Gene for reducing ineffective tillering and improving crop yield and nitrogen fertilizer utilization efficiency and application thereof

Abstract

The invention discloses application of a DUP3 gene and an allele thereof in reduction of ineffective tillering, increase of yield and / or improvement of nitrogen fertilizer utilization efficiency in crops. The invention further discloses a method for reducing ineffective tillering and improving crop yield and / or nitrogen fertilizer utilization efficiency by improving expression quantity of DUP3 or allele thereof, and the method can be used for genetic improvement of rice.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to DUP3 gene and its alleles DUP3 Japonica New application in reducing ineffective tiller number, improving earing rate, yield and nitrogen use efficiency of crops. BACKGROUND

[0002] Nitrogen is an important component of proteins, amino acids, nucleic acids and chlorophyll, and is the most demanded mineral element for plants, which is essential for plant growth and development (Dechorgnat et al., 2011; Yaseen et al., 2013).

[0003] According to statistics, since the 1960s, the global nitrogen fertilizer application has increased by about five times from 20 million tons per million hectares to 108 million tons per million hectares (Lu and Tian, 2017), while the absorption and utilization rate of nitrogen by crops is only about 40.2% (Li et al., 2021). Moreover, the nitrogen utilization rate in China is only about 30% of the world average, while the nitrogen fertilizer application is more than three times the world average. This means that a large amount of nitrogen that is not absorbed and utilized by crops is lost into rivers, lakes and seas or released into the atmosphere through denitrification, causing a series of environmental problems such as water eutrophication, soil acidification and air pollution (Erisman et al., 2013; Steffen et al., 2015), and the production of nitrogen fertilizer is a typical high-energy and high-pollution industry, which is heavily dependent on non-renewable resources such as coal, natural gas and oil (Zhang et al., 2020). Therefore, how to reduce nitrogen input in agricultural production and continuously improve grain yield has become a major problem to be solved in the sustainable development of agriculture (Liu et al., 2022).

[0004] As early as the 1960s, scientists found that ineffective tillering was common in wheat, barley and rye crops (Motzo et al., 2004; Nuruzzaman et al., 2000). In 1990, Yamamoto and Aihara found that the tillering earing rate of the 2nd to 6th node of rice could reach 100%, but the survival rate of tillers gradually decreased as the node increased, especially after the large-scale promotion of semi-dwarf rice varieties and indica-japonica hybrid varieties, the accumulation of ineffective tillers was more serious (Nuruzzaman et al., 2000; Yamamoto and Aihara, 1990).

[0005] In 2000, Nuruzzaman et al. conducted a correlation analysis between the number of ineffective tillers and the average dry matter accumulation, nitrogen content, and carbon content per tiller from the time of maximum tillering to heading in 14 rice varieties. The results showed that the number of ineffective tillers was significantly negatively correlated with all three indicators, suggesting that tiller survival is related to the amount of dry matter accumulated (Nuruzzaman et al., 2000). Current research indicates that tillers with low biomass, insufficient photosynthetic area, and underdeveloped root systems are unable to produce the nutrients they need for survival and are therefore easily converted into ineffective tillers. The time of tiller death generally occurs around the jointing stage of rice. This is mainly because during the jointing stage, the main stem grows rapidly and is itself a strong energy demand reservoir. Therefore, most of the nutrients produced by the main stem are used for its own growth, reducing the proportion of carbohydrates supplied to the small tillers. In addition, the rapid growth of the main stem's vegetative body blocks part of the light of the small tillers, resulting in the phenomenon that a large number of ineffective tillers begin to die during the jointing stage.

[0006] In the 1960s, the Green Revolution dwarfing breeding addressed the challenges of high-fertilizer tolerance and lodging in crops, significantly increasing harvest index and yield (Khush, 1999; Zeigler and Mohanty, 2010). Green Revolution varieties (GRVs) tolerated high fertilizer levels and showed strong lodging resistance, leading to an increase in tillering. However, there was also a significant accumulation of ineffective tillers, resulting in a decrease in the productive tiller percentage (PPT). A 2008 study showed that increasing nitrogen fertilization increased both the maximum number of tillers and the number of effective panicles in rice, but the rate of increase in effective panicle number was significantly lower than that of the maximum number of tillers. This suggests that while high nitrogen fertilization significantly increased the maximum number of tillers, its effect on the number of effective panicles was weaker. This suggests that while high nitrogen fertilizer application increases the number of tillers, many of these tillers eventually become ineffective, ultimately reducing the productive tiller percentage (Rashid and Khan, 2008). To date, the molecular basis and intrinsic regulatory mechanism of ineffective tillering have not been elucidated. How to reduce ineffective tillering of crops under nitrogen application conditions and improve crop yield and nitrogen utilization efficiency remains a major issue facing the current sustainable development of agriculture. Summary of the Invention

[0007] The present invention relates to new functions and applications of known genes. Specifically, the present invention relates to new functions and applications of the known hexose transporter MST4 (monosaccharide transporter 4) encoded by the DUP3 gene in reducing ineffective tillering of crops and synergistically improving crop yield and nitrogen fertilizer use efficiency.

[0008] Cloning of a gene DUP3 (nucleotide sequences are shown in SEQ ID NO. 1 and 3) controlling rice panicle formation rate by genome-wide association study (GWAS) using 727 rice germplasm resources with rich genetic diversity planted in field, and using the ratio of tiller number at jointing stage (i.e. maximum tiller number) and effective panicle number at maturity as the panicle formation rate, and the gene DUP3 encodes a hexose transporter protein MST4 (amino acid sequence is shown in SEQ ID NO. 5); near-isogenic lines and transgenic studies prove that the DUP3 gene has a new function of controlling the number of ineffective tillers, and the allele DUP3 Japonica (nucleotide sequences are shown in SEQ ID NO. 2 and 4, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 6) can reduce the number of ineffective tillers of crops, increase the number of effective panicles, and significantly improve the yield of crops under different nitrogen fertilizer application rates, especially the yield of crops under low fertilizer application rate. Therefore, the DUP3 Japonica gene and its allele DUP3

[0009] Rice variety Huanghuazhan (HHZ) is a variety with strong tillering ability but slightly more ineffective tillers, and its panicle formation rate is relatively low, about 70% on average. The inventors applied the DUP3 gene to HHZ, which significantly reduced the number of ineffective tillers of HHZ and finally improved the yield of HHZ. Therefore, the DUP3 gene and its allele DUP3 Japonica are important genetic resources for reducing ineffective tillers of crops, improving crop yield and nitrogen-efficient breeding.

[0010] In summary, the present application provides a new function of the DUP3 gene in reducing ineffective tillers of crops and synergistically improving yield and nitrogen use efficiency, and its application. The purpose of the present application is to clone genes that reduce ineffective tillers of crops, to reveal the molecular basis and regulatory mechanism of ineffective tiller formation, and to provide theoretical basis and technical support for synergistically improving crop yield and nitrogen use efficiency.

[0011] Specifically, the present application mainly relates to the following aspects:

[0012] In a first aspect, a gene DUP3 that reduces the number of unproductive tillers, improves crop yield and nitrogen use efficiency is cloned, which is a known function gene MST4. The MST4 gene is known to have the function of transporting monosaccharides such as glucose, fructose and mannose, but there is no report on the regulation of crop agronomic traits by the gene (Wang et al., 2007). The inventors determined that DUP3 has a new function of reducing the number of unproductive tillers, improving yield and nitrogen use efficiency, and the allele from japonica rice increases the expression amount of itself, thereby showing the crop breeding application potential of high yield and high nitrogen efficiency.

[0013] In one embodiment, GWAS analysis is performed on 727 germplasm resources, and a quantitative trait locus (QTL) for controlling the tiller-to-ear rate of rice is successfully located, which is named qDUP3, and the QTL only contains 4 ORFs (ORF1, ORF2, ORF3 and ORF4) Figure 2 A, B). Further, part of the varieties with extremely high earing rate and part of the varieties with extremely low earing rate are randomly selected from the germplasm resources, and the expression levels of the 4 genes are analyzed by qRT-PCR method. The results show that the expression level of ORF1 is higher in the high earing rate varieties and lower in the low earing rate varieties Figure 2 C), and it is speculated that ORF1 may be a candidate gene for controlling the earing rate, which is named DUP3 (Deletion of unproductive tiller 3, DUP3). The gene is a known gene, which encodes a hexose transporter protein MST4 (Monosaccharide transporter gene 4, MST4).

