Application of PDIL2;3 gene in regulating grain shape and chalkiness traits in rice

Abstract

The application discloses application of a PDIL2;3 gene in regulation of rice grain type and chalkiness. The application provides, for the first time, important regulation of a protein disulfide isomerase coding gene PDIL2;3 on the grain type and chalkiness of rice, and a spike becomes larger, a grain becomes longer, a grain width becomes wider, the thousand-grain weight increases and the chalkiness increases by knocking out or destroying the normal expression level of the PDIL2;3 gene, so as to obtain an ideal rice grain type, improve the rice quality, provide a new approach for high-yield and high-quality breeding of rice, and have a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and particularly to... PDIL2;3 Application of genes in regulating rice grain shape and chalkiness traits. Background Technology

[0002] Rice is one of the most important food crops in my country and other Asian countries. High yield and high quality have always been the two most important goals of rice application and basic research in my country. Since the development and utilization of dwarf rice breeding and hybrid rice in the 1950s-1970s, my country's total grain output has experienced a qualitative leap, and high-yield breeding has reached a world-leading level. With the improvement of people's living standards and changes in dietary structure, rice quality is receiving increasing attention. People's demand for high-quality rice with good appearance, taste, and nutrition is growing. Against this backdrop, breeding new rice varieties that are high-quality, high-yielding, highly nutritious, and more popular with consumers has become a goal for breeders in recent years. The evaluation of rice quality by rice breeders, sellers, and consumers is basically consistent both domestically and internationally, generally including four main aspects: appearance quality, processing quality, cooking and eating quality, and nutritional quality. Among these, the appearance quality of rice mainly depends on grain size, chalkiness, and translucency, and their quality directly affects the commercial quality of rice, making them the most important rice quality traits and evaluation standards.

[0003] Chalkiness is a white, opaque part caused by insufficient grain filling in rice, resulting in loose endosperm tissue and scattered light. Chalky rice has reduced transparency, loose texture, and is easily broken during processing, directly affecting the yield of polished rice. Furthermore, chalky rice has low amylose content, poor eating and nutritional quality, and reduced commercial value, making it one of the main limiting factors restricting the rate of high-quality rice production in my country.

[0004] Rice grain shape is typically represented by three sets of data: grain length, grain width, and grain thickness. Generally, a slender grain shape is beneficial for grain filling, reducing chalkiness and chalky rice rate, thus significantly improving the appearance quality of rice. Some cloned grain shape QTLs have shown a concomitant effect of chalkiness, such as qTGW6, GW2, GS2, qSW5, GW7, and GW8. These research results indicate that grain shape and chalkiness are the most important indicators determining the appearance quality of rice, and that rice grain shape has a significant impact on chalkiness formation. Grain shape genes have broad application prospects in regulating high-yield and high-quality rice breeding. Currently, research on genes related to rice quality, such as chalkiness rate, transparency, and texture, is relatively limited, restricting the application of technologies such as gene editing to improve rice quality. Even when candidate genes related to grain shape and chalkiness are discovered, their functional validation and practical application are insufficient, lacking extensive field validation experience. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a... PDIL2;3 Application of genes in regulating rice grain shape and chalkiness traits. PDIL2;3 After the gene mutation, the rice grains exhibited a phenotype of larger panicles, longer grains, wider grains, and increased thousand-grain weight, but also a significant increase in chalkiness. This provides excellent genetic material for studying the regulation of rice grain shape and chalkiness, and can be applied to the genetic improvement of rice yield and appearance quality. It provides technical support and scientific basis for ensuring the continuous increase of rice production in agriculture, and has important production application value.

[0006] This invention provides PDIL2;3 The application of genes in regulating grain shape and / or chalkiness traits in rice, the aforementioned PDIL2;3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

[0007] In some embodiments, the particle shape is particle length, particle width, and / or particle weight.

[0008] In some implementations, the application specifically refers to: controlling the grain width, grain weight, or chalkiness of rice.

[0009] In some implementations, the regulation specifically includes any one of the following:

[0010] (i) By inhibiting or reducing PDIL2;3 The activity of gene-encoded proteins or their expression levels can increase grain length, grain width, grain weight, and / or chalkiness in rice.

