A PDIL2; application of 3 gene in regulation and control of rice grain shape and chalkiness character

Abstract

The present invention discloses a PDIL2; the invention also discloses application of the gene 3 in regulation of rice grain shape and chalkiness character. The invention provides the protein disulfide isomerase coding gene PDIL2 for the first time; 3, an important regulation effect on the grain shape and chalkiness character of rice grains is achieved, and PDIL2 is knocked out or destroyed; the germplasm with enlarged panicle, increased grain length, widened grain width, increased thousand grain weight and increased chalkiness can be obtained according to the normal expression level of the genes 1, 2, 3, so that the purposes of obtaining ideal rice grain shape and improving rice quality are achieved, a brand new way is provided for high-yield and high-quality breeding work of rice, and the application prospect is wide.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to the application of PDIL2;3 gene in regulating rice grain shape and chalky traits. Background Art

[0002] Rice is one of the important food crops in my country and other Asian countries. High yield and high quality of rice have always been the two most important goals of rice application and basic research in my country. Since the development and utilization of dwarf breeding and hybrid rice in the 1950s to 1970s, my country's total grain output has made a qualitative leap, and high-yield breeding has been at the world's leading level. With the improvement of people's living standards and changes in dietary structure, rice quality has received more and more attention and concern. People's demand for high-quality rice with good appearance, good taste and good nutrition is increasing. Under this realistic background, breeding new rice varieties with high quality, high yield, high nutritional value and more popular with the people has become the goal of breeders in recent years. The evaluation of rice quality by rice breeders, sellers and consumers is basically the same at home and abroad, generally including four main aspects: appearance quality, processing quality, cooking and eating quality and nutritional quality. Among them, the appearance quality of rice mainly depends on grain size, chalkiness and translucency, which directly affects the commercial quality of rice and is the most important rice quality trait and evaluation standard.

[0003] Chalkiness is the white opaque part due to insufficient filling of rice grains, which results in loose filling of endosperm tissue and scattered light. Chalkiness reduces the transparency of rice, makes the rice loose, and is easy to break during processing, which directly affects the yield of polished rice after processing. At the same time, chalkiness has a low content of amylose, poor taste and nutritional quality, and reduces the commerciality and market value of rice, which is one of the main limiting factors restricting the high-quality rice quality in my country.

[0004] The grain shape of rice is usually represented by three sets of data: grain length, grain width, and grain thickness. Generally speaking, a slender grain shape is conducive to grain filling, reducing chalkiness and chalky rice rate, thereby greatly improving the appearance quality of rice. Some cloned grain shape QTLs show a concomitant effect of chalkiness, such as qTGW6, GW2, GS2, qSW5, GW7, and GW8. These research results show that grain shape and chalkiness are the most important indicators that determine the appearance quality of rice. Rice grain shape has an important influence on the formation of chalkiness. Grain shape genes have broad application prospects in regulating high-yield and high-quality rice breeding. At present, there are relatively few studies on genes related to rice quality, such as chalkiness rate, transparency, texture, etc., which limits the application of technologies such as gene editing to improve rice quality. Or candidate genes related to grain shape and chalkiness have been found, but their functional verification and practical application are relatively insufficient, and there is a lack of extensive field verification application experience. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes an application of the PDIL2;3 gene in regulating rice grain shape and chalkiness. After the PDIL2;3 gene mutation, the rice grains show a phenotype of larger panicles, longer grain length, wider grain width, increased thousand-grain weight, but also significantly increased chalkiness, which provides very good genetic material for studying rice grain shape and chalkiness regulation, and can be applied to the genetic improvement of rice yield and appearance quality, providing technical guarantee and scientific basis for ensuring the continuous increase in agricultural rice crop yield, and has important production application value.

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

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

[0008] In some embodiments, the application is specifically: regulating grain width, grain weight or chalkiness of rice.

[0009] In some embodiments, the regulation is specifically any one of the following:

[0010] (i) increasing the grain length, grain width, grain weight and / or chalkiness of rice by inhibiting or reducing the activity of the protein encoded by the PDIL2;3 gene or the expression level thereof;

[0011] (ii) Increasing the grain length, grain width, grain weight and / or chalkiness of rice by knocking out, inhibiting or silencing the expression of the PDIL2;3 gene.