[0014] In one embodiment, a CRISPR / Cas9 knockout vector is constructed, and a rice variety WYJ7 is transformed by using the agrobacterium-mediated method to obtain a DUP3 knockout strain WYJ7-dup3, and the mutation type is insertion of base T at the 3239th position of DUP3, which causes a code disorder from the 165th amino acid, and encodes a mutant protein of 518 amino acids. The WYJ7-dup3 plant has a weak overall growth potential Figure 3 A), the number of unproductive tillers increases Figure 3 B), the number of productive ears decreases Figure 3 C), and the number of grains per ear decreases Figure 3 D). Therefore, the loss of function of DUP3 increases the unproductive tillers of rice and inhibits the growth and development of rice.

[0015] In an embodiment, a pActin::DUP3-Flag vector is constructed and introduced into the rice variety Huanghuazhan by Agrobacterium-mediated method. According to the National Rice Data Center, Huanghuazhan is a late-maturing indica rice carrying the "Green Revolution" gene sd1 bred by the Rice Research Institute of Guangdong Academy of Agricultural Sciences. It has strong tillering ability, but has more ineffective tillers, resulting in a panicle formation rate of only about 70%. Field trait statistics show that overexpression of DUP3 can significantly reduce the number of ineffective tillers of Huanghuazhan Figure 4 B), significantly increase the number of effective panicles of Huanghuazhan Figure 4 C) and the yield per plant Figure 4 D).

[0016] In an embodiment, according to the filtering criteria of minimum allele frequency greater than 0.05 and genotype missing rate less than 0.02, single nucleotide polymorphisms (SNPs) located in the promoter region of DUP3 (2 kb upstream of the start codon ATG) and nonsynonymous mutations located in the coding region in germplasm resources are extracted. Haplotype analysis of DUP3 using these SNPs found that the gene is mainly divided into three haplotypes, and shows obvious indica-japonica differentiation. Haplotype 1 (Hap. 1) exists only in tropical japonica and temperate japonica, Hap. 2 exists mainly in indica and a small number of AUS subgroups, and Hap. 3 exists mainly in AUS subgroups and indica Figure 6 A). Significant difference analysis of the panicle formation rate phenotypes of rice varieties with different haplotypes found that the panicle formation rate of rice varieties carrying Hap. 1 was significantly higher than that of rice varieties carrying Hap. 2 or Hap. 3, indicating that Hap. 1 can improve the panicle formation rate of rice. It is named DUP3 Japonica ( Figure 6 B).

[0017] In an embodiment, the high-yield indica rice variety Huajiangxian 74 (from South China Agricultural University, also available on the market, HJX74) is used as the recipient and japonica rice as the donor to construct near-isogenic lines HJX74 and HJX74-DUP3 Japonica by hybridization and backcrossing. Field phenotype statistical results show that, compared with HJX74, HJX74-DUP3 Japonica has reduced ineffective tiller number Figure 7 C), increased effective panicle number Figure 7 D), increased grain number per panicle Figure 7 E), and increased yield Figure 7 F). Therefore, the japonica-type DUP3 Japonica can reduce the number of ineffective tillers of indica rice varieties and improve rice yield.

[0018] In one embodiment, HJX74 and HJX74-DUP3 Japonica Planted in the field with different nitrogen application rates (60kg / ha, 120kg / ha, 210kg / ha and 300kg / ha). Statistical field phenotypic results showed that compared with HJX74, HJX74-DUP3 Japonica The number of ineffective tillers was relatively small ( Figure 8 B), the output has increased to varying degrees ( Figure 8 C), especially at lower nitrogen rates, HJX74-DUP3 Japonica Therefore, the superior allele DUP3 from japonica rice Japonica It has the potential to reduce the number of ineffective tillers of crops, reduce weight and increase yield.

[0019] In a second aspect, the present invention relates to a gene DUP3 and an allele DUP3 that reduces the number of ineffective tillers and improves crop yield and nitrogen use efficiency. Japonica Specifically, the DUP3 gene (nucleotide sequence is preferably SEQ ID NO. 1, 3, and the amino acid sequence of the protein encoded is preferably SEQ ID NO. 5) and its alleles DUP3 Japonica The expression level of the gene (preferably SEQ ID NO. 2, 4 for the nucleotide sequence and SEQ ID NO. 6 for the encoded protein) is increased, which can reduce the number of ineffective tillers of crops, and increase yield and nitrogen utilization efficiency.

[0020] In some embodiments, a gene DUP3 and its allele DUP3 are provided for reducing the number of ineffective tillers of crops, improving yield and nitrogen use efficiency. Japonica , which is an isolated polynucleotide, the amino acid sequence of the protein encoded by it is shown in any one of the following:

[0021] 1) the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6;

[0022] 2) an amino acid sequence that differs from the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6 due to substitution, deletion and / or insertion of one or more amino acid residues;

[0023] 3) an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and especially at least 95% or 98% identical to the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6;

[0024] 4) An active fragment of the amino acid sequence described in (1) or (2) or (3).

[0025] said DUP3 and its alleles DUP3 Japonica nucleotide sequence as shown in any one of the following:

[0026] 1) the nucleotide sequence as shown in SEQ ID NO. 1, 2, 3, 4;

[0027] 2) a nucleotide sequence which differs from the nucleotide sequence as shown in SEQ ID NO. 1, 2, 3, 4 by one or more substitutions, deletions and / or insertions of nucleotides, but which codes for a protein having the same activity as the protein coded for by the nucleotide sequence as shown in SEQ ID NO. 5, 6;

[0028] 3) a nucleotide sequence which has at least 70%, preferably at least 80%, more preferably at least 90% identity with the nucleotide sequence as shown in SEQ ID NO. 1, 2, 3, 4, and which codes for a protein having the same activity as the protein coded for by the nucleotide sequence as shown in SEQ ID NO. 5, 6;

[0029] 4) a nucleotide sequence which differs from the nucleotide sequence as shown in SEQ ID NO. 1, 2, 3, 4 due to the degeneracy of the genetic code;

[0030] 5) an active fragment comprising a nucleotide sequence according to any one of 1) to 4);

[0031] 6) a nucleotide sequence comprising a sequence complementary to the nucleotide sequence according to any one of 1) to 5) which hybridizes under medium stringency conditions, preferably high stringency hybridization conditions; or

[0032] 7) a nucleotide sequence comprising a sequence complementary to the nucleotide sequence according to any one of 1) to 5).

[0033] compared to the DUP3 protein (SEQ ID NO. 5) coded by the DUP3 gene, its alleles DUP3 Japonica coding for DUP3 Japonica protein (SEQ ID NO. 6) has one amino acid difference.

[0034] The present application also relates to the promoter sequence of the gene DUP3 and its alleles DUP3 Japonica which reduces the number of ineffective tillers, increases crop yield and nitrogen use efficiency, the nucleotide sequence of which is shown in any one of the following:

[0035] (1) the nucleotide sequence as shown in SEQ ID NO. 13 and 14;

[0036] (2) a nucleotide sequence which hybridizes to the complement of the sequence of SEQ ID NO. 13 and 14 under medium stringency conditions, preferably high stringency hybridization conditions;

[0037] (3) a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% or 98% identity with the sequence of SEQ ID NO. 13 and 14;

[0038] (4) an active fragment of the nucleotide sequence of any one of (1) to (3); or

[0039] (5) a nucleotide sequence complementary to the nucleotide sequence of any one of (1) to (4).

[0040] In a third aspect, there is provided a recombinant construct comprising the polynucleotide sequence of the gene DUP3 or its allele for reducing the number of ineffective tillers, increasing the yield and nitrogen use efficiency of crops, and / or comprising the promoter sequence as described herein. The recombinant construct can be a cloning vector or an expression vector for expressing the polynucleotide.