[0011] (ii) By knocking out, suppressing or silencing PDIL2;3 Gene expression levels are used to increase grain length, grain width, grain weight, and / or chalkiness in rice.

[0012] In some implementations, the regulation specifically includes any one of the following:

[0013] (a) By improving PDIL2;3 The activity of gene-encoded proteins or their expression levels can be used to reduce grain width, grain weight, and / or chalkiness in rice.

[0014] (b) By improving PDIL2;3 The expression level of genes is used to reduce grain length, grain width, grain weight and / or chalkiness in rice.

[0015] In some embodiments, the PDIL2;3 The nucleotide sequence of the gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0016] This invention also provides a method for adjusting rice grain shape and / or chalkiness traits, adjusting... PDIL2;3 The expression or activity of a gene or its encoded protein; PDIL2;3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

[0017] In some embodiments, the rice quality refers to the grain length, grain width, and / or grain weight of the rice.

[0018] In some implementations, the method is selected from any of the following:

[0019] (1) Downward adjustment PDIL2;3 Gene expression levels, PDIL2;3 The expression level of the gene-encoded protein or PDIL2;3 The activity of gene-encoded proteins increases the grain length, grain width, grain weight, and / or chalkiness of rice.

[0020] (2) Upward adjustment PDIL2;3 Gene expression levels, PDIL2;3 The expression level of the gene-encoded protein or PDIL2;3 The activity of gene-encoded proteins reduces grain length, grain width, grain weight, and / or chalkiness in rice.

[0021] In some implementations, the upward adjustment in (2) PDIL2;3 Gene expression levels, PDIL2;3 The expression level of the gene-encoded protein or PDIL2;3 The activity of the gene-encoded protein specifically includes any of the following:

[0022] (a) will PDIL2;3 Gene expression constructs or vectors are transferred into rice or rice cells;

[0023] (b) containing PDIL2;3 Gene expression constructs or vectors are transferred into rice or rice cells.

[0024] This invention also provides a method for breeding rice, comprising the following steps: improving or inhibiting... PDIL2;3 Gene expression levels, PDIL2;3 The expression level of the gene-encoded protein or PDIL2;3 The active substance of a gene-encoded protein is introduced into rice or rice cells to obtain selectively bred rice; PDIL2;3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

[0025] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0026] 1. This invention provides for the first time a gene encoding a protein disulfide isomerase. PDIL2;3 Application in regulating rice grain shape and chalkiness traits; PDIL2;3 Genes play an important role in regulating the grain shape and chalkiness of rice grains, providing a new approach for high-yield and high-quality rice breeding and having broad application prospects.

[0027] 2. This invention utilizes gene editing technology to knock out or disrupt... PDIL2;3 The goal of obtaining rice with larger panicles, longer grains, wider grains, increased thousand-grain weight, and increased chalkiness is to achieve the ideal rice grain shape and improve rice quality by obtaining the normal expression level of genes.

[0028] 3. Discovery of this invention PDIL2;3 The knockout mutant exhibits larger ears, longer grains, wider grains, and increased thousand-grain weight, proving that... PDIL2;3 This study plays an important role in rice grain shape formation and yield regulation, enriching our understanding of rice grain shape formation and providing a theoretical basis for the cultivation of high-yield rice. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 As in Embodiment 1 of the present invention PDIL2;3 Gene expression pattern diagram; Figure A is PDIL2;3 The expression profiles of genes in various tissues and organs of rice 'Zhonghua 11' are shown in Figures B-L. AB represents lateral buds, L represents leaves, LS represents leaf sheaths, R represents roots, S represents stems, 0.5P-25P represents young panicles 0.5 cm-25 cm in length, and En3-En24 represents endosperm 3-24 days after fertilization. Figures B-L show the expression profiles of genes expressed in various tissues and organs of rice 'Zhonghua 11'. PDIL2;3Expression in various tissues and organs; the scale bar for Figures B-E and I-M is 0.2 cm; the scale bar for F is 0.1 cm; and the scale bar for G-H is 1 cm.