[0012] In some embodiments, the regulation is specifically any one of the following:

[0013] (a) reducing grain width, grain weight and / or chalkiness of rice by increasing the activity of a protein encoded by the PDIL2;3 gene or its expression level;

[0014] (b) Reducing the grain length, grain width, grain weight and / or chalkiness of rice by increasing the expression level of the PDIL2;3 gene.

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

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

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

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

[0019] (1) down-regulating the expression level of the PDIL2;3 gene, the expression level of the protein encoded by the PDIL2;3 gene, or the activity of the protein encoded by the PDIL2;3 gene, thereby increasing the grain length, grain width, grain weight and / or chalkiness of rice;

[0020] (2) Up-regulating the expression level of the PDIL2;3 gene, the expression level of the protein encoded by the PDIL2;3 gene, or the activity of the protein encoded by the PDIL2;3 gene, thereby reducing the grain length, grain width, grain weight and / or chalkiness of rice.

[0021] In some embodiments, the up-regulation of the expression level of the PDIL2;3 gene, the expression level of the protein encoded by the PDIL2;3 gene, or the activity of the protein encoded by the PDIL2;3 gene in (2) is any one of the following:

[0022] (a) transferring the expression construct or vector of PDIL2;3 gene into rice or rice cells;

[0023] (b) Transforming the expression construct or vector containing the PDIL2;3 gene into rice or rice cells.

[0024] The present invention also provides a rice breeding method, comprising the following steps: introducing a substance that increases or inhibits the expression level of the PDIL2;3 gene, the expression level of the protein encoded by the PDIL2;3 gene, or the activity of the protein encoded by the PDIL2;3 gene into rice or rice cells to obtain the bred rice; the amino acid sequence encoded by the PDIL2;3 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. The present invention provides for the first time the application of the protein disulfide isomerase encoding gene PDIL2;3 in regulating rice grain shape and chalky traits; the PDIL2;3 gene can play an important regulatory role in the grain shape and chalky traits of rice grains, providing a new approach for high-yield and high-quality rice breeding, and has broad application prospects.

[0027] 2. The present invention utilizes gene editing technology to knock out or destroy the normal expression level of PDIL2;3 gene to obtain germplasm with larger panicles, longer grains, wider grain widths, increased 1000-grain weight and increased chalkiness, so as to achieve the purpose of obtaining an ideal rice grain shape and improving rice quality.

[0028] 3. The present invention found that the panicle of the PDIL2;3 knockout mutant became larger, the grain length became longer, the grain width became wider, and the 1000-grain weight increased, proving that PDIL2;3 plays an important role in the formation of rice grain shape and yield regulation. This not only enriches the understanding of rice grain shape formation, but also provides a theoretical basis for the cultivation of high-yield rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 It is an expression pattern diagram of the PDIL2;3 gene of Example 1 of the present invention; Figure A is the expression spectrum of the PDIL2;3 gene in various tissues and organs of rice "Zhonghua 11", wherein AB is lateral buds, L is leaf blade, LS is leaf sheath, R is root, S is stem, 0.5P~25P is young panicle with a length of 0.5cm~25cm, En3~En24 is endosperm 3~24 days after fertilization; Figures B~L are GUS staining to detect the expression of PDIL2;3 in various tissues and organs; the scale bars of Figures B~E and I~M are 0.2cm; the scale bar of F is 0.1cm, and the scale bar of G~H is 1cm.

[0031] Figure 2 The creation of the PDIL2;3 mutant of Example 2 of the present invention and the related sequencing results; Figure A is a schematic diagram of the recognition position of sgRNA at the PDIL2;3 gene locus; Figure B is a quantitative RT-PCR detection of the expression level of PDIL2;3 in wild-type and PDIL2;3-cri T2 transgenic plants; Figure C is a sequencing analysis of the mutation of PDIL2;3-cri1 and PDIL2;3-cri2 at the PDIL2;3 gene locus; Figure D is the mutation of the amino acid sequence encoded by the PDIL2;3-cri mutant, wherein red represents the mutated amino acid sequence.