[0041] In a fourth aspect, there is provided a recombinant host cell comprising the vector as described in the third aspect, or having integrated in its genome the polynucleotide sequence of the gene DUP3 or its allele for reducing the number of ineffective tillers, increasing the yield and nitrogen use efficiency of crops as described herein. The host cell can be an animal cell, a plant cell or a microbial cell, such as an E. coli cell or an Agrobacterium cell, preferably a plant cell, more preferably a rice, wheat, maize cell, most preferably a rice cell. The cell can be isolated, in vitro, cultured or part of a plant.

[0042] In a fifth aspect, there is provided a method for reducing the number of ineffective tillers, increasing the yield and nitrogen use efficiency of crops, the method comprising: cultivating a crop plant comprising the polynucleotide sequence of the gene DUP3 or its allele for reducing the number of ineffective tillers, increasing the yield and nitrogen use efficiency of crops as described herein, or a related recombinant construct. The method can comprise: transfecting a crop plant with a recombinant Agrobacterium cell comprising the polynucleotide sequence of the gene DUP3 or its allele for reducing the number of ineffective tillers, increasing the yield and nitrogen use efficiency of crops as described herein, to obtain a transgenic plant; or crossing a crop plant with another plant comprising the polynucleotide sequence of the gene DUP3 or its allele for reducing the number of ineffective tillers, increasing the yield and nitrogen use efficiency of crops as described herein, to obtain a hybrid plant; or using other methods to increase the expression of DUP3 or its allele in the crop (e.g. using CRISPR / Cas9 gene editing method). The use of the method for increasing the earing rate, yield and nitrogen use efficiency of crops is not limited to the above. Japonica The use of the method for increasing the earing rate, yield and nitrogen use efficiency of crops is not limited to the above.

[0043] In one embodiment, the overexpression vector pActin::DUP3-Flag was introduced into the indica rice variety HHZ via Agrobacterium-mediated transgenic plants overexpressing DUP3, and phenotypic statistical analysis showed that compared with the control HHZ, the transgenic plants pActin::DUP3-Flag had fewer ineffective tillers and increased effective panicle number and yield per plant ( Figure 4 Therefore, in indica rice, increasing the transcription level of the DUP3 gene can reduce the number of ineffective tillers and increase the number of effective panicles and yield per plant.

[0044] In one embodiment, DUP3 is expressed by hybridization and backcrossing combined with marker-assisted selection. Japonica When introduced into the indica rice variety HJX74, the number of ineffective tillers of HJX74 was significantly reduced, and the number of effective panicles, number of grains per panicle and yield were increased ( Figure 7 ), especially the yield under low nitrogen fertilizer planting conditions ( Figure 8 ).

[0045] The following are definitions of some terms used in the present invention. Unless otherwise specified, the terms used in the present invention have the meanings known to those skilled in the art.

[0046] Ineffective tillers are those that die during growth and therefore fail to produce seeds and contribute to yield. Ineffective tillers are defined as those with fewer than four leaves during the rice's peak tillering period (generally around the jointing stage).

[0047] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably, the RNA is then translated in an organism to produce a protein.

[0048] A "nucleic acid molecule" or "nucleic acid sequence" is a linear fragment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, preferably, a nucleic acid molecule is a DNA fragment. A "nucleic acid molecule" is also called a polynucleotide molecule.

[0049] A "plant" is any plant at any stage of development, in particular a seed plant.

[0050] A "promoter" is an untranslated DNA sequence upstream of a coding region that contains a binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region may also contain other elements that act as regulators of gene expression.

[0051] The phrase "substantially identical" in the context of two nucleic acid or protein sequences refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90%, even more preferably 95% and most preferably at least 99% of nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the sequence will be substantially identical over a region of at least about 50 residues, more preferably over a region of at least about 100 residues, and most preferably over a sequence of at least about 150 residues. In a particularly preferred embodiment, the sequences are substantially identical over the entire length of the coding region. Furthermore, substantially identical nucleic acid or protein sequences have substantially the same function.

[0052] For sequence comparison, typically one sequence acts as the reference sequence, to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the program parameters.

[0053] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel et al., infra).

[0054] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words buried in the sequence of the query aligned with words in the database sequence, which have scores above a positive, typically zero, threshold value (T). T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the

[0055] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm includes the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0056] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specific hybridization" refers to when a molecule binds to a particular nucleotide sequence, forming a double helix or hybridizing, under stringent conditions, when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Substantial binding" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and includes minor mismatches that can be tolerated by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0057] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence dependent, and are different under different ambient parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the art of nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Typically, high stringency hybridization and wash conditions will be selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Under "stringent conditions" a probe will hybridize to its target subsequence, but not to other sequences.

[0058] Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with the protein encoded by the second nucleic acid or binds specifically to the second protein. Thus, a protein is typically substantially identical to a second protein, e.g., where the two proteins differ only by conservative substitutions.

[0059] "Transformation" is the process of introducing a heterologous nucleic acid into a host cell or organism, in particular, "transformation" means the stable integration of a DNA molecule into the genome of the organism of interest.

[0060] "Transformed" refers to a host organism, such as a bacterium or a plant, that has been introduced with a heterologous nucleic acid molecule. The nucleic acid molecule can be stably integrated into the host genome or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such an extrachromosomal molecule can be self-replicating. A transformed cell, tissue, or plant is understood to include not only the final product of the transformation process but also transgenic progeny thereof.

[0061] As used herein, the terms "polynucleotide," "polynucleotide molecule," "polynucleotide sequence," "coding sequence," and the like include single- and double-stranded DNA and RNA molecules, which can contain one or more of the following: prokaryotic sequences, cDNA sequences, genomic DNA sequences including exons and introns, chemically synthesized DNA and RNA sequences, and sense and corresponding antisense strands.

[0062] Methods for producing and manipulating the polynucleotide molecules disclosed herein are known to those of skill in the art and can be accomplished in accordance with described recombinant techniques (see Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Innis et al. (eds.), 1995, PCR Protocols, Academic Press, Inc., San Diego; and Erlich (ed.), 1992, PCR Technology, Oxford University Press, New York).

[0063] "Plant transformation" refers to the expression of at least one foreign gene in a plant for the purpose of conferring one or more desirable phenotypic traits on the transformed plant.

[0064] Plants transformed in accordance with the present application can be monocotyledonous or dicotyledonous plants, including but not limited to corn, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugar cane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as conifers and deciduous trees. Particularly preferred are rice, wheat, barley, corn, oat, or rye.

[0065] It can be desirable to transform a desired nucleotide sequence into a particular plant species, to propagate it in that species or to transfer it into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques.

[0066] Preferably, the nucleotide sequences of the application are expressed in transgenic plants, thereby causing the biosynthesis of the corresponding control phosphorus fertilizer use efficiency and yield traits proteins in the transgenic plants. In this way, transgenic plants with improved traits can be produced. In order to express the nucleotide sequences of the application in transgenic plants, the nucleotide sequences of the application can need to be modified and optimized. All organisms have specific codon usage biases, which are known in the art, and the codons of the nucleotide sequences described in the application can be changed to conform to the plant bias while maintaining the amino acids encoded by the nucleotide sequences of the application. Also, high levels of expression in plants can be best achieved from coding sequences having at least about 35%, preferably more than about 45%, more preferably more than 50%, most preferably more than about 60% GC content. Although the preferred gene sequences can be expressed adequately in both monocotyledonous and dicotyledonous plant species, the sequences can be modified to accommodate the specific codon bias and GC content bias of monocotyledons or dicotyledons, as these biases have been shown to be different (Murray et al., Nucl. Acids Res. 17:477-498 (1989)). In addition, the nucleotide sequences can be screened for the presence of unconventional splice sites that cause message truncation. All changes needed in the nucleotide sequences, such as those described above, can be made using site-directed mutagenesis techniques, PCR and synthetic gene construction well known in the art, using the methods described in published patent applications EP 0 385 962 (Monsanto), EP 0 359 472 (Lubrizol) and WO 93 / 07278 (Ciba-Geigy). BRIEF DESCRIPTION OF DRAWINGS

[0067] The above features and advantages of the present application will become more apparent from the following detailed description, taken in conjunction with the accompanying drawings, in which:

[0068] Figure 1 Figure 1 shows that the "Green Revolution" gene sd1 suppresses rice heading rate. (A), comparison of heading rate of indica rice germplasm carrying SD1 and sd1 loci, respectively, orange and green represent rice varieties carrying SD1 locus and sd1 locus, respectively. (B), comparison of heading rate of "Green Revolution" gene near-isogenic lines in different backgrounds, including rice variety Nanjing 6 (NJ6), HJX74, 9311. Values in the figure are mean ± standard error (n=10), asterisks on the column chart are the results of significant difference analysis using Student's t-test. *** represents p<0.001.