[0031] Figure 2 This is Example 2 of the present invention. PDIL2;3 The creation of mutants and related sequencing results; Figure A shows the sgRNA in... PDIL2;3 Schematic diagram of gene locus identification location; Figure B shows the quantitative RT-PCR detection of wild-type and... PDIL2;3-cri T2 generation transgenic plants PDIL2;3 Expression levels; Figure C shows sequencing analysis. PDIL2;3-cri1 and PDIL2;3-cri2 exist PDIL2;3 Mutation patterns at gene loci; Figure D shows... PDIL2;3-cri The mutation status of the amino acid sequence encoded by the mutant, where red represents the mutated amino acid sequence.

[0032] Figure 3 This is a map of the empty GUS clone vector DX2181 in Example 2 of the present invention.

[0033] Figure 4 This is Example 2 of the present invention. PDIL2;3 Results of yield-related phenotypic identification of mutants; Figure A shows T2 generation gene-edited plants. PDIL2;3-cri Figure B shows the mature plant form of the wild-type plant, with a scale bar of 15 cm; Figure B shows the T2 generation. PDIL2;3-cri The spike type of wild-type plants is shown on a scale bar of 5 cm; Figures C-H represent the T2 generation. PDIL2;3-cri Grain shape similar to wild type, scale bar is 2cm; Figure I shows T2 generation. PDIL2;3-cri Compared with the wild type, the grain length and width are compared on a scale bar of 10 mm; Figures J~S represent the T2 generation. PDIL2;3- cri The statistical results of tiller number, plant height, number of primary branches, number of secondary branches, number of grains per ear, grain length, grain width, grain thickness, and thousand-grain weight of wild-type plants were presented. P A value <0.01 indicates a highly significant difference.

[0034] Figure 5 This is a map of the overexpression vector in Example 3 of the present invention.

[0035] Figure 6 Overexpression in Example 3 of the present invention PDIL2;3 Results of yield-related phenotypic identification of transgenic plants; Figure A shows T2 generation transgenic positive plants. PDIL2;3-OE Figure B shows the plant type of wild-type and negative-type plants, with a scale bar of 15 cm; Figure B shows the plant type of wild-type and negative-type plants detected by quantitative RT-PCR. PDIL2;3-OE In transgenic plants PDIL2;3 Expression levels; Figure C shows T2 generation transgenic positive plants. PDIL2;3-OEFigures D-E show the ear shape of T2 generation transgenic positive and negative plants, with a scale bar of 5 cm; Figures F-K show the ear shape of T2 generation transgenic positive and negative plants, with a scale bar of 5 mm; Figure LU shows the statistical results of tiller number, plant height, number of primary branches, number of secondary branches, number of grains per ear, grain length, grain width, grain thickness, and thousand-grain weight for T2 generation transgenic positive and negative plants. P A value <0.01 indicates a highly significant difference.

[0036] Figure 7 The wild type of embodiment 4 of the present invention, PDIL2;3-cri and PDIL2;3-OE Results of chalky grain identification in mature seeds; Figure A shows T2 generation gene-edited plants. PDIL2;3-cri Phenotypic comparison with mature polished rice strains from wild-type families. Scale bar: 3 mm; Figures B-D represent the T2 generation. PDIL2;3-cri Phenotypic comparison of mature kernel cross-sections with wild-type families, scale bar: 0.5 mm; Figures E-G represent T2 generation. PDIL2;3-cri Scanning electron microscope (SEM) images of the endosperm cross-section of mature seeds from wild-type families, scale bar: 50 μm; Figures H-I represent T2 generation. PDIL2;3-cri Comparison of grain chalkiness rate (PGWC), chalkiness degree (DEC), and milling rate with wild-type families. Data are expressed as mean ± standard deviation (n=10), with each replicate containing at least 300 seeds. P <0.01 indicates a highly significant difference; K~M in the figure represent wild type, PDIL2;3-cri and PDIL2;3-OE Determination of total starch, amylose and protein content in mature grains.

