Use of rice gene OsABCA3 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 OsABCA3 for regulating rice grain shape and its mutants. The coding sequence of the rice gene OsABCA3 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 OsABCA3 for regulating rice grain shape. The constructed rice modified plants containing the modified rice gene OsABCA3 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.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop breeding, and relates to the use of the rice gene OsABCA3 for regulating rice grain shape and its mutants. Background Art

[0002] With the continuous growth of the population and the prosperity of the economy in the 21st century, the global population is expected to exceed 9 billion by 2050. The contradiction between population growth and the reduction of arable land leads to food shortages, which will become a major challenge faced by each country. Rice is one of the most important food crops in the world. The size and weight of rice grains are two major determinants of rice yield. Grain development involves cell proliferation and expansion, which affects its shape and size. Grain size is an important trait and has been the focus of many studies to discover more genes regulating rice grain shape and to analyze the potential regulatory mechanisms. Grain size not only affects the adaptability of plants under natural conditions but also is the direction of human long-term selection. Therefore, studying the molecular mechanism regulating rice grain shape is of great significance. 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. Rice grain shape mainly includes grain length, grain width, and grain thickness. Generally, changes in rice grain shape will affect the grain weight of rice, and thus will affect the final yield of rice.

[0003] Rice grain shape is a prerequisite for determining rice grain weight and an important basis for the composition of rice yield. Appropriately increasing rice grain shape is beneficial to high rice yield. 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.

[0004] During the domestication process of cultivated rice over thousands of years, many different grain shapes have been formed under different rice genetic backgrounds, providing a large number of germplasm resources for exploring the regulation of rice grain shape. Research has found that several genes regulating rice grain shape have been applied to rice breeding to cultivate high-yield rice varieties with ideal grain shape.

[0005] However, current research on rice grain shape cannot form a regulatory network of these genes for rice grain shape. Therefore, excavating and studying more rice grain shape regulatory genes helps to further understand how rice regulates grain shape, and thus improve the appearance and yield of rice. Summary of the Invention

[0006] The primary object of the present invention is to provide the use of the rice gene OsABCA3 for regulating rice grain shape, so as to be better used for the regulation of rice grain shape.

[0007] To achieve this purpose, in a basic embodiment, the present invention provides the use of the rice gene OsABCA3 (LOC_Os08g30740) for regulating rice grain shape, wherein the coding sequence of the rice gene OsABCA3 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.

[0008] The second object of the present invention is to provide a modified rice gene OsABCA3, which can significantly increase the grain length, 1000-grain weight and yield of rice.

[0009] To achieve this purpose, in a basic embodiment, the present invention provides a modified rice gene OsABCA3, and the coding sequence of the modified rice gene OsABCA3 is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0010] The third object of the present invention is to provide a method for constructing a modified rice plant containing the modified rice gene OsABCA3 as described above, so as to be able to construct a modified rice plant containing the modified rice gene OsABCA3 as described above, and this modified rice plant can significantly increase the grain length, 1000-grain weight and yield of rice.

[0011] To achieve this purpose, in a basic embodiment, the present invention provides a method for constructing a modified rice plant containing the modified rice gene OsABCA3 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 culturing, a modified rice plant containing the modified rice gene OsABCA3 as described above is obtained.

[0012] The present invention uses a CRISPR / Cas9 gene editing vector to cause a frameshift mutation in the rice gene OsABCA3, 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.

[0013] Preferably, the construction of the above expression vector may include: screening and designing the OsABCA3 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, etc., to obtain the CRISPR / Cas9 gene editing vector of the OsABCA3 gene.

[0014] 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 OsABCA3 gene, and obtaining positive plants of the OsABCA3 gene editing vector through steps such as hygromycin screening, differentiation of resistant callus, and rooting culture. Collect the leaves of each positive plant, isolate the genomic DNA of single-plant rice by the CTAB method, and identify whether the obtained positive plants with OsABCA3 gene editing meet the modification requirements through processes such as PCR, agarose gel electrophoresis, recovery of target fragments, and sequencing analysis. Transplant the OsABCA3 gene editing plants that meet the requirements to the field, and analyze and statistically analyze the grain length, 1000-grain weight, and yield of the rice at the rice maturity stage to determine the changes in the grain length, 1000-grain weight, and yield traits of the OsABCA3 gene editing plants compared with the wild-type japonica rice Zhonghua 11.