[0069] Figure 2, showing GWAS analysis of panicle setting rate regulation genes in rice. (A), Manhattan plot of the results of genome-wide association analysis, the horizontal black dotted line represents the significance threshold calculated by GEC software, and the green dot represents the most significant QTL in the region. (B), Linkage disequilibrium blocks composed of SNPs with -log10 (p) greater than 3 in the 300 kb flanking the most significant SNP in the qDUP3 signal interval and all candidate genes in this interval. Blue boxes in the ORF represent exons, arrows represent the direction of gene transcription, and gray boxes represent 5' UTR and 3' UTR. In the inverted triangle plot, each black line represents a SNP, and the color gradually changes from white to red, representing the strength of linkage of the SNP. (C), Comparison of the relative expression of ORF1 in the leaves of high and low panicle setting rate varieties.

[0070] Figure 3 , showing that loss of function of DUP3 leads to an increase in the number of ineffective tillers in crops. (A), Plant type of gene knockout line dup3, scale is 20 cm. (B), Comparison of ineffective tiller number. (C), Comparison of effective panicle number. (D), Comparison of grain number per panicle.

[0071] Figure 4 , showing that DUP3 positively regulates the panicle setting rate of rice. (A), Plant type of DUP3 transgenic line, scale is 20 cm. (B), Comparison of ineffective tiller number. (C), Comparison of effective panicle number. (D), Comparison of grain yield per plant.

[0072] Figure 5 , showing that the expression of DUP3 is reduced in sd1 type materials.

[0073] Figure 6 , showing haplotype analysis of DUP3. (A), Haplotype analysis results of DUP3, Num. represents the number of varieties, black rectangles represent exons, TEJ represents temperate japonica rice, TRJ represents tropical japonica rice, and IND represents indica rice. (B), Comparison of panicle setting rates of different haplotype types of DUP3, different letters in the violin plot represent the results of significance difference analysis using Tukey's HSD test.

[0074] Figure 7 , showing the comparison of agronomic traits between HJX74 and HJX74-DUP3 Japonica . (A), Comparison of plant type, scale is 20 cm. (B), Comparison of ear type, scale is 1 cm. (C), Comparison of ineffective tiller number. (D), Comparison of effective panicle number. (E), Comparison of grain number per panicle. (F), Comparison of grain yield per plant.

[0075] Figure 8 , showing the comparison of agronomic traits between HJX74 and HJX74-DUP3 JaponicaComparison of agronomic traits under different nitrogen application rates. (A) Plant type comparison, scale is 20 cm. (B) Comparison of ineffective tillering numbers. (C) Comparison of yield per plant. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further explained below with reference to specific embodiments and accompanying drawings. It should be understood that the embodiments are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.

[0077] The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The reagents, kits, and experimental instruments used in the experiments can all be purchased from biological instrument and reagent companies unless otherwise specified. Unless otherwise specified, the relevant data involved in the present invention are based on the following methods:

[0078] Phenotyping of ear formation rate: Studies have shown that the maximum number of tillers in rice generally occurs during the jointing phase. Based on differences in the rice growth period, this peak is typically concentrated between 45 and 60 days after transplanting (Ma et al., 2020; Rashid and Khan, 2008; Shahidullah et al., 2009). Therefore, the present inventors used the number of rice tillers 45 to 60 days after transplanting as the maximum tiller number (MTN), the number of panicles at maturity as the effective panicle number (PN), and the ratio of PN to MTN as the percentage of productive tiller (PPT).

[0079] All documents mentioned in this invention are incorporated herein by reference. After reading the contents of this invention, any changes or modifications made to this invention are deemed equivalent to the present invention and fall within the scope of the appended claims.

[0080] Example 1: Rice "Green Revolution" gene sd1 reduces rice panicle formation rate

[0081] On the one hand, the inventors compared the ear formation rates of indica rice varieties carrying SD1 and sd1 allele variant types in 727 germplasm resources. It was found that among the indica rice germplasm resources, the average ear formation rate of indica rice varieties carrying the sd1 allele was about 0.57, and the average ear formation rate of indica rice varieties carrying the wild-type SD1 allele was about 0.64. The Student's t-test was used to conduct a significant difference analysis, and the p value was 0.04, which indicated that the "Green Revolution" gene sd1 reduced the ear formation rate of rice. Further plotting the dot plot of the ear formation rate of indica rice varieties of germplasm resources also clearly showed that the ear formation rate of rice varieties carrying the sd1 allele was generally lower than that of rice varieties carrying the wild-type SD1 allele ( Figure 1 A). To further demonstrate that sd1 reduces the panicle formation rate of rice, the present inventors constructed three pairs of near-isogenic lines from the NJ6, 9311, and HJX74 backgrounds by hybridization and backcrossing (the hybridization and backcrossing methods are conventional methods known in the art): NJ6-SD1 and NJ6-sd1, 9311-SD1 and 9311-sd1, and HJX74-SD1 and HJX74-sd1. NJ6-sd1 was obtained by hybridizing NJ6 and Zhenshan 97B, followed by continuous backcrossing with NJ6 as the recurrent parent; 9311-SD1 was obtained by hybridizing 9311 and NJ6, followed by continuous backcrossing with 9311 as the recurrent parent; and HJX74-SD1 was obtained by hybridizing HJX74 and NJ6, followed by continuous backcrossing with HJX74 as the recurrent parent. NJ6, 9311, and HJX74 are all commercially available. These materials were used to analyze the effect of the "Green Revolution" gene sd1 on the panicle formation rate of rice. The results showed that the panicle formation rate of SD1 type materials in different backgrounds was higher than that of sd1 type materials in their corresponding rice backgrounds ( Figure 1 B) The above results confirmed that sd1 negatively regulates rice panicle formation rate.

[0082] Semi-dwarf varieties carrying the "Green Revolution" gene sd1 have been reported to have excellent characteristics of high fertilizer tolerance, lodging resistance and high yield, but they also have the disadvantage of low nitrogen fertilizer utilization efficiency (Li et al., 2018). This example further determined that sd1 increased the number of ineffective tillers of rice, resulting in a decrease in the panicle formation rate of semi-dwarf varieties, suggesting that the decrease in panicle formation rate may be another reason for the decrease in nitrogen fertilizer utilization efficiency of semi-dwarf varieties, and the phenotype of reduced nitrogen fertilizer utilization in semi-dwarf varieties can be further compensated by increasing the panicle formation rate, which has the potential to further increase the yield of semi-dwarf varieties. Combined with Example 5, the "Green Revolution" gene sd1 inhibits the gene expression of DUP3, in order to study the DUP3 gene and allele DUP3 Japonica It provides a research basis for its application in crops containing the "Green Revolution" gene sd1.

[0083] Example 2: DUP3 controls rice panicle rate

[0084] The inventors selected 727 materials from the 3,000 rice genome project, which were all resequenced, had high-quality SNP information, and were distributed in different rice planting areas around the world, fully representing the genetic diversity of rice (Zheng et al., 2015), including 308 Indica, 297 Tropical japonica, 47 Temperate japonica, 31 Aus, 10 Basmati, and 34 Admixed materials. GWAS analysis of the panicle rate of the 727 materials located the QTL qDUP3 Figure 2 A) controlling the panicle rate of rice. Further linkage disequilibrium analysis of the qDUP3 locus located it on chromosome 3, and by detecting the expression levels of genes in varieties with different panicle rates, it was found that an open reading frame (ORF1) had a higher expression level in high-panicle-rate varieties and a lower expression level in low-panicle-rate varieties, which led to the speculation that ORF1 might be a candidate gene controlling the panicle rate, which was named DUP3, encoding a known monosaccharide transporter Figure 2 C).