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

[0033] Figure 43-cri and the wild-type plants at maturity, with a scale of 15 cm; FIG. B is the panicle type of the T2 generation PDIL2;3-cri and the wild-type plants, with a scale of 5 cm; FIG. C to H are the grain types of the T2 generation PDIL2;3-cri and the wild-type, with a scale of 2 cm; FIG. I is a comparison of grain length and width between the T2 generation PDIL2;3-cri and the wild-type, with a scale of 10 mm; FIG. J to S are the statistical results of the number of tillers, plant height, number of primary stalks, number of secondary stalks, number of grains per panicle, grain length, grain width, grain thickness and 1000-grain weight of the T2 generation PDIL2;3-cri and the wild-type plants, **P<0.01 indicates an extremely significant difference.

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

[0035] Figure 6 3-OE and negative plants, with a scale of 15 cm; FIG. B is a quantitative RT-PCR test of the expression level of PDIL2;3 in wild-type and PDIL2;3-OE transgenic plants; FIG. C is an ear type of T2 transgenic positive plants PDIL2;3-OE and negative plants, with a scale of 5 cm; FIG. D to E are grain types of T2 transgenic positive plants and negative plants, with a scale of 5 mm; FIG. F to K are grain types of T2 transgenic positive plants and negative plants, with a scale of 2 cm; FIG. LU is a statistical result of the number of tillers, plant height, number of primary stalks, number of secondary stalks, number of grains per ear, grain length, grain width, grain thickness and 1000-grain weight of T2 transgenic positive plants and negative plants, **P<0.01 indicates extremely significant difference.

[0036] Figure 7The chalky trait identification results of the wild-type, PDIL2; 3-cri and PDIL2; 3-OE mature grains of Example 4 of the present invention; Figure A is a phenotypic comparison of polished rice of the T2 generation gene-edited plant PDIL2; 3-cri and the wild-type family mature strain, scale: 3mm; Figures B to D are phenotypic comparisons of cross-sections of mature grains of the T2 generation PDIL2; 3-cri and the wild-type family, scale: 0.5mm; Figures E to G are T2 generation PDIL2; 3-cri and wild-type family mature seed embryos Scanning electron microscope image of milk cross section, scale: 50μm; Figures H~I are comparisons of grain chalkiness rate (PGWC), chalkiness degree (DEC) and polished rice rate between T2 generation PDIL2;3-cri and wild-type family strains, data are expressed as mean ± standard deviation (n=10), each replicate was no less than 300 seeds, **P<0.01 indicates extremely significant difference; Figures K~M are determinations of total starch, amylose and protein contents in mature grains of wild-type, PDIL2;3-cri and PDIL2;3-OE.

[0037] Figure 8 The scanned images of the hull cells of the wild type, PDIL2; 3-cri and PDIL2; 3-OE of Example 5 of the present invention; Figure A is an electron microscope scanned image of the mature sun-dried grains of the wild type, PDIL2; 3-cri and PDIL2; 3-OE, with a scale of 1 mm; Figure B is a local magnified electron microscope scanned image of the grains of the wild type, PDIL2; 3-cri and PDIL2; 3-OE, with a scale of 100 μm; Figures C to D are statistical results of the average length of the longitudinal and transverse protrusions of the hulls of the wild type, PDIL2; 3-cri and PDIL2; 3-OE; Figures E to F are statistical results of the longitudinal and transverse cell numbers of the hulls of the wild type, PDIL2; 3-cri and PDIL2; 3-OE, NS represents no significant difference, and letters a, b, c represent significant differences (P<0.01) ; Figure G is the glume picture of the wild type, PDIL2;3-cri and PDIL2;3-OE before flowering, with a scale of 1 mm; Figures H to I are paraffin sections of the glume of the wild type, PDIL2;3-cri and PDIL2;3-OE before flowering, with a scale of 400 μm for H and 100 μm for I; Figures J to K are the statistical results of the number and area of ​​peripheral cells of the glume of the wild type, PDIL2;3-cri and PDIL2;3-OE before flowering; Figure L is the result of flow cytometry detection of cell ploidy of young panicles of the wild type, PDIL2;3-cri and PDIL2;3-OE; Figure M is the proportion of 2C and 4C cells in young panicles of the wild type, PDIL2;3-cri and PDIL2;3-OE, and the letters a, b, and c represent significant differences (P<0.01).