[0037] Figure 8 This is the wild type of Embodiment 5 of the present invention. PDIL2;3-cri and PDIL2;3-OE Scanned images of glumes cells; Figure A shows wild-type glumes. PDIL2;3-cri and PDIL2;3-OE Scanning electron microscopy image of mature, sun-dried seeds, scale bar 1 mm; Figure B shows wild-type seeds. PDIL2;3-cri and PDIL2;3-OE Partial magnified scanning electron microscope image of the grains, scale bar 100 µm; Figures C-D show wild-type grains. PDIL2;3-cri and PDIL2;3-OE Statistical results of the average length of the longitudinal and transverse protrusions of the glumes; Figures E-F represent the wild type, PDIL2;3-cri and PDIL2;3-OE The statistical results of the longitudinal and transverse cell numbers of the glumes are shown in the figure. NS represents no significant difference, and the letters a, b, and c represent significant differences. P <0.01); Figure G represents the wild type, PDIL2;3-cri and PDIL2;3-OE Image of glumes before flowering, scale bar is 1 mm; Images H~I represent wild type.PDIL2;3-cri and PDIL2;3-OE Paraffin sections of glumes before flowering, with a scale bar of 400 μm for H and 100 μm for I; Figures J-K represent wild type. PDIL2;3-cri and PDIL2;3-OE Statistical results of the number and area of ​​peripheral cells of the glumes before flowering; Figure L shows the flow cytometry results for wild-type and... PDIL2;3-cri and PDIL2;3-OE Results of spikelet cell ploidy; Figure M shows wild type, PDIL2;3-cri and PDIL2;3-OE The ratio of 2C and 4C cells in the young spikelet, where letters a, b, and c represent significant differences ( P <0.01).

[0038] Figure 9 Wild type and wild type of embodiment 6 of the present invention PDIL2;3-cri Statistical chart of cell cycle-related gene expression levels in young spikelets. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] This invention is the first to discover an important gene in rice that can regulate grain shape and chalkiness. PDIL2;3 CRISPR / Cas9 specific knockout PDIL2;3 This invention yields new rice germplasm characterized by larger panicles, longer and wider grains, increased thousand-grain weight, and increased chalkiness rate. The invention utilizes CRISPR / Cas9 gene editing technology to target the rice protein disulfide isomerase encoding gene. PDIL2;3 Edit and identify PDIL2;3 Gene-specific knockout frameshift mutants were demonstrated through analysis of grain morphology and yield. PDIL2;3 Gene mutations affect the division and elongation of glume cells, thus influencing grain morphology. They also affect the chalkiness rate by impacting the synthesis of endosperm contents, ultimately affecting rice yield and quality. PDIL2;3 The gene sequence is shown in SEQ ID NO.1. PDIL2;3 The coding sequence of the gene is shown in SEQ ID NO.2.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0042] Rice varieties:

[0043] Zhonghua 11 (ZH11): Originated from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. Zhonghua 11 was developed by the Institute of Crop Science in 1979 using Jingfeng No. 5 / Tetepu / Fujin varieties for flowering cultivation. Zhonghua 11 is documented in the following literature: Ni Pichong. New Flowering Rice Variety—Zhonghua 11 Crop Variety Resources, 1989, Issue 04. Zhonghua 11 plants are also known as wild-type plants, denoted by WT.

[0044] Example 1 Rice PDIL2;3 Gene expression pattern analysis

[0045] For analysis PDIL2;3 The spatiotemporal expression patterns of genes were detected using quantitative RT-PCR technology in this embodiment. PDIL2;3 The expression profiles of genes in various tissues and organs of the rice variety "Zhonghua 11" were determined through the following steps:

[0046] Samples of Zhonghua 11, a variety planted in Wuhan during the peak growing season, were taken from its roots, stems, leaves, buds, leaf sheaths, young spikelets of different lengths, and endosperm at different grain-filling days. The specific procedures for total RNA extraction, reverse transcription, and quantitative RT-PCR were performed according to the experimental method of Li et al. (Li et al., OsSPL14 acts upstream of...). OsPIN1b and PILS6b to modulate axillary budoutgrowth by fine-tuning auxin transport in rice. Plant J, 2022, 111: 1167-1182).