[0085] Example 3: Loss of function of DUP3 leads to increased ineffective tillering in crops

[0086] The inventors used the method of gene editing (CRISPR / Cas9) to create DUP3 gene knockout rice lines. The construction and transformation method of the CRISPR / Cas9 vector is as follows:

[0087] According to the gDNA sequence of DUP3 gene, the following primers were designed:

[0088] DUP3-CRISPR-F1:

[0089] CATCATCTCCTGCATCATGGGTTTTAGAGCTAGAAAT (SEQ ID NO. 7)

[0090] DUP3-CRISPR-R1:

[0091] CCATGATGCAGGAGATGATGCGGCAGCCAAGCCAGCA (SEQ ID NO. 8)

[0092] The method for constructing the plant dual-expression pYLCRISPR / Cas9 vector was described in an article published by Professor Liu Yaoguang of South China Agricultural University (A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. (2015) Molecular Plant, 8(8): 1274-1284). The correctly sequenced pYLCRISPR / Cas9 vector was transformed into the Agrobacterium tumefaciens strain AGL1 (Beijing Bomade Gene Technology Co., Ltd.) to obtain recombinant host cells with Agrobacterium as the host. After single clones were grown on the plate, the callus tissue of the japonica rice variety WYJ7 was infected with Agrobacterium to obtain the DUP3 gene knockout strain dup3 ( Figure 3 A). A T was inserted at base 3239 of the genomic DNA sequence at the editing target site, resulting in garbled amino acid sequence at position 165, encoding a mutant protein of 518 amino acids. Compared with WYJ7, dup3 plants had reduced biomass and increased ineffective tillers ( Figure 3 B), effective ear number ( Figure 3 C) and the number of grains per ear decreased ( Figure 3 D).

[0093] Example 4: DUP3 overexpression reduces ineffective tillering in crops

[0094] The present inventors created transgenic rice materials with overexpression of DUP3 gene.

[0095] The primers were designed based on the CDS sequence and genomic sequence of the DUP3 gene as follows:

[0096] DUP3-F1:

[0097] ACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGGAGGGTTCTCTGTG(SEQ ID NO.9)

[0098] DUP3-R1:

[0099] ACCACTTTGTACAAGAAAGCTGGGTCTTAAACGGTGGCGCCGTTGC(SEQ ID NO.10)

[0100] Using Nipponbare cDNA as a template, primers DUP3-F1 and DUP3-R1 were used to amplify the CDS fragment of DUP3. The CDS fragment of DUP3 was ligated into the pCAMBIA2300-pActin-GWR-Flag vector using Gateway cloning technology (the pCAMBIA2300 vector backbone was purchased from CAMBIA, Australia. Our laboratory added the rice constitutive promoter Actin and the Flag tag to this backbone and transformed it into a new vector suitable for Gateway cloning, named pCAMBIA2300-pActin-GWR-Flag). The ligation product was transformed into DH5α Escherichia coli. In the bacterial cells, recombinant host cells with Escherichia coli cells as the host were obtained. After a single colony grew on the plate, the single colony was picked and sequenced to obtain the correctly sequenced pCAMBIA2300-pActin-DUP3-Flag overexpression vector, which was transformed into the Agrobacterium tumefaciens strain AGL1 (Beijing Bomade Gene Technology Co., Ltd.) to obtain recombinant host cells with Agrobacterium as the host. After a single colony grew on the plate, the callus tissue of the indica rice variety HHZ was infected with Agrobacterium to obtain the homozygous overexpression strain HHZpActin::DUP3-Flag. Phenotypic analysis showed that overexpression of DUP3 can significantly reduce the ineffective tillering of HHZ ( Figure 4 B), increase the effective number of HHZ spikes ( Figure 4 C), ultimately increasing the yield per plant ( Figure 4 D). Therefore, DUP3 can reduce ineffective tillers and increase the number of effective ears and yield.

[0101] Example 5: The "Green Revolution" gene sd1 inhibits gene expression of DUP3

[0102] To investigate the effect of the "Green Revolution" gene sd1 on the DUP3 gene, three pairs of near-isogenic lines, HJX74-sd1 and HJX74-SD1, NJ6-sd1 and NJ6-SD1, and 9311-sd1 and 9311-SD1, were cultured in a greenhouse. The expression of the DUP3 gene was identified in the aerial parts of the three-week-old plants. RNA was extracted from the aerial parts of the three-week-old plants using the Trizol method and reverse transcribed into cDNA. The expression of DUP3 was identified by real-time quantitative PCR (qRT-PCR) using actin as an internal reference.

[0103] The results showed that the expression levels of DUP3 in SD1 types under different backgrounds were higher than those in sd1 types under the corresponding rice backgrounds. This indicates that the "Green Revolution" gene sd1 leads to a decrease in the expression level of the DUP3 gene ( Figure 5 ).

[0104] Example 6: DUP3 has obvious indica-japonica differentiation

[0105] According to the filtering criteria of minimum allele frequency greater than 0.05 and genotype missing rate less than 0.02, the inventors extracted SNPs located in the promoter region of DUP3 (2 kb upstream of the start codon ATG) and non-synonymous mutations located in the coding region, and found that most of the SNPs of the gene exist in the promoter region, and only one non-synonymous mutation exists in the coding region, which is aspartic acid mutated to glycine from the reference genome. Using these SNPs for haplotype analysis of DUP3, it was found that the gene is mainly divided into three haplotypes, and shows obvious indica-japonica difference. Haplotype 1 (Hap. 1) only exists in tropical japonica and temperate japonica, haplotype 2 (Hap. 2) mainly exists in indica and a small number of AUS subgroup varieties, and haplotype 3 (Hap. 3) exists in AUS and indica varieties Figure 6 A). Significant difference analysis of the phenotypes of rice varieties carrying different haplotypes showed that the rice varieties carrying Hap. 1 had a significantly higher earing rate than the rice varieties carrying Hap. 2 or Hap. 3. Therefore, DUP3 has obvious indica-japonica differentiation, and japonica varieties carrying haplotype Hap. 1 (also referred to as DUP3 Japonica , the CDS sequence of which is shown as SEQ ID NO: 4 or the genomic nucleotide sequence of which is shown as SEQ ID NO: 2) have a higher earing rate Figure 6 B).

[0106] Example 7: Allele DUP3 Japonica Reducing invalid tillers and improving crop yield

[0107] The inventors used indica variety HJX74 (carrying haplotype Hap. 2) as the receptor parent and japonica (carrying haplotype Hap. 1) as the donor parent for foreground selection and background selection. Among them, the foreground selection marker uses the published genomes of Nipponbare and 9311 to develop specific differential markers near the DUP3 interval, and the genomes of HJX74 and japonica are verified. Those markers that show differences between the genomes of HJX74 and japonica are used as foreground selection markers, and the specific sequences are SEQ ID NO. 11 and SEQ ID NO. 12. The background selection marker selects simple sequence repeat molecular markers (Simple Sequence Repeat, SSR) from the national rice database (see Guo Xia, Qin Wanglan, Zhang Guiquan, 2014), and the HJX74 rice material HJX74-DUP3 derived from haplotype 1 (japonica) is obtained by successive backcrossing combined with molecular marker assisted selection.Japonica ( Figure 7 A). The specific steps of the generation-by-generation backcrossing combined with molecular marker-assisted selection are briefly described as follows: first, the two parents are hybridized to obtain the first generation of hybrids, genomic DNA of individual plants of the first generation of hybrids is extracted, true hybrids are determined using foreground selection markers, true hybrid plants are selected and backcrossed with HJX74 to obtain the first generation of backcrosses, genomic DNA of all backcross plants is extracted, the genotypes of the foreground selection markers and the background selection markers are identified, and individual plants showing the HJX74 genotype for most of the background selection markers and the japonica genotype for the foreground selection markers (SEQ ID NO.11 and SEQ ID NO.12) are selected, and backcrossed with HJX74 to obtain the second generation of backcrosses, and the above steps are repeated until individual plants showing the HJX74 genotype for most of the genome and the japonica genotype in the DUP3 region are selected as HJX74-DUP3 Japonica 10 non-side row plants were selected for field phenotypic statistics. The results showed that compared with HJX74, HJX74-DUP3 Japonica The number of ineffective tillers decreased by about one on average ( Figure 7 C), the number of effective ears increased by about 2, ( Figure 7 D), the ears became larger, and the number of grains per ear increased from about 240 to about 260 ( Figure 7 E), the yield per plant increased from 38g to about 43g ( Figure 7 F). Therefore, DUP3 Japonica It can reduce ineffective tillering and has the potential to increase yield.