[0038] Fig. 9This is a statistical graph of the expression levels of cell cycle-related genes in the wild-type and PDIL2;3-cri panicles of Example 6 of the present invention. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0040] The present invention first discovered the important gene PDIL2;3 that can regulate rice grain shape and chalky traits in rice. New rice germplasm with larger panicle, longer grain length, wider grain width, increased thousand-grain weight and increased chalky rate was obtained by specifically knocking out PDIL2;3 through CRISPR / Cas9. The present invention edited the rice gene protein disulfide isomerase encoding gene PDIL2;3 through CRISPR / Cas9 gene editing technology, and identified the frameshift mutant of the PDIL2;3 gene specific knockout. The grain appearance morphology and yield analysis proved that the PDIL2;3 gene mutation affects the division and extension of the husk cells, thereby affecting the morphology of the grain, and also affects the chalky rate by affecting the synthesis of endosperm contents, and finally affects the rice yield and quality. Among them, the sequence of the PDIL2;3 gene is shown in SEQ ID NO.1, and the coding sequence of the PDIL2;3 gene is shown in SEQ ID NO.2.

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

[0042] Rice Varieties:

[0043] Zhonghua 11 (ZH11): Originated from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. Zhonghua 11 was cultivated by the Institute of Crop Science, Chinese Academy of Agricultural Sciences in 1979 using Jingfeng 5 / Tetepu / Fujin. Zhonghua 11 is recorded in the following document: Ni Pichong. New Rice Varieties Cultivated by Flower Culture—Crop Variety Resources of Zhonghua 11, Issue 4, 1989. Zhonghua 11 plants are also called wild-type plants, represented by WT.

[0044] Example 1 Analysis of the expression pattern of rice PDIL2;3 gene

[0045] In order to analyze the spatiotemporal expression pattern of the PDIL2;3 gene, this example uses quantitative RT-PCR technology to detect the expression profile of the PDIL2;3 gene in various tissues and organs of the rice variety "Zhonghua 11", and the specific steps are as follows:

[0046] Zhonghua 11 planted in Wuhan during the mid-season was selected, and roots, stems, leaves, buds, leaf sheaths, panicles of different lengths and endosperms of different filling days were sampled. The specific steps of total RNA sample extraction, reverse transcription and quantitative RT-PCR experiments were carried out according to the experimental methods of Li et al. (Li et al., OsSPL14 acts upstream of OsPIN1b and PILS6b to modulate axillarybud outgrowth by fine-tuning auxin transport in rice. Plant J, 2022, 111: 1167-1182).

[0047] The qRT-PCR primer sequences for PDIL2;3 and 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 A, the results of qRT-PCR showed that the PDIL2;3 gene was expressed to varying degrees in roots, stems, leaves, leaf sheaths, and developing young panicles and endosperms during the vegetative period. Moreover, the expression level of PDIL2;3 gradually increased with the development of panicles and embryos, while it was almost not expressed in lateral buds, indicating that the PDIL2;3 gene may play a role in the development of panicles and seeds.

[0053] In order to further clarify the role of PDIL2;3 gene in the development of rice panicle and seed embryo, the 1.9kb promoter of PDIL2;3 gene was amplified and promoter-driven GUS transgenic plants were constructed. β-glucosidase staining of multiple tissues was performed on 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 1As shown in B to L, PDIL2;3 is expressed in nutritional organs, panicles and endosperm, especially in young panicles and endosperm, which further verifies that PDIL2;3 presents a specific expression pattern in young panicles, indicating that it may play a role in the development of rice panicles and seeds.

[0058] Example 2 Acquisition and characterization of PDIL2;3-cri mutant strains

[0059] In order to analyze the effect of PDIL2;3 gene function loss on rice development, this example uses CRISPR / Cas9 technology to create PDIL2;3 mutants. The specific steps are as follows:

[0060] like Figure 2 As shown in A, a gRNA target site was designed in the region near PDIL2; 3ATG, and the target site was constructed into the CRISPR / Cas9 binary vector pYLCRISPR / Cas9Pubi-H using U6a-PDIL2; 3-F / R primers. The plasmid map is shown in Figure 3 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 by Agrobacterium tumefaciens-mediated method, and the genomic DNA of the transgenic individual plants was extracted by the CTAB method. PCR amplification was performed with the upstream and downstream specific primers crispr-PDIL2-3F / R of the PDIL2;3 gene target site, respectively. After amplification, the PCR products were recovered and sequenced. The PDIL2;3 knockout was determined by comparing the sequencing peak graph, and the sequencing peak graph was analyzed by DSDecodeM to determine the mutation type. The expression level of PDIL2;3 in the transgenic plants was then detected by qRT-PCR.