[0047] in, PDIL2;3 The qRT-PCR primer sequences for the internal reference gene are as follows (5'-3'):

[0048] PDIL2;3 -F: SEQ ID NO.4;

[0049] PDIL2;3 -R: SEQ ID NO.5;

[0050] qRT-Ubq-F: SEQ ID NO.6;

[0051] qRT-Ubq-R: SEQ ID NO.7.

[0052] The results are as follows Figure 1 As shown in Figure A, the qRT-PCR detection results indicate that... PDIL2;3The gene is expressed to varying degrees in the roots, stems, leaves, leaf sheaths during the vegetative stage, as well as in the developing young spike and endosperm, and its expression increases as the spike and embryo develop. PDIL2;3 Expression levels gradually increased, while almost no expression was observed in lateral buds, indicating... PDIL2;3 Genes may play a role in the development of ears and seeds.

[0053] To further clarify PDIL2;3 The role of genes in the development of rice panicles and seed embryos is revealed through amplification. PDIL2;3 A 1.9kb promoter was used to construct promoter-driven GUS transgenic plants. β-glucosidase staining was performed on multiple tissues of the transgenic positive plants.

[0054] The primer sequences are as follows (5'-3'):

[0055] Gus-PDIL2;3-F: SEQ ID NO.8;

[0056] Gus-PDIL2;3-R: SEQ ID NO.9.

[0057] The results are as follows Figure 1 As shown in B~L, it displays PDIL2;3 It is expressed in vegetative organs, spikelets, and endosperm, with particularly high expression levels in young spikelets and endosperm, further validating its efficacy. PDIL2;3 The unique expression pattern observed in young panicles suggests that it may play a role in panicle and seed development in rice.

[0058] Example 2 PDIL2;3-cri Obtaining and identifying mutant strains

[0059] For analysis PDIL2;3 The effects of gene loss of function on rice development were studied in this embodiment using CRISPR / Cas9 technology. PDIL2;3 The specific steps for obtaining the mutant are as follows:

[0060] like Figure 2 As shown in A, near PDIL2;3 A gRNA target site was designed from the ATG region. Using U6a-PDIL2;3-F / R primers, the target site was constructed into the CRISPR / Cas9 binary vector pYLCRISPR / Cas9Pubi-H. The plasmid diagram is shown below. Figure 3 As shown.

[0061] The primer sequences are as follows (5'-3'):

[0062] U6a-PDIL2;3-F: SEQ ID NO.10;

[0063] U6a-PDIL2;3-R: SEQ ID NO. 11.

[0064] The CRISPR / Cas9 vector was transformed into the rice recipient material ZH11 using Agrobacterium tumefaciens-mediated transformation, and genomic DNA was extracted from transgenic single plants using the CTAB method. PDIL2;3 PCR amplification was performed using upstream and downstream specific primers crispr-PDIL2-3F / R at the gene target site. The PCR products were then recovered and sequenced. The gene target site was determined by comparing sequencing peak profiles. PDIL2;3 In cases of knockout, the sequencing peak patterns were analyzed using DSDecodeM to determine the mutation type. Subsequently, qRT-PCR was used to detect the mutation in the transgenic plants. PDIL2;3 The amount of expression.

[0065] The primer sequences are as follows (5'-3'):

[0066] crispr-PDIL2-3-F: SEQ ID NO.12;

[0067] crispr-PDIL2-3-R: SEQ ID NO. 13.

[0068] like Figure 2 As shown in C and D, the T0 generation transformed plants PDIL2;3-cri1 , PDIL2;3-cri2 exist PDIL2;3 Editing occurred at the target site of the gene, in which PDIL2;3-cri1 The deletion of two bases at the target site causes a frameshift mutation starting from amino acid position 21, which terminates prematurely at amino acid position 105. PDIL2;3-cri2 The deletion of 8 bases at the target site causes a frameshift mutation starting from amino acid position 21, which terminates prematurely at amino acid position 102. Figure 2 B shows that, compared with the wild type, transgenic single plants PDIL2;3-cri1 , PDIL2;3-cri2 middle PDIL2;3 The expression level decreased significantly. The results indicate that this embodiment achieved [the desired result] through CRISPR / Cas9 gene editing technology. PDIL2;3 Specific knockout of different allelic mutants resulted in premature termination of the encoded proteins.