[0108] Example 8: DUP3 Japonica Reduce ineffective tillering of crops, increase crop yield and nitrogen use efficiency

[0109] The inventors combined HJX74 and HJX74-DUP3 Japonica Planted in the field with different urea application rates (60kg / ha, 120kg / ha, 210kg / ha and 300kg / ha). The results of field agronomic trait analysis showed that HJX74-DUP3 Japonica The number of ineffective tillers of HJX74 was lower than that of HJX74 ( Figure 8 B) The yield per plant was higher than that of HJX74 ( Figure 8 C), especially at lower nitrogen rates, HJX74-DUP3 Japonica The yield-increasing effect is more obvious. Japonica It has the potential to reduce ineffective tillering and further reduce weight and increase yield.

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[0130] Sequence Listing

[0131]

[0132] SEQ ID No. 1 gDNA sequence of DUP3 (derived from HJX74)

[0133] gttcatcgtctccatctcctgcttgctcttggtagctagctagtggagatcgccggagaggagagagacgacgagagagagagaggggg

[0134] caagatggcgggagggttctcggtgtcggggtccggggtggagttcgaggcgaagatcacgccgatcgtcatcatctcctgcatcatggcg

[0135] gccaccggcggcctcatgttcggctacgacgtcggtatctcaggtatatctacgcacggccggccgccagcgatgtccgaacatggccat

[0136] ggccggctttgatgtccttctgttgattagttggttcatctcgattattcatattggtgtctcctgcttgaaattgagtgagattcctcttgcacgtgtgt

[0137] gttatcggtttgatctgtctacatatttgtgttatcttgctatggataagcacacataagcgcaaagattaaagcgaaacggatgattttatccttgtc

[0138] attaaatacctaccatttacagaggatcaaaaaagatcttgccattttattgagtccatttggtgcaattttatcctttttcattagtttccggcttgtagt

[0139] GCTCCTACAGTCCAGCTAGTAGTATGATTTTCTTGAGGATCCCAAAGCATTAAAATTTTAGATGTTATTTTAGAGAAGGAATCGAGTGTATGAGCTA

[0140] GCCACCTGCTTTGACAGGCTGGTAAGTCAATGCATTTGTGTTATCCACGGATCACGTTTCTTGC AACTCTCTCAGCTTTCTTTCTTAATT TTGTGTGGCCA

[0141] TGTTCCCTTTTTACATCTAAGCCAATAAATTCCAATTAAAACGGAAAAAAAATCTTATCCACTCTCTGATTCAACCTTTTCCATGGC GCCTAACACAA

[0142] CATTTTAAAACATTCCTAGTTCTATACCTATACATGCCATTTTTGCCACAATAGATGACGAAC TTTACGACTTATGAAACTAAATAATATCCGTTGAAAAT

[0143] CTTGTTAATTTCCTTTTCTTGGTTAGGCAGAGTGACATCGATGGATGACTTCCTGC GCGAGTTCTTCCC GACG GTGCTGAAGAAGACG CAGG

[0144] ACAAGGAGAGCAACTACTGCAAGTATGACAACCAGGGCCTGCAGCTGTTCA CCTCGTCGCTGTACCTCGCCGGCCTCACGGCCACCTTCTTCG

[0145] CCTCCTACACCAC TCGCCGGCTCGGCCGCCGCCTCACC ATGCTCATCGCCGGCGTCTTCTTCTATCGTCGGCGT CATCTTCAACGGCGCCGCCC

[0146] AGAACCCTGCCATGCTCATAGTCGGCAGGATCCTGCTCGGCTGTGGCGTCGGCTTCG CCAACCAGCAAGCAACTTTCCAATAATCTTATCGTT

[0147] acaggcttacagcattatatcatttcatataataagctatggagttaaacaaagtcattacgccttatactattgttttcactcataatttgcacggata

[0148] tttccatgttccttttttcgtcctgtactttcttcgcaacatcatcttctttgattcatatattattgactagtactaataataaactgatactactcacttgag

[0149] tccaaaagtcttagcacagagtttaagtcaaacatataaagcggtggctggcttgagaatttgcatcacaagcaagccgaattttctgcttccatg

[0150] aaaacgacgtgcgttcctgcctgcctaatactcctatatgaaccaccgtcctgattagaatttttatactcgggtcgagcatatacattactaaaaat

[0151] tatatttttatacgtatttcttatggcgtcggatactggggtagttagtactgtacttaaacacggggccctgtgtaattgccgtaatttggggtcaa

[0152] acgcgtagaagtatgggcatatatatacatggatttatggacacctcggccggtgccgagagagtcttgatcgtgcatgacttgtgttcgtctac

[0153] gttgacatcttccattggcagcagcatgcgtgtgataaactttttaagttccaagattccgatgtcctcgtgactctcctagtttaggattagccacg

[0154] cttcttaattaatccttactgaacctggctaaattacccatttttgttgtccaagtttgtagtcgagacgtctgaagaagttgcagccaaccaagag

[0155] gcgataggtgtgcatgattttacagccggtagttttgcgtgtggctgcctgctatctggccgtcgcagttgttatcaacggaggaatcgacctgtg

[0156] tagtgtagcaacagacttgtgtttgtcacgtttgttggagattggtcagctttgttgactgatctttacagcaactgctagctagttacagccacaac

[0157] tgtcaattcctcaatggatgcagcttggaatacacacatgctgttgcagctttggcttgctctggtttgatttttaagcagaggctgctatggctgtc

[0158] cacgcatatgccacacgggtcgcaatatgtagacctttggttcactgagttttgggctgcaaagtactagttggctgctagtgtgcttgattggta

[0159] ggagagtaggagtagtacttaatttgcttggcttcttctgaatttattcgtgtctaatagatgaaacagtagtttacatattacgcacactcgccaac

[0160] atatgggacgtctagactcaattatgaagctgccaagacaataaactaaatatgcaagaggtttagtacaggacagagttcatgtgatcagttatt

[0161] cagtcaccacctaacaagactttgatggaggcctaggttcaacaaacattagagtggtagcagcagtcagcagacactatcgtactcgtagta

[0162] gtagtctaatactaccatatgtacaactactacggtttgttaattccacctctaccgatcagcccccttgattatggttaagataacaaaaactatctt

[0163] Ctctgaataacaaaactatctacataattcaagggtcatgccgtattgccatgtgattaggtattaggttcaacgcctaacctagcttgggttgttatt

[0164] Atactcagaaatacgcacggggaatttattggcagagttggtgcctttgtaagtttattattactactgaatgatgagctaggtatctgcacgctaa

[0165] Acaaaggcgaatcaagtgtcactttgggcctaatgggcctcgtcaagcccatctgaatttaataacagaggtgaatacgtgcaggccgttccat

[0166] Tgttcctgtcggagatcgcgccgacgaggatccgcggcgggctcaacatcctcttccagctcaacgtcaccatcggcatcctcttcgccaacc

[0167] Tcgtcaactacggcaccgccaagtacgtcacacatcatcatacacacacattcagatacaaatgcaatgcattgtacgtatgatgatgaactgac

[0168] Atgagctgcatcattttacgtacgcatgcaggatccacccgtggggatggcggctgtcgctgtcgctggccggcatcccggcggcgctgctg

[0169] Acgctgggcgcgctgttcgtggtggacacgcccaacagcctgatcgagcgcgggaggctggaggaagggaaggcggtgctgaggaaga

[0170] Tccgggggacggacaacgtggagccggagttcaacgagatcgtggaggcgagccgcgtggcgcaggaggtgaagcacccgttccgcaa