[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 2As shown in C and D, the T0 generation transformed plants PDIL2;3-cri1 and PDIL2;3-cri2 underwent editing at the target site of the PDIL2;3 gene. The deletion of two bases at the target site of PDIL2;3-cri1 caused a frameshift mutation in the amino acid sequence encoded by it starting from the 21st amino acid and terminating prematurely at the 105th amino acid. The deletion of 8 bases at the target site of PDIL2;3-cri2 caused a frameshift mutation in the amino acid sequence encoded by it starting from the 21st amino acid and terminating prematurely at the 102nd amino acid. Figure 2 B shows that compared with the wild type, the expression of PDIL2;3 in the transgenic individual PDIL2;3-cri1 and PDIL2;3-cri2 was significantly reduced. The results show that in this example, PDIL2;3 specific knockout mutants of different alleles were obtained by CRISPR / Cas9 gene editing technology, and the proteins encoded by them all terminated prematurely.

[0069] Subsequently, the above-mentioned PDIL2;3 gene-edited T1 plants were self-pollinated and mature T2 family seeds were harvested to comprehensively compare the phenotypic differences between the wild type and PDIL2;3-cri1 and PDIL2;3-cri2 mutants.

[0070] Figure 4 The results of J and K showed that compared with the wild type, the tiller number and plant height of PDIL2;3-cri1 and PDIL2;3-cri2 in the vegetative stage were not significantly different from those of the wild type, but the ears in the mature stage were significantly larger than those of the wild type, and the plant height was slightly higher than that of the wild type ( Figure 4 A, B and L). Figure 4 M, N and S showed that compared with the wild type, the number of primary branches, secondary branches and grains per ear of PDIL2;3-cri1 and PDIL2;3-cri2 increased significantly. In addition, PDIL2;3-cri1 and PDIL2;3-cri2 also had significant changes in grain morphology ( Figure 4 C~I). In terms of grain length, PDIL2;3-cri1 and PDIL2;3-cri2 mutants were 12.24% and 10.64% longer than the wild type, respectively. In terms of grain width, PDIL2;3-cri1 and PDIL2;3-cri2 mutants were 6.89% and 6.99% longer than the wild type. In terms of grain thickness, there was no significant difference between PDIL2;3-cri1 and PDIL2;3-cri2 mutants and the wild type. In terms of thousand-grain weight, PDIL2;3-cri1 and PDIL2;3-cri2 mutants were 5.89% and 5.12% higher than the wild type, respectively ( Figure 4The above results indicate that the PDIL2;3 gene plays an important role in the development of rice panicles and grains, and the grain width, thickness and weight of the PDIL2;3 mutant rice are significantly increased.

[0071] Example 3 Obtaining and characterizing PDIL2;3 overexpressing plants

[0072] In this example, genetic analysis was performed by creating materials that overexpress PDIL2;3. First, the primer sequence PDIL2;3-OE-F / R was designed, and the full-length cDNA fragment of PDIL2;3 was amplified using the cDNA of ZH11 young ears (2mm-5mm) as a template. The homologous recombination method was used to connect the T5 nuclease to the overexpression vector pU1301 treated with KpnI-BamHI. The plasmid map is shown in FIG. Figure 5 As shown, the vector with the correct enzyme digestion was named pU1301-PDIL2;3 and sequenced for verification. Subsequently, the vector with the correct sequence was selected and electroporated into the Agrobacterium tumefaciens EHA105 strain. The overexpression vector was introduced into the callus tissue of ZH11 by Agrobacterium-mediated genetic transformation (the induction of callus tissue was carried out by conventional methods), and PDIL2;3 overexpression plants under the ZH11 background were obtained.