[0069] The above will then be PDIL2;3 Gene-edited T1 generation plants were self-pollinated, and mature T2 generation family seeds were harvested. A comprehensive comparison was made between wild-type and... PDIL2;3-cri1 , PDIL2;3-cri2 Phenotypic differences between mutants.

[0070] Figure 4 The J and K results showed that, compared with the wild type, PDIL2;3-cri1, PDIL2;3-cri2 The number of tillers and plant height during the vegetative stage were not significantly different from those of the wild type, but the number of ears at maturity was significantly larger than that of the wild type, and the plant height was slightly higher. Figure 4 (A, B, and L). Figure 4 The M, N, and S values ​​show that, compared to the wild type, PDIL2;3-cri1 , PDIL2;3-cri2 The number of primary branches, secondary branches, and grains per ear increased significantly. Furthermore, PDIL2;3-cri1 , PDIL2;3-cri2 There are also significant changes in grain morphology. Figure 4 (C~I). Regarding particle length, PDIL2;3-cri1 , PDIL2;3-cri2 The mutants were 12.24% and 10.64% longer than the wild type, respectively. Regarding grain width, PDIL2;3-cri1 , PDIL2;3-cri2 The mutants showed increases of 6.89% and 6.99% compared to the wild type. Regarding grain thickness, PDIL2;3-cri1 , PDIL2;3-cri2 The mutant showed no significant difference compared to the wild-type. Regarding the thousand-grain weight... PDIL2;3-cri1 , PDIL2;3-cri2 The mutant strain showed increases of 5.89% and 5.12% compared to the wild type, respectively. Figure 4 The above results indicate that... PDIL2;3 Genes play an important role in the development of rice panicles and grains. PDIL2;3 The mutant rice showed a significant increase in grain width, grain thickness, and grain weight.

[0071] Example 3 PDIL2;3 Obtaining and identifying traits of overexpression plants

[0072] This embodiment creates overexpression PDIL2;3 Genetic analysis was performed on the materials. First, the PDIL2;3-OE-F / R primer sequence was designed, and the full-length cDNA fragment of PDIL2;3 was amplified using ZH11 young spikelets (2 mm–5 mm) as a template. Homologous recombination was then used to ligate the fragment into T5 restriction endonuclease. Kpn I- BamH The plasmid was implanted onto the I-treated overexpression vector pU1301, and the plasmid image is shown below. Figure 5 As shown, the correctly digested vector was named pU1301-PDIL2;3 and sequenced to verify it. Subsequently, the vector with the correct sequence was selected and electroporated into Agrobacterium (…). Agrobacterium tumefaciens In strain EHA105, the overexpression vector was introduced into ZH11 callus using Agrobacterium-mediated genetic transformation (callus induction was performed using conventional methods) to obtain ZH11-based callus. PDIL2;3 Overexpressing plants.

[0073] The primer sequences are as follows (5'-3'):

[0074] PDIL2;3 -OE-F: SEQ ID NO.14;

[0075] PDIL2;3 -OE-R: SEQ ID NO.15.

[0076] Figure 6 Gene expression analysis of the B-generation T0 showed that, compared with the wild type (i.e., non-transgenic), transgenic positive single plants PDIL2;3-OE1 , PDIL2;3-OE2 middle PDIL2;3 The expression level was significantly upregulated. Figure 6 Phenotypic identification of A, L, M, and N showed that PDIL2;3-OE1 and PDIL2;3-OE2 The plants showed no significant changes in plant height and number of tillers compared to the wild type, and there were no significant differences in ear type, number of branches, and number of grains per ear at maturity. Figure 6 (C, O, P and U), but the grain morphology has changed significantly ( Figure 6 (D~K). Figure 6 Statistics on Q~T in China show that, PDIL2;3-OE1 and PDIL2;3-OE2 The grain thickness remained unchanged, but the grain length, grain width, and thousand-grain weight were all significantly reduced. This result further validates... PDIL2;3 It plays an important role in the regulation of rice grain shape and yield, and overexpression... PDIL2;3 This will lead to smaller particle size and reduced yield.

