Use of rice gene OsPOK1 for regulating rice grain shape and its mutants

Abstract

The present invention belongs to the technical field of crop breeding, and relates to the use of the rice gene OsPOK1 for regulating rice grain shape and its mutants. 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. The present invention discovers a new use of the rice gene OsPOK1 for regulating rice grain shape. 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. Such improvement of rice grain shape and yield provides new gene resources for rice breeding.

Description

Technical Field

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

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

[0003] Rice yield is mainly determined by three factors: the number of grains per panicle, the number of panicles 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, and they jointly regulate the grain weight of rice.

[0004] Rice grain shape is a prerequisite for determining rice grain weight and an important basis for rice yield composition. Appropriately increasing rice grain shape is beneficial to high rice yields. Therefore, discovering and utilizing functional genes regulating rice grain shape plays an extremely important role in aspects such as rice molecular-assisted breeding, cultivating high-yield varieties, and enriching the rice regulation network.

[0005] During the domestication of cultivated rice over more than 10,000 years, 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. Research has found that several genes regulating rice grain shape have been applied to modern rice breeding to cultivate new rice varieties with high yields and ideal grain shapes.

[0006] However, current research on rice grain shape cannot yet form a regulatory network for these genes related to rice grain shape. Therefore, excavating and studying more genes regulating rice grain shape helps to further understand how rice regulates grain shape, and thus improve the appearance and yield of rice. Summary of the Invention

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

[0008] For this purpose, 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 as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded thereby is as 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, 1000-grain weight and yield of rice.

[0010] For this purpose, in a basic embodiment, the present invention provides a modified rice gene OsPOK1, and the coding sequence of the modified rice gene OsPOK1 is as 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 modified rice plant containing the modified rice gene OsPOK1 as described above, so as to be able to construct a modified rice plant containing the modified rice gene OsPOK1 as described above, and this modified rice plant can significantly increase the grain length, 1000-grain weight and yield of rice.

[0012] For this purpose, 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. Based on the principle of CRISPR / Cas9 gene editing, a gene carrying the Cas9 protein and an expression vector carrying sgRNA are transferred into the original rice plant, and after screening and cultivation, a 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 causing the protein sequence of SEQ ID NO.2 to stop translation prematurely, resulting in the loss of the protein function of SEQ ID NO.2.

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

[0015] Preferably, the acquisition of the above-mentioned genetically modified rice plants may include: using the Agrobacterium-mediated method to directly transform the callus / cells of japonica rice Zhonghua 11 with the above-constructed CRISPR / Cas9 gene editing vector of the OsPOK1 gene. Through steps such as hygromycin screening, resistant callus differentiation, and rooting culture, positive plants of the OsPOK1 gene editing vector are obtained. Collect the leaves of each positive plant, isolate the genomic DNA of individual rice plants by the CTAB method, and identify whether the obtained positive plants with OsPOK1 gene editing meet the modification requirements through processes such as PCR, agarose gel electrophoresis, recovery of target fragments, and sequencing analysis. Transplant the OsPOK1 gene editing plants that meet the requirements to the field, and analyze and count the grain length, 1000-grain weight, and yield of the rice at the maturity stage of the rice to determine the changes in the grain length, 1000-grain weight, and yield traits of the OsPOK1 gene editing plants compared with the wild-type japonica rice Zhonghua 11.

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

[0017] In a preferred embodiment, the present invention provides a method for constructing a genetically modified rice plant containing the above-mentioned modified rice gene OsPOK1, 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 genetically modified rice plant containing the above-mentioned modified rice gene OsPOK1, wherein the expression vector is transformed into the original rice plant by the Agrobacterium-mediated method.

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

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

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

[0022] For this purpose, in a basic implementation, 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 effects of the present invention are as follows. The present invention discovers a new use of the rice gene OsPOK1 in regulating rice grain shape. 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 new gene resources for rice breeding.

[0024] The modified rice gene OsPOK1 of the present invention and the rice modified plants containing it are of great significance for studying the rice grain shape and yield regulation pathways, providing a brand-new breeding scheme for reasonably and appropriately using the OsPOK1 gene to cultivate large-grain and high-yield rice varieties, and having a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the plasmid vector maps of pOsU6a and pCRISPR / Cas9-related plasmids. Among them 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 for expressing the ampicillin resistance gene sequence, Ori represents the plasmid replication origin, BsaI and BsmBI are the restriction enzyme sites used for constructing the vector; Figure 1 In B, CaMV 35S promoter is the 35S promoter sequence of cauliflower mosaic 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 respectively represent the left and right border sequences, 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 termination sequence, ccdB represents a gene sequence that is toxic to Escherichia coli and is used to improve the screening of positive clones; Figure 1 C and 1D are schematic diagrams of the insertion of two target sequences of OsPOK1 into the pOsU6a vector respectively; Figure 1Schematic diagrams of the insertion of two target sequence expression cassettes of OsPOK1 into the pCRISPR / Cas9 vector, respectively, for E and 1F.