[0015] In a preferred embodiment, the present invention provides a method for constructing a genetically modified rice plant containing the above-mentioned genetically modified rice gene OsABCA3, wherein the target sequence of the sgRNA is as shown in SEQ ID NO.5 or SEQ ID NO.6.

[0016] In a preferred embodiment, the present invention provides a method for constructing a genetically modified rice plant containing the above-mentioned genetically modified rice gene OsABCA3, wherein the expression vector contains a hygromycin resistance gene and / or a kanamycin resistance gene.

[0017] In a preferred embodiment, the present invention provides a method for constructing a genetically modified rice plant containing the above-mentioned genetically modified rice gene OsABCA3, wherein the expression vector is transformed into the original rice plant by the Agrobacterium-mediated method.

[0018] In a preferred embodiment, the present invention provides a method for constructing a genetically modified rice plant containing the above-mentioned genetically modified rice gene OsABCA3, wherein the original rice plant is japonica rice Zhonghua 11.

[0019] In a preferred embodiment, the present invention provides a method for constructing a genetically modified rice plant containing the above-mentioned genetically modified rice gene OsABCA3, 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.

[0020] The fourth object of the present invention is to provide the use of the above-mentioned genetically modified rice gene OsABCA3 for regulating rice grain shape and / or yield.

[0021] For this purpose, in a basic implementation, the present invention provides the use of the modified rice gene OsABCA3 as described above for regulating rice grain shape and / or yield.

[0022] The beneficial effects of the present invention are as follows: The present invention discovers a new use of the rice gene OsABCA3 in regulating rice grain shape. The constructed rice modified plants containing the modified rice gene OsABCA3 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.

[0023] The modified rice gene OsABCA3 of the present invention and the rice modified plants containing the same 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 OsABCA3 gene to cultivate large-grain and high-yield rice varieties, and having a very broad application prospect. Brief Description of the Drawings

[0024] 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 OsABCA3 into the pOsU6a vector respectively; Figure 1Schematic diagrams of the expression cassettes of two target sequences of OsABCA3 inserted into the pCRISPR / Cas9 vector, respectively, for E and 1F.

[0025] Figure 2 Schematic diagrams of the target sequences for gene editing of OsABCA3 and the sequences after editing of the positive plants for OsABCA3 gene editing. 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 inserted bases at that position, and the red font represents the PAM sequence.

[0026] Figure 3 Results graphs of the grain phenotypes, grain shapes, and yield-related traits of the OsABCA3 gene-edited plants, where Figure 3 A shows the observed results of the grain length phenotype of the plants at the mature stage, and the scale bar is 10 mm; Figure 3 B shows the statistical results of the grain length of the plants at the mature stage; Figure 3 C shows the statistical results of the 1000-grain weight of the plants at the mature stage; Figure 3 D shows the statistical results of the yield per plant of the plants at the mature stage; Figure 3 E shows the statistical results of the plant height of the plants. Figure 3 In B, 3C, 3D, and 3E, *: significant difference with p < 0.05. WT: Japonica rice Zhonghua 11 (wild type); M1-1: the plant obtained after gene editing of the OsABCA3 target sequence 1; M2-1: the plant obtained after gene editing of the OsABCA3 target sequence 2. Specific implementation methods

[0027] The following further illustrates the specific implementation methods 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 Biological Engineering Co., Ltd.

[0028] Example 1: Construction of the gene editing vector of OsABCA3 and its genetic transformation in rice

[0029] The coding sequence of the rice OsABCA3 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 2 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; for Target-2, relevant primer sequences were designed. 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.

[0030] pOsU6a uses pUC57 as the vector backbone. First, the BsmBI and BsaI restriction enzyme digestion sites in pUC57 were subjected to point mutations 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, and its vector structure is as Figure 1 shown in A.

[0031] pCRISPR / Cas9 uses pCABMBIA1300 as the backbone. First, the Bsa1 restriction enzyme digestion site in pCABMBIA1300 was subjected to point mutations 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, and its vector structure is as Figure 1 shown in B.