[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 T0 generation in B showed that the expression level of PDIL2;3 was significantly upregulated in the transgenic positive individuals PDIL2;3-OE1 and PDIL2;3-OE2 compared with the wild type (i.e. non-transgenic). Figure 6 The A, L, M and N phenotypes of PDIL2;3-OE1 and PDIL2;3-OE2 showed no significant changes in plant height and tiller number compared with the wild type, and there were no significant differences in panicle type, number of branches and number of grains per panicle at maturity ( Figure 6 C, O, P and U), but the grain morphology changed significantly ( Figure 6 (middle D~K). Figure 6The statistics of Q-T showed that the grain thickness of PDIL2;3-OE1 and PDIL2;3-OE2 plants remained unchanged, but the grain length, grain width and 1000-grain weight were significantly reduced. This result further verified that PDIL2;3 plays an important role in regulating rice grain shape and yield. Overexpression of PDIL2;3 will lead to smaller grain shape and reduced yield.

[0077] Example 4 PDIL2;3 affects the formation of grain chalkiness and the accumulation of endosperm storage substances

[0078] In order to analyze whether the PDIL2;3 gene is involved in the formation of rice chalkiness, the mature grains of the wild type, PDIL2;3-cri mutant and PDIL2;3-OE family were observed and analyzed in this example. It was found that the polished rice of the WT and PDIL2;3-OE families was transparent and waxy, while the polished rice of the PDIL2;3-cri mutant showed an opaque phenotype with obvious chalkiness accumulation ( Figure 7 A). The seeds of each family were then harvested and dried for half a year to count the chalkiness. It was found that compared with the wild type, the chalkiness rate and chalkiness of PDIL2;3-cri grains increased significantly. However, the chalkiness rate and chalkiness of PDIL2;3-OE grains decreased significantly ( Figure 7 HI). The head rice rate is a grading index for rice processing quality and is greatly affected by chalkiness. Compared with the wild type, the head rice rate of the PDIL2;3-cri transgenic line was significantly reduced, while the head rice rate of the PDIL2;3-OE line was significantly increased ( Figure 7 J).

[0079] Further, the cross-section of each family's grains was observed using a stereomicroscope to explore the cytological basis of chalkiness in the mutants, such as Figure 7 As shown in Figures B to D, the endosperm of WT and PDIL2;3-OE was full and compact, while the endosperm of grains from the PDIL2;3-cri family had large areas of chalky accumulation.

[0080] To further observe the chalkiness in the endosperm, the cross-sections of mature grains of the wild type, PDIL2;3-cri mutant, and PDIL2;3-OE were observed using a scanning electron microscope. It was found that the starch granules in the endosperm of the wild type and PDIL2;3-OE were of uniform size, densely arranged, and squeezed to form a regular polyhedral structure; while the starch granules in the endosperm of PDIL2;3-cri were irregular small spheres, loosely arranged, and with large gaps between the granules, and could not be tightly arranged to form a polygonal shape ( Figure 7 E~G). This indicates that the mutation of PDIL2;3 gene affects the shape and arrangement of endosperm storage substances, and the seeds show an undesirable phenotype of increased chalkiness.

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

[0082] Figure 7 The K-M plot results showed that compared with the wild type, the total starch and amylose content of the PDIL2;3-cri mutant grains decreased, while the protein content increased significantly; the results of PDIL2;3-OE were opposite to those of PDIL2;3-cri mutants. It can be seen that the PDIL2;3 gene plays a role in the development of rice endosperm and the accumulation of its contents.

[0083] Example 5 Effect of PDIL2;3 on the division of husk cells

[0084] Seed size is mainly determined by husk size, and husk size is closely related to the development of husk cells. In order to study the changes in husk cells of the PDIL2;3 transgenic family, this example observed the husk by scanning electron microscopy and paraffin sectioning. The specific steps are as follows:

[0085] The mature and sun-dried seeds of the PDIL2;3-cri1 mutant, PDIL2;3-OE1 and wild-type plants were sampled and sprayed with gold, and then observed under a scanning electron microscope (S570, Hitachi, Tokyo, Japan, Electron Microscope Laboratory of Huazhong Agricultural University).

