upstream and downstream of the target sequence (the forward primer sequence of the first pair of primers is such as SEQ ID NO.15, and the reverse primer sequence is such as SEQ ID NO.16; the forward primer sequence of the second pair of primers is such as SEQ ID NO.17, and the reverse primer sequence is such as SEQ ID NO.18). Using the genomic DNA of japonica rice Zhonghua 11 (wild type) and the positive plant obtained in Example 1 as PCR templates, PCR amplification of the OsPOK1 target region was performed, and then the fragments were recovered and sequenced to identify whether the OsPOK1 gene of the T0 generation positive plant was edited. As a result, the M1-1 plant had 7 bases missing at the target site sequence, while the M2-1 plant had 22 bases missing at the target site sequence. Plants M1 and M2 with missing bases in the OsPOK1 target sequence and premature termination of the encoded amino acid sequence were used as plants for subsequent research. The progeny of the OsPOK1 candidate plants were separated to obtain the T-DNA-free OsPOK1 gene-edited plants M1-1 and M2-1 for subsequent phenotypic identification.

[0044] The target sequence of the OsPOK1 gene editing and the edited sequence of the OsPOK1 gene editing positive plant are as follows: Figure 2 shown.

[0045] Example 3: Grain shape-related detection of OsPOK1 gene-edited plants

[0046] Rice OsPOK1 gene-edited plants and wild-type japonica rice Zhonghua 11 (WT) were planted in the field at the same time (100 plants each, 10 rows, 10 plants per row, 25 cm between plants and 30 cm between rows), and the differences between them were observed throughout the growth period.

[0047] During the rice maturity period, the observation results are as follows Figure 3 A. The statistical results are as follows Figure 3 B. Figure 3 C. Figure 3 D. Figure 3 E. After comparison and statistical analysis, it was found that:

[0048] In terms of grain length, M1-1 and M2-1 were significantly larger than the wild type Zhonghua 11. The average grain length of M1-1 was 8.27 mm, which was 10.56% higher than the wild type's average grain length of 7.48 mm. The average grain length of M2-1 was 8.17 mm, which was 9.22% higher than the wild type's average grain length of 7.48 mm.

[0049] In terms of thousand-grain weight, M1-1 and M2-1 were significantly larger than the wild type Zhonghua 11. The average thousand-grain weight of M1-1 was 29.28g, which was 12.62% higher than the wild type's average thousand-grain weight of 26.00g. The average thousand-grain weight of M2-1 was 28.84g, which was 10.92% higher than the wild type's average thousand-grain weight of 26.00g.

[0050] In terms of single-plant yield, M1-1 and M2-1 were significantly greater than the wild-type japonica rice Zhonghua 11. The average single-plant yield of M1-1 was 46.71 g, which was 13.24% higher than the wild-type average single-plant yield of 41.25 g. The average single-plant yield of M2-1 was 46.00 g, which was 11.52% higher than the wild-type average single-plant yield of 41.25 g.

[0051] In terms of plant height, there was no significant difference between M1-1 and M2-1 and the wild type japonica rice Zhonghua 11. The average plant height of WT was 112.78 cm, the average plant height of M1-1 was 113.46 cm, and the average plant height of M2-1 was 112.12 cm.

[0052] It can be seen that the grain shape and yield traits of OsPOK1 gene-edited plants are significantly higher than those of wild-type plants, which proves that the OsPOK1 gene is involved in regulating rice grain shape and yield.

[0053] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above embodiments or implementations are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described implementations should be considered illustrative rather than restrictive in any respect. The scope of the present invention should be described by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. Use of the rice gene OsPOK1 for regulating rice grain shape, wherein the coding sequence of the rice gene OsPOK1 is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.

2.

2. A modified rice gene OsPOK1, characterized in that: The coding sequence of the modified rice gene OsPOK1 is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. A method for constructing a rice modified plant containing the modified rice gene OsPOK1 according to claim 2, characterized in that: The construction method is based on the principle of CRISPR / Cas9 gene editing, and the gene carrying the Cas9 protein and the expression vector carrying the sgRNA are transferred into the original rice plant. After screening and cultivation, the modified rice plant containing the modified rice gene OsPOK1 according to claim 2 is obtained.

4. The construction method according to claim 3, characterized in that: The target sequence of the sgRNA is shown in SEQ ID NO.5 or SEQ ID NO.

6.

5. The construction method according to claim 3, characterized in that: The expression vector contains a hygromycin resistance gene and / or a kanamycin resistance gene.

6. The construction method according to claim 3, characterized in that: The expression vector is transformed into the original rice plant through Agrobacterium-mediated method.

7. The construction method according to claim 3, characterized in that: The original rice plant is japonica rice Zhonghua 11.

8. The construction method according to claim 3, characterized in that: The screening and cultivation include callus induction, Agrobacterium activation and infection, co-cultivation, resistant callus screening and differentiation, rooting culture, positive plant identification, seedling hardening and field transplanting.

9. Use of the modified rice gene OsPOK1 according to claim 2 for regulating rice grain shape and / or yield.

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