[0026] Figure 2 Schematic diagrams of the target sequences for gene editing of OsPOK1 and the sequences after editing of positive plants with gene-edited OsPOK1. In the figure, the black boxes represent exons, the black lines represent introns, ATG represents the start codon, TGA represents the stop codon, the bold and green-filled font indicates the deletion of bases at that position, and the red font represents the PAM sequence.

[0027] Figure 3 Results graphs of the grain phenotypes, grain shapes, and yield-related traits of gene-edited OsPOK1 plants, where Figure 3 A shows the observed results of the grain length phenotype of plants at the mature stage, with the scale bar being 10 mm; Figure 3 B shows the statistical results of the grain length of plants at the mature stage; Figure 3 C shows the statistical results of the 1000-grain weight of plants at the mature stage; Figure 3 D shows the statistical results of the yield per plant of plants at the mature stage; Figure 3 E shows the statistical results of the plant height of plants. Figure 3 In B, 3C, 3D, and 3E, *: significant difference with 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. Specific implementation manners

[0028] The following further illustrates the specific implementation manners of the present invention through examples. The experimental methods involved in the examples are all conventional experimental methods unless otherwise specified. The synthesis of primer sequences and the sequencing analysis of DNA fragments were all completed by Shanghai Sangon Biotech Co., Ltd.

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

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

[0031] pOsU6a uses pUC57 as the vector backbone. First, point mutations were made to the BsmBI and BsaI restriction enzyme digestion sites contained in pUC57 to eliminate these two restriction enzyme digestion sites. Then, the vector was linearized using the restriction enzyme EcoRV, and the U6a-sgRNA expression cassette sequence was inserted into the pUC57 linearized sequence to construct an intermediate vector for the Cas9 expression cassette. Its vector structure is as Figure 1 shown in A.

[0032] pCRISPR / Cas9 uses pCABMBIA1300 as the backbone. First, a point mutation was made to the Bsa1 restriction enzyme digestion site contained in pCABMBIA1300 to eliminate this restriction enzyme digestion site. Then, the vector was linearized using the restriction enzymes KpnI and HindIII, and the Ubiquitin promoter sequence, Cas9 protein sequence, and ccdB sequence were inserted into the pCABMBIA1300 linear sequence to construct the pCRISPR / Cas9 gene editing vector. Its vector structure is as Figure 1 shown in B.

[0033] First, dissolve the primer pairs of target 1 and target 2 separately in sterile ultrapure water to prepare 100 μM stock solutions. Then, prepare the target adapters according to the reaction system in Table 1 (the reaction is carried out in a 1.5 mL centrifuge tube; incubate at 95 °C for 30 s, place at room temperature for 5 min, and the adapter preparation is completed). Then, incubate the target adapters 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 perform 5 reaction cycles). Finally, transform the reaction products into DH5α Escherichia coli competent cells, place on ice for 30 min, heat shock at 42 °C for 30 s, then place on ice for 2 min, add 500 μL of LB liquid medium, incubate in a shaker at 37 °C and 220 rpm for 30 min, and finally spread the products evenly on an LB solid culture dish containing AMP (50 mg / L). Perform colony PCR with specific primers (SEQ ID NO.11 and SEQ ID NO.8; or SEQ ID NO.11 and SEQ ID NO.10), pick positive monoclonal colonies for sequencing analysis, and name the vectors with correct sequencing as pOsU6a-Target1 and pOsU6a-Target2, and their vector structures are respectively as Figure 1 C and Figure 1 D.

[0034] Table 1 Reaction system for target adapters

[0035]

[0036] Table 2 Reaction system for target adapters and pOsU6a plasmid vector

[0037]

[0038] Digest pOsU6a-Target1 and pOsU6a-Target2 with Bsa1 respectively, and recover the gene editing expression cassette fragments (about 500 bp) by agarose gel electrophoresis. Incubate the recovered fragments 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 perform 10 reaction cycles). Finally, transform the reaction products into DH5α Escherichia coli competent cells respectively. Perform colony PCR with specific primers (SEQ ID NO.12 and SEQ ID NO.8; or SEQ ID NO.12 and SEQ ID NO.10), pick positive monoclonal colonies for shaking culture, extract plasmids respectively, and the vectors with correct sequencing are the OsPOK1 gene editing vectors, named pCRISPR / Cas9-OsPOK1-Target1 and pCRISPR / Cas9-OsPOK1-Target2 respectively, and their vector structures are respectively as Figure 1 E and Figure 1F。