[0086] The electron microscopy results are as follows Figure 8 As shown, Figure 8 In A, the PDIL2;3-cri1 mutant seeds were significantly larger than the wild type, while the PDIL2;3-OE1 seeds were smaller than the wild type. The cells in the middle of the outer glume were then magnified and observed, and the distance between the small protrusions between the glume cells was measured using ImageJ software ( Figure 8 B). Figure 8 The results of C showed that in terms of grain length, there was no significant difference in the length of husk cells between the PDIL2;3-cri1 mutant, PDIL2;3-OE1 and the wild type. Figure 8 The results of E showed that the number of cells in the longitudinal direction of the PDIL2;3-cri1 mutant was significantly higher than that of the wild type, while that of PDIL2;3-OE1 was lower than that of the wild type, indicating that the change in the grain length of the PDIL2;3 transgenic lineage was caused by the change in the number of cells / divisions in the longitudinal direction. In the grain width direction, compared with the wild type, the transverse cell length of the PDIL2;3-cri1 mutant increased significantly, while the transverse cell length of the PDIL2;3-OE1 decreased significantly ( Figure 8D). The results of cell number statistics showed that there was no significant difference in the number of cells between the PDIL2;3-cri1 mutant and PDIL2;3-OE1 compared with the wild type ( Figure 8 F), suggesting that the change in grain width in the PDIL2;3 transgenic lines is caused by changes in cell size / extension in the horizontal direction. The above results indicate that PDIL2;3 can negatively regulate the division of longitudinal cells in the husk and the extension of transverse cells, thereby controlling grain length and grain width, respectively.

[0087] The husks of the PDIL2;3-cri1 mutant, PDIL2;3-OE1 and wild type at the heading stage were taken and immersed in 70% FAA (70% anhydrous ethanol, 5% acetic acid, 3.7% formalin), evacuated in a vacuum pump for 30 minutes, and fixed at room temperature for 24 hours. The fixed biological materials were transferred to 70% ethanol, dehydrated with a series of alcohols, stained with eosin overnight, and further dehydrated with gradient alcohols, transparentized with xylene, and paraffin-infiltrated with gradient wax. After 3-4 days of pure paraffin embedding, the slices were sectioned (the slicer was Yidi YD-1508A rotary slicer), and the slice thickness was 8μm. The cut wax strips were spread, dried, dewaxed, stained, and sealed, and then observed under a microscope ( Figure 8 H~I).

[0088] The results showed that the average area of ​​the outermost layer of cells in the glume thin-walled cell layer of the PDIL2;3-cri1 mutant increased, while the number of cells did not change significantly ( Figure 8 JK). This further indicates that the change in grain width in the PDIL2;3 transgenic line is caused by the change in cell size / extension in the horizontal direction. The results of flow cytometry detection of cell ploidy showed that the proportion of 4C cells in the young panicles of the PDIL2;3-cri1 mutant was significantly higher than that of the wild type, while that of PDIL2;3-OE1 was lower than that of the wild type, indicating that the cell division in the young panicles of the mutant was more vigorous. The above results indicate that the longitudinal division of the husk cells and the strengthening of the lateral extension are the reasons for the enlargement of the husk of the mutant.

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

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

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

[0092] Table 1 Fluorescence quantitative PCR primers

[0093] Upstream primer name Upstream primer sequence Downstream primer name Downstream primer sequence qRT-CDKA1;1-F SEQ ID NO.16 qRT-CDKA1;1-R SEQ ID NO.17 qRT-CDKA2;1-F SEQ ID NO.18 qRT-CDKA2;1-R SEQ ID NO.19 qRT-CDKB2;1-F SEQ ID NO.20 qRT-CDKB2;1-R SEQ ID NO.21 qRT-CycA1;1-F SEQ ID NO.22 qRT-CycA1;1-R SEQ ID NO.23 qRT-CycA2;1-F SEQ ID NO.24 qRT-CycA2;1-R SEQ ID NO.25 qRT-CycA3;2-F SEQ ID NO.26 qRT-CycA3;2-R SEQ ID NO.27 qRT-CycB1;1-F SEQ ID NO.28 qRT-CycB1;1-R SEQ ID NO.29 qRT-CycB1;4-F SEQ ID NO.30 qRT-CycB1;4-R SEQ ID NO.31 qRT-CycB2.1-F SEQ ID NO.32 qRT-CycB2.1-R SEQ ID NO.33 qRT-CycB2;2-F SEQ ID NO.34 qRT-CycB2;2-R SEQ ID NO.35 qRT-CycD3;1-F SEQ ID NO.36 qRT-CycD3;1-R SEQ ID NO.37 qRT-CycD4;1-F SEQ ID NO.38 qRT-CycD4;1-R SEQ ID NO.39 qRT-CycD4;2-F SEQ ID NO.40 qRT-CycD4;2-R SEQ ID NO.41 qRT-CycD6;1-F SEQ ID NO.42 qRT-CycD6;1-R SEQ ID NO.43 qRT-CycD7;1-F SEQ ID NO.44 qRT-CycD7;1-R SEQ ID NO.45 qRT-CyclaZm-F SEQ ID NO.46 qRT-CyclaZm-R SEQ ID NO.47 qRT-CycT1;3-F SEQ ID NO.48 qRT-CycT1;3-R SEQ ID NO.49 qRT-EF2-F SEQ ID NO.50 qRT-EF2-R SEQ ID NO.51 qRT-MCM2-F SEQ ID NO.52 qRT-MCM2-R SEQ ID NO.53 qRT-MCM3-F SEQ ID NO.54 qRT-MCM3-R SEQ ID NO.55 qRT-MCM4-F SEQ ID NO.56 qRT-MCM4-R SEQ ID NO.57 qRT-MCM5-F SEQ ID NO.58 qRT-MCM5-R SEQ ID NO.59