[0171] CCTGCTCCAGCGCCGGAACCGGCCGCAGCTGGTGATCGCCGTGCTGCTGCAGATATTC CAGCAGTTCACGGGCATCAACGCCATCATGTTCTA

[0172] CGCGCCC GTGCTGTTCAACACGCTCGGTTCAAGACC GACGC GTCGCTCTACTCCGCGGTGATCACGGGGGCCGTGAACGTGCTGTCCACG

[0173] CTGGTGTCCGTCTACTCGGTGGACAGGGTGGGGAGGCGGATGCTGCTGCTGGAGGCGGC GTGCAGATGTT CCTGT CGCAGGTGGCCATCG

[0174] CCGTCGTGCTGGGGATCAAGGTGACGGACC GGT CGGACAACCTCGGCCACGGGTGGGC GATCATGGTG GTGGTGATGGTGTGCACGTT CGT

[0175] GTCCTCCTTCGC GTGGTCGTGGGGCCCTCTCGGTTGGCTCATCCCCAGCGAGACGTTCCCGCTGGA GACGCGGTCGGCGGGCAGAGCGTC

[0176] ACC GTGTGC GTCAACCTGCTCTTCACCTTCGT CATCGCGCAGGCGTTCCTCTCCATGCTCTGCCACCTCAAGTACGCCATCTTCGCCTTCTTCTCCG

[0177] C GTGGGTCGTCGT CATGTCGCTCTTCGTGCTCTTCTTCCTGCCCGAGACCAAGAACATCCCCATCGAGGAGATGACCGAGAGGGTGTGGAAGC

[0178] AGC ACTGGTTCTGGAAGCGCTT CATGGACGGCGCCGACAACACCACGTCGTCCCCAATGGCGGSAAGAGCAACGGCGCCACCGTTTAATT

[0179] tagtcgctgaactgttctatatatatgtcactgtctcactgatcaaatcacggttgagaaactacaccgatgctatgtatcagctcatgcgtggcca

[0180] tgtgagattgatcgataagcagctagcgacggcatgcaaacttaagggtagtgtagcaatttcggctctgtacgtactgcaagcaatggcgagt

[0181] atttcaattcggagtgcttcttgagtttgtagtttgttgtatgagttgtatccttttatgtttctccgagcaagcctgacacgagttggcgcctcagtgt

[0182] aacttgcagaacagaatgccatatatctgcacctttttttgcc

[0183] SEQ ID No.2DUP3 Japonica 的gDNA序列(来源于粳稻)

[0184] gttcatcgtctccatctcctgcttgctcttggtagctagctagtggagatcgccggagaggagagagacgacgagagagagagagaggggg

[0185] caagatggcgggagggttctctgtgtcggggtccggggtggagttcgaggcgaagatcacgccgatcgtcatcatctcctgcatcatggcgg

[0186] ccaccggcggcctcatgttcggctacgacgtcggtatctcaggtatatatctacgcacggccggccgccggcgatgtccgaacatggccatg

[0187] gccggctttgatgtccttctgttgattagttggttcatctcgattattcatattggtgtctcctgcttgaaattgagtgagattcctcttgcacgtgtgtgt

[0188] TATCGGT TTGATCTGTCT ACATATT TGTGTTATCTTGCTATGGATAAGCACATATAAGCGCAAAGATTA AAGCGAAACGGATGATTTTATCCTTGTCA TT

[0189] AAATACCTACCATT TACAGAGGATCAAAAAAGATCTTGCCATTTTATTGAGTCCATTTGGTGCAATTTTATCCTTTTTCA TTAGTTTCCGGCTTGTA GTGCT

[0190] CCTACAGTCCAGCTAGTAGTATGATTTTCTTGAGGATCCCAAAGCATTAAAATTTTAGATGTTATTTTAGAGAAGGAATCGAGTGTATGAGCTAGC

[0191] CACCTGCTTTGACAGGCTGGTAAGTCAATGCATTTGT TTATCCACGGATCACGTTTCTTGCAACTCTCTCAGCTTTCTTTCCTAATTTTGTGTGGCCATGT

[0192] TCCCTTTTTACATCTAAGACAATAAATTCCAATTAAAACGGAAAAAAAATCTTATCCACTCTCTGATTCAACCTTTTCCATGGC GCACTAACACAACAT

[0193] TTTAAAACATTC C AAGTT C ATACTT AT AC ATGCC ATTTTTTGCCACAATAGATGACGAAC TTTACGACTTATGAAACTAAATA TATCCGT T GAA AATCTT

[0194] GTGAATTTCTTTTTCTTGGTTAGGC GGAGTGACATCGATGGATGACTTCCTGC GCGAGTTCTTCCC GACGGTGCTGAAGAAGAAGCACGAGGACA

[0195] AGGAGAGCACTACTGCAAGTATGACAACCAGGGCCTGCAGCTGTT CACCTCGTCGCTGTACCTCGCCGGCCTCACGGCCACCTTCTTCGCCT

[0196] CCTACACCAC TCGCCGGCTC GGCCGCCGCC TCACC ATGCTCATCGCCGGC GTCTTCTTCA TCgtcggcgtcatcttcaacggcgccgcccaga

[0197] ACCTCGCCAT GCTCATAGTC GGCAGGATCC TGCTCGGCTG TGGCgtcggctttgccaaccaggcaagcaactttccaataatcttatcgttacag

[0198] GCTTACAGCA TTATATCATT Tcatataataagctatggagttaaacaaagtcattacgccttatactattgttttcactcataatttgcacggatatttcc

[0199] ATGTTCCTTTTTTCGTCCTGTACTTTCTTCGCAACATCATCTTCTTTGATTCCATATATTATTGACTAGTACTAATAAACTGATACTACTC ACTTGAGTCCA

[0200] AAAGTCTTAGCACAGAGTTTAAGTCAACATATAAAGCGGTGGCTGGTTTGAGAATTTGCA TCACAAGCAAGCCGAATTTCTGCTTCCATGAAAA

[0201] CGACGTGCGTTCCTGCCTGCCTAATACTCCTATATGAACCACCgtcctgattagaatttttatactcgagtcgagcatatacattactaaaaattatat

[0202] TTTTTACGTATTTCttatggcgtcggatactggggtagttagtactgtacttaaacacggggcccttcgtaattgccgtaattttggggtcaaacgc

[0203] GTAGAAGTATGGGCATATATATACATGGATTTATGGACACCTCGGCCGgtgccgagagagtcttgatcgtgcatgacttgtgttcgtctacgttga

[0204] Catcttccattggcagcagcatgcgtgtgataaactttttaagttccaagattccgatgtcctcgtgactctcctagtttaggattagccacgcttctt

[0205] Aattaatccttactgaacctggctaaattacccattttttgttgtccaagtttgtagtcgagacgtctgaagaagttgcagccaaccaagaggcgat

[0206] Aggtgtgcatgattttacagccggtagttttgcgtgtggctgcctgctatctggccgtcgcagttgttatcaacggaggaatcgacctgtgtagtg

[0207] Tagcaacagacttgtgtttgtcacgtttgttggagattggtcagctttgttgactgatctttacagcaacttctagctagttacagccacaactgtcaa

[0208] Gtcctcaatggatgcagcttggaatacacacatgctgttgcagctttggcttgctctggtttgatttttaagcagaggctgctatggctgtccacgc

[0209] Atatgccacacgggtcgcaatatgtagacctttggtttggttcactgagttttgggctgcaaagtactagttggctgctagtgtgcttgattggtag

[0210] Gagagtaggagtagtacttaatttgcttggcttcttctgaatttattcgtgtctaatagatgaaacagtagtttacatattacgcacactcgccaacat

[0211] Atgggacgtctagactcaattatgaagctgccaagacaataaactaaatatgcaagaggtttagtacaggacagagttcatgtgatcagttattc

[0212] agtcaccacctaacaagactttgatggaggcctaggttcaacaaacattagagtggtagcagcagtcagcagacactatcgtactcgtagtagt

[0213] agtctaatactaccatatgtacaactactacggtttgttaattccacctctaccgatcagcccccttgattatggttaagataacaaaaactatcttct

[0214] ctgaataacaaaactatctacataattcaagggtcatgccgtattgccatgtgattaggtattaggttcaacgcctaacctagcttgggttgttattat