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

[0040]

[0041] Based on the Agrobacterium-mediated method, japonica rice Zhonghua 11 (wild type WT) was transformed. Through callus induction, Agrobacterium activation and infection, co-cultivation, resistant callus screening and differentiation, rooting culture, positive plant identification, acclimatization and transplanting to the field, etc., 10 genetically transformed T0 rice positive plants were obtained respectively. The specific operation refers to the corresponding part (paragraphs 0043-0119) of Example 1 in the specification of Chinese Patent Application CN118086362A.

[0042] Example 2: Molecular identification of the genomic sequence modification of OsPOK1

[0043] According to the target sequence information of the OsPOK1 gene, two pairs of specific primers were designed about 100-200 bp upstream and downstream of the target sequence (the forward primer sequence of the first pair of primers is as SEQ ID NO.15, and the reverse primer sequence is as SEQ ID NO.16; the forward primer sequence of the second pair of primers is as SEQ ID NO.17, and the reverse primer sequence is as SEQ ID NO.18). Using the genomic DNA of japonica rice Zhonghua 11 (wild type) and the positive plants obtained in Example 1 as PCR templates, the target region of OsPOK1 was amplified by PCR, and then the recycled fragments were subjected to sequencing analysis to identify whether the OsPOK1 gene of the T0 generation positive plants was edited. The results showed that the M1-1 plant had a 7-base deletion at the target site sequence, while the M2-1 plant had a 22-base deletion at the target site sequence. Plants M1 and M2 with deletions in the OsPOK1 target sequence and premature termination of the encoded amino acid sequence were used as plants for subsequent research. The OsPOK1 candidate plants were subjected to progeny segregation to obtain OsPOK1 gene-edited plants M1-1 and M2-1 without T-DNA for subsequent phenotype identification.

[0044] The above-mentioned target sequence of OsPOK1 gene editing and the edited sequence of the OsPOK1 gene-edited positive plants are as Figure 2 shown.

[0045] Example 3: Detection related to grain shape of OsPOK1 gene-edited plants

[0046] The OsPOK1 gene-edited plants of rice 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, plant spacing 25 cm, row spacing 30 cm), and the differences between them were observed during the whole growth period.

[0047] At the mature stage of rice, the observation results are asFigure 3 A, the statistical results are as follows Figure 3 B, Figure 3 C, Figure 3 D, Figure 3 E. Through comparison and statistical analysis, it is found that:

[0048] In terms of grain length, M1-1 and M2-1 are significantly greater than the wild-type japonica rice Zhonghua 11. The average grain length of M1-1 is 8.27 mm, which is 10.56% higher than the average grain length of the wild type (7.48 mm). The average grain length of M2-1 is 8.17 mm, which is 9.22% higher than the average grain length of the wild type (7.48 mm).

[0049] In terms of 1000-grain weight, M1-1 and M2-1 are significantly greater than the wild-type japonica rice Zhonghua 11. The average 1000-grain weight of M1-1 is 29.28 g, which is 12.62% higher than the average 1000-grain weight of the wild type (26.00 g). The average 1000-grain weight of M2-1 is 28.84 g, which is 10.92% higher than the average 1000-grain weight of the wild type (26.00 g).

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

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

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

[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 equivalent technologies, the present invention also intends to include these modifications and variations. The above embodiments or implementation manners are only illustrative examples of the present invention. The present invention can 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 implementation manners should be regarded as illustrative rather than restrictive in any aspect. The scope of the present invention should be defined by the appended claims, and any changes equivalent to the intention and scope of the claims should also be included within the scope of the present invention.

Claims

1. 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.

2. A method for constructing a rice modified plant containing the modified rice gene OsPOK1 according to claim 1, characterized in that: The construction method is based on the principle of CRISPR / Cas9 gene editing, and the gene encoding Cas9 protein and the expression vector carrying 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 1 is obtained, and the original rice plant is japonica rice Zhonghua 11.

3. The construction method according to claim 2, characterized in that: The target sequence of the sgRNA is shown in SEQ ID NO.5 or SEQ ID NO.

6.

4. The construction method according to claim 2, characterized in that: The expression vector contains a hygromycin resistance gene and / or a kanamycin resistance gene.

5. The construction method according to claim 2, characterized in that: The expression vector is transformed into the original rice plant through Agrobacterium-mediated method.

6. The construction method according to claim 2, 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.

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