[0077] Example 4 PDIL2;3 Influences the formation of chalky grains and the accumulation of endosperm storage substances.

[0078] For analysis PDIL2;3 Whether the gene is involved in the formation of chalkiness in rice, this example focuses on wild-type, PDIL2;3-cri mutants and PDIL2;3-OE Mature kernels from the family line were observed and analyzed. WT and... PDIL2;3-OE The rice from this family is translucent and waxy, while PDIL2;3-cri The milled rice of the mutant exhibited an opaque phenotype with obvious chalky accumulation. Figure 7 (A). Subsequently, seeds from each family were harvested and dried for six months. The chalkiness was then assessed, revealing that compared to the wild type, PDIL2;3-cri The chalkiness rate and chalkiness of the grains increased significantly. PDIL2;3-OE The chalkiness rate and chalkiness of the grains were significantly reduced. Figure 7 (HI). Head rice yield is a grading indicator for rice processing quality and is significantly affected by chalkiness. Compared to wild type, PDIL2;3-cri The head rice yield of transgenic lines was significantly reduced. PDIL2;3-OEThe head rice percentage of the grains increased significantly. Figure 7 J).

[0079] Further investigation was conducted using stereomicroscopy to observe the cross-sections of grains from various families to explore the cellular basis of chalkiness formation in mutants, such as... Figure 7 As shown in B~D, WT and PDIL2;3-OE The endosperm is fully filled and has a dense structure, while PDIL2;3-cri The endosperm of the family grains has a large area of ​​chalky accumulation.

[0080] To further observe the chalkiness in the endosperm, scanning electron microscopy was used to examine the wild-type and... PDIL2;3-cri mutants and PDIL2;3-OE Cross-section observation of mature kernels revealed wild-type and... PDIL2;3-OE The starch granules in the endosperm are uniform in size, densely packed, and compressed together to form a regular polyhedral structure; while PDIL2;3-cri The starch granules in the endosperm are irregularly shaped small spheres, loosely arranged, with large gaps between the granules, and cannot be tightly packed to form a polygonal shape. Figure 7 (E~G). This indicates... PDIL2;3 Gene mutations affect the shape and arrangement of endosperm storage substances, resulting in seeds exhibiting an undesirable phenotype of increased chalkiness.

[0081] Increased chalkiness in rice endosperm is usually accompanied by changes in the content of storage substances. This embodiment also tested wild-type rice. PDIL2;3-cri mutants and PDIL2;3-OE The content of starch, amylose, and total protein in the endosperm.

[0082] Figure 7 The K~M plot results show that, compared with the wild type, PDIL2;3-cri The mutant grains had reduced total starch and amylose content, but significantly increased protein content; PDIL2;3-OE Results and PDIL2;3-cri The mutant detection results were contradictory. Therefore, it can be concluded that... PDIL2;3 Genes play a role in the development of rice endosperm and the accumulation of its contents.

[0083] Example 5 PDIL2;3 Effects on cell division in glume

[0084] Seed size is primarily determined by the size of the glume, which in turn is closely related to the development of glume cells. For research... PDIL2;3 The changes in glume cells in transgenic families were observed in this example using scanning electron microscopy and paraffin sections. The specific steps are as follows:

[0085] Pick PDIL2;3-cri1 mutants PDIL2;3-OE1The seeds of the wild-type plant were dried and then subjected to steps such as gluing and gold spraying before being observed using a scanning electron microscope.

[0086] Electron microscopy observation results as follows Figure 8 As shown, Figure 8 In A, PDIL2;3-cri1 The mutant seeds were significantly larger than the wild type, while PDIL2;3-OE1 The seeds are smaller than those of the wild type. Subsequently, the cells in the middle part of the outer glume were magnified for observation, and the distance between the small protuberances between the glume cells was measured using ImageJ software. Figure 8 B). Figure 8 The C results show that, in the grain length direction, PDIL2;3- cri1 mutants PDIL2;3-OE1 There was no significant difference in glume cell length between the wild type and the wild type. Figure 8 The E results show that PDIL2;3-cri1 The mutant had a significantly higher number of cells in the longitudinal direction than the wild type. PDIL2;3-OE1 This indicates that it is lower than the wild type. PDIL2;3 The changes in grain length in transgenic families are due to longitudinal changes in cell number / division. In the grain width direction, compared to the wild type, PDIL2;3-cri1 The mutant showed a significant increase in transverse cell length. PDIL2;3-OE1 The transverse cell length is significantly reduced ( Figure 8 (D). Cell count results showed that, PDIL2;3-cri1 mutants and PDIL2;3-OE1 There was no significant difference in cell number compared to the wild type. Figure 8 (F), implying PDIL2;3 The altered grain width in transgenic families is due to changes in transverse cell size / elongation. These results indicate that... PDIL2;3 Grain length and grain width can be controlled by negatively regulating the division of longitudinal cells and the extension of transverse cells in the glume.