[0032] First, dissolve the primer pairs of target 1 and target 2 separately in sterile ultrapure water to prepare 100 μM mother 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 react for 5 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 at 37 °C on a shaker at 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.

[0033] Table 1 Reaction system for target adapters

[0034]

[0035] Table 2 Reaction system for target adapters and pOsU6a plasmid vector

[0036]

[0037] Use Bsa1 to digest pOsU6a-Target1 and pOsU6a-Target2 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 react for 10 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 OsABCA3 gene editing vectors, named pCRISPR / Cas9-OsABCA3-Target1 and pCRISPR / Cas9-OsABCA3-Target2 respectively, and their vector structures are respectively as Figure 1 E andFigure 1 F.

[0038] Table 3 Reaction system of the recovered fragment and pCRISPR / Cas9 vector

[0039]

[0040] Based on the Agrobacterium-mediated method, japonica rice Zhonghua 11 (wild type WT) was transformed. After callus induction, Agrobacterium activation and infection, co-culture, resistant callus screening and differentiation, rooting culture, positive plant identification, hardening off and transplanting to the field, etc., 10 genetically transformed T0 rice positive plants were obtained respectively. The specific operations refer to the corresponding part (paragraphs 0043 - 0119) of Example 1 in the specification of Chinese Patent Application CN118086362A.

[0041] Example 2: Molecular identification of the modification of the OsABCA3 genomic sequence

[0042] According to the target sequence information of the OsABCA3 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 shown in SEQ ID NO.15, and the reverse primer sequence is as shown in SEQ ID NO.16; the forward primer sequence of the second pair of primers is as shown in SEQ ID NO.17, and the reverse primer sequence is as shown in 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 OsABCA3 was amplified by PCR, and then the recovered fragment and sequencing analysis were carried out to identify whether the OsABCA3 gene of the T0 generation positive plants was edited. The results showed that 1 base was inserted at the target site sequence of plants M1 - 1 and M2 - 1 respectively. Plants M1 and M2 with inserted bases in the OsABCA3 target sequence and premature termination of the encoded amino acid sequence were used as plants for subsequent research. The OsABCA3 candidate plants were subjected to progeny segregation to obtain OsABCA3 gene-edited plants M1 - 1 and M2 - 1 without T-DNA for subsequent phenotypic identification.

[0043] The above-mentioned target sequence of OsABCA3 gene editing and the edited sequence of the OsABCA3 gene-edited positive plants are as Figure 2 shown.

[0044] Example 3: Detection related to grain shape of OsABCA3 gene-edited plants

[0045] The OsABCA3 gene-edited plants of rice and wild type japonica rice Zhonghua 11 (WT) were planted in the field simultaneously (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.

[0046] At the mature stage of rice, the observation results are as Figure 3 shown in Figure 3 Figure Figure 3 A, and the statistical results are as Figure 3 shown in Figure 3 Figures

[0047] 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 7.99 mm, which is 7.83% higher than the average grain length of the wild type (7.41 mm). The average grain length of M2-1 is 7.96 mm, which is 7.42% higher than the average grain length of the wild type (7.41 mm).

[0048] 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 27.50 g, which is 5.36% higher than the average 1000-grain weight of the wild type (26.10 g). The average 1000-grain weight of M2-1 is 27.40 g, which is 4.98% higher than the average 1000-grain weight of the wild type (26.10 g).

[0049] 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 44.11 g, which is 5.63% higher than the average yield per plant of the wild type (41.76 g). The average yield per plant of M2-1 is 43.89 g, which is 5.10% higher than the average yield per plant of the wild type (41.76 g).

[0050] 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 110.55 cm, the average plant height of M1-1 is 111.28 cm, and the average plant height of M2-1 is 111.62 cm.

[0051] Therefore, it can be seen that the grain shape and yield traits of the OsABCA3 gene-edited plants are significantly higher than those of the wild-type plants, thus proving that the OsABCA3 gene is involved in regulating the grain shape and yield of rice.

[0052] 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 is also intended 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. Use of rice gene OsABCA3 for regulating rice grain length, wherein the coding sequence of the rice gene OsABCA3 is as shown in SEQ ID NO.1, the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.2, and the use is achieved by making the protein function of SEQ ID NO.2 deficient.

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