[0094] Figure 9 The results showed that the expression levels of many cell cycle regulatory genes in the PDIL2;3-cri mutant were significantly higher than those in the wild type, such as CYCT1, CDKA2, CDKE, CDKF, etc. Based on the above results, it can be inferred that the changes in cell morphology and number in the PDIL2;3-cri mutant are caused by the abnormal expression of cell cycle-related genes in the PDIL2;3-cri 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

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

3.

2. The use according to claim 1, characterized in that: The grain shape is grain length, grain width, and / or grain weight.

3. The use according to claim 2, characterized in that: The regulation is specifically any one of the following: (i) increasing the grain length, grain width, grain weight and / or chalkiness of rice by inhibiting or reducing the activity of the protein encoded by the PDIL2;3 gene or the expression level thereof; (ii) Increasing the grain length, grain width, grain weight and / or chalkiness of rice by knocking out, inhibiting or silencing the expression of the PDIL2;3 gene.

4. The use according to claim 2, characterized in that: The regulation is specifically any one of the following: (a) reducing the grain length, grain width, grain weight and / or chalkiness of rice by increasing the activity of the protein encoded by the PDIL2;3 gene or the expression level thereof; (b) Reducing the grain length, grain width, grain weight and / or chalkiness of rice by increasing the expression level of the PDIL2;3 gene.

5. The use according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the PDIL2;3 gene is shown as SEQ ID NO.1 or SEQ ID NO.

2.

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

3.

7. The method according to claim 6, characterized in that The rice grain shape refers to the rice grain length, grain width and / or grain weight.

8. The method according to claim 7, characterized in that The method is selected from any one of the following: (1) down-regulating the expression level of the PDIL2;3 gene, the expression level of the protein encoded by the PDIL2;3 gene, or the activity of the protein encoded by the PDIL2;3 gene, thereby increasing the grain length, grain width, grain weight and / or chalkiness of rice; (2) Up-regulating the expression level of the PDIL2;3 gene, the expression level of the protein encoded by the PDIL2;3 gene, or the activity of the protein encoded by the PDIL2;3 gene, thereby reducing the grain length, grain width, grain weight and / or chalkiness of rice.

9. The method according to claim 8, characterized in that In (2), the up-regulation of the expression level of the PDIL2;3 gene, the expression level of the protein encoded by the PDIL2;3 gene, or the activity of the protein encoded by the PDIL2;3 gene is specifically any one of the following: (a) transferring the expression construct or vector of PDIL2;3 gene into rice or rice cells; (b) Transforming the expression construct or vector containing the PDIL2;3 gene into rice or rice cells.

10. A rice breeding method, characterized in that: The method comprises the following steps: introducing a substance that increases or inhibits the expression level of the PDIL2;3 gene, the expression level of the protein encoded by the PDIL2;3 gene, or the activity of the protein encoded by the PDIL2;3 gene into rice or rice cells to obtain the bred rice; the amino acid sequence encoded by the PDIL2;3 gene is shown in SEQ ID NO.3.

Images