[0087] Take another PDIL2;3-cri1 mutants PDIL2;3-OE1 Wild-type heading glumes were immersed in 70% FAA (70% anhydrous ethanol, 5% acetic acid, 3.7% formalin) under vacuum for 30 min and fixed at room temperature for 24 h. The fixed biological material was then transferred to 70% ethanol for serial alcohol dehydration, eosin staining overnight, further dehydration with a gradient of alcohols, xylene clearing, and gradient paraffin embedding. After embedding in pure paraffin for 3-4 days, sections were prepared (using a Yidi YD-1508A rotary microtome) to a thickness of 8 µm. The prepared paraffin sections were spread, dried, dewaxed, stained, and mounted before being observed under a microscope. Figure 8 H~I).

[0088] The results showed that PDIL2;3-cri1The mutant showed an increase in the average area of ​​the outermost layer of cells in the glume parenchyma cell layer, while the number of cells did not change significantly. Figure 8 (JK). Further explanation PDIL2;3 The altered granule width in transgenic families is due to changes in transverse cell size / elongation. Flow cytometry analysis of cell ploidy results showed that... PDIL2;3-cri1 The proportion of 4C cells in the young spikelets of the mutant was significantly higher than that of the wild type, and PDIL2;3-OE1 The results showed that the mutant spikelet cell division was more vigorous than that of the wild type. These results indicate that the enhanced longitudinal division and transverse extension of glume cells are the reasons for the increased glume size in the mutant.

[0089] Example 6 PDIL2;3 Effects on the expression of cell cycle-related genes

[0090] The number of cells is regulated by cell cycle-related genes. In this example, quantitative real-time PCR was used to measure the cell number. PDIL2;3-cri The expression of cell cycle-related genes in mutant and wild-type young spikelets was analyzed.

[0091] The primer sequences are shown in Table 1 (5'-3'):

[0092] Table 1 Primers for Quantitative Real-Time PCR

[0093]

[0094] Figure 9 The results show that PDIL2;3-cri The expression levels of many cell cycle regulatory genes in the mutant were significantly higher than those in the wild type, such as CYCT1 , CDKA2 , CDKE , CDKF Based on the above results, it can be inferred that... PDIL2;3-cri The changes in the morphology and number of mutant cells are due to the presence of cell cycle-related genes. PDIL2;3-cri This is caused by abnormal expression in the mutant, which leads to an increase in the number of cortical cells in the mutant.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. PDIL2;3 The application of genes in regulating grain shape and / or chalkiness traits in rice, the aforementioned PDIL2;3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The particle shape refers to particle length, particle width, and / or particle weight.

3. The application according to any one of claims 1 to 2, characterized in that, The PDIL2;3 The nucleotide sequence of the gene is shown in SEQ ID NO.1 or SEQ ID NO.

2.

4. A method for regulating rice grain shape and / or chalkiness traits, characterized in that, adjust PDIL2;3 The expression or activity of a gene or its encoded protein; The PDIL2;3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3.

5. The method according to claim 4, characterized in that, The rice grain shape refers to the grain length, grain width, and / or grain weight of the rice.

6. A method for breeding rice, characterized in that, Includes the following steps: suppress PDIL2;3 Gene expression levels, PDIL2;3 The expression level of the gene-encoded protein or PDIL2;3 The active substance of a gene-encoded protein is introduced into rice or rice cells to obtain selectively bred rice; PDIL2;3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

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