[0215] actcagaaatacgcacggggaatttattggcagagttggtgcctttgtaagtttattattactattgaatgatgagctaggtatctgcacgctaaac

[0216] aaaggcgaatcaagtgtcactttgggcctaatgggcctcgtcaagcccatctgaatttaataacagaggtgaatacgtgcaggccgttccattgt

[0217] tcctgtcggagatcgcgccgacgaggatccgcggcgggctcaacatcctcttccagctcaacgtcaccatcggcatcctcttcgccaacctcg

[0218] tcaactacggcaccgccaagtacgtcacacatcatcatacacacacattcagatacaaatgcaatgcattgtacgtatgatgatgaactgacatg

[0219] agctgcatcattttacgtacgcatgcaggatccacccgtggggatggcggctgtcgctgtcgctggccggcatcccggcggcgctgctgacg

[0220] ctgggcgcgctgttcgtggtggacacgcccaacagcctgatcgagcgcgggaggctggaggaagggaaggcggtgctgaggaagatcc

[0221] gggggacggacaacgtggagccggagttcaacgagatcgtggaggcgagccgcgtggcgcaggaggtgaagcacccgttccgcaacct

[0222] gctccagcgccggaaccggccgcagctggtgatcgccgtgctgctgcagatattccagcagttcacgggcatcaacgccatcatgttctacg

[0223] cgcccgtgctgttcaacacgctcgggttcaagaccgacgcgtcgctctactccgcggtgatcacgggggccgtgaacgtgctgtccacgctg

[0224] gtgtccgtctactcggtggacagggtggggaggcggatgctgctgctggaggccggcgtgcagatgttcctgtcgcaggtggccatcgccg

[0225] tcgtgctggggatcaaggtgacggaccggtcggacaacctcggccacgggtgggcgatcatggtggtggtgatggtgtgcacgttcgtgtc

[0226] ctccttcgcgtggtcgtggggcccgctcgggtggctcatccccagcgagacgttcccgctggagacgcggtcggcggggcagagcgtcac

[0227] cgtgtgcgtcaacctgctcttcaccttcgtcatcgcgcaggcgttcctctccatgctctgccacctcaagtacgccatcttcgccttcttctccgcg

[0228] TGGGTCGTCG TCGATGTCGC TCTTCGTGCT CTTCTTCCTG CCCGAGACCA AGAACATCCC CATCGAGGAG ATGACCGAAG GGCGTGGAAG CAG

[0229] CCTGTTTCTG GAGCGCTTC ATGGACGACG CCCTCAAACA CCACGTGGCC CCAATGGCGG AAGAGCAACG GCACCGTTTA ATTTAC

[0230] GTCGCTGAAC TGTTCCTATA TATGTCACTG TCTCCTGATC AAATCACCGT TGAGAACTAC ACCGATGCTA TGTATCAGCT CATGCCTGGC CATG

[0231] TGAGATTGAT CGAAGCAGCT AGCGACGGCA TGCAAACCTA AGGGTAGTGT AGCAATTTTC GGCTCTGTAC GTACTGCAAG CAATGGCGAGT AT

[0232] TTCAATTCGG AGTGCTTCTT GAGTTTGTTG TTGTTGTATG AGTTGTATCC TTTTATGTTC TCCGAGCAAG CCTGACACGA GTTGGCGCCT CAGTGTAAT

[0233] CTTGCAGAAC AGAATGCCAT ATATCTGCAC CTTTTTTGCC

[0234] SEQ ID No. 3 CDS sequence of DUP3 (derived from HJX74)

[0235] ATGGCGGGAG GTTCTCGGTG TCGGGGTCCG GGGTGGAGTT CGAGGCAGAA GATCACGCCG ATCGTCATCA TCTCCTGCAT CATGGCGGCC A

[0236] CCGGCGGCCT CATGTTCGGC TACGACGTCG GTATCTCAGG CGGAGTGACA TCGATGGATG ACTTCCTGCG CGAGTTCTTC CCACGGTGCT GA

[0237] agaagaagcacgaggacaaggagagcaactactgcaagtatgacaaccagggcctgcagctgttcacctcgtcgctgtacctcgccggcct

[0238] cacggccaccttcttcgcctcctacaccactcgccggctcggccgccgcctcaccatgctcatcgccggcgtcttcttcatcgtcggcgtcatc

[0239] ttcaacggcgccgcccagaacctcgccatgctcatagtcggcaggatcctgctcggctgtggcgtcggcttcgccaaccaggccgttccatt

[0240] gttcctgtcggagatcgcgccgacgaggatccgcggcgggctcaacatcctcttccagctcaacgtcaccatcggcatcctcttcgccaacct

[0241] cgtcaactacggcaccgccaagatccacccgtggggatggcggctgtcgctgtcgctggccggcatcccggcggcgctgctgacgctggg

[0242] cgcgctgttcgtggtggacacgcccaacagcctgatcgagcgcgggaggctggaggaagggaaggcggtgctgaggaagatccggggg

[0243] acggacaacgtggagccggagttcaacgagatcgtggaggcgagccgcgtggcgcaggaggtgaagcacccgttccgcaacctgctcca

[0244] gcgccggaaccggccgcagctggtgatcgccgtgctgctgcagatattccagcagttcacgggcatcaacgccatcatgttctacgcgcccg

[0245] tgctgttcaacacgctcgggttcaagaccgacgcgtcgctctactccgcggtgatcacgggggccgtgaacgtgctgtccacgctggtgtcc

[0246] gtctactcggtggacagggtggggaggcggatgctgctgctggaggccggcgtgcagatgttcctgtcgcaggtggccatcgccgtcgtgc

[0247] tggggatcaaggtgacggaccggtcggacaacctcggccacgggtgggcgatcatggtggtggtgatggtgtgcacgttcgtgtcctccttc

[0248] gcgtggtcgtggggccctctcgggtggctcatccccagcgagacgttcccgctggagacgcggtcggcggggcagagcgtcaccgtgtgc

[0249] gtcaacctgctcttcaccttcgtcatcgcgcaggcgttcctctccatgctctgccacctcaagtacgccatcttcgccttcttctccgcgtgggtcg

[0250] tcgtcatgtcgctcttcgtgctcttcttcctgcccgagaccaagaacatccccatcgaggagatgaccgagagggtgtggaagcagcactggtt

[0251] ctggaagcgcttcatggacggcgccgacaaacaccacgtcgtccccaatggcgggaagagcaacggcgccaccgtttaa

[0252] SEQ ID No.4DUP3 Japonica 的CDS序列(来源于粳稻)

[0253] atggcgggagggttctctgtgtcggggtccggggtggagttcgaggcgaagatcacgccgatcgtcatcatctcctgcatcatggcggccac

[0254] cggcggcctcatgttcggctacgacgtcggtatctcaggcggagtgacatcgatggatgacttcctgcgcgagttcttccccgacggtgctgaa

[0255] gaagaagcacgagcaaggagagcaactactgcaagtatgacaaccagggcctgcagctgttcacctcgtcgctgtacctcgccggctc

[0256] acggccaccttcttcgctcctacaccactcgccggctcggccgccgcctcaccatgctcatcgccggcgtcttcttcatcgtcggcgtcatctt

[0257] caacggcgccgcccagaacctcgccatgctcatagatcggcaggatcctgctcggctgtggcgtcggctttgccaaccaggccgttccattgtt

[0258] cctgtcggagatcgcgccgacgaggatccgcggcgggctcaacatcctcttccagctcaacgtcaccatcggcatcctcttcgccaacctcgt

[0259] caactacggcaccgccaagatccacccgtggggatggcggctgtcgctgtcgctggccggcatcccggcggcgctgctgacgctggggcg

[0260] cgctggttcgtggtggacacgcccaacagcctgatcgagcgcgggagggctgagaagggaagggcggtgctgaggaagatccggggac

[0261] ggaacgtggagccggagttcaacgagatcgtggaggcgagccgcgtggcgcaggaggtgaagcaccccgttccgcaacctgctccagc

[0262] gccggaaccggccgcagctggtgatcgccgtgctgctgcagatattccagcagttcacgggcatcaacgccatcatgttctacgcgcccgtg

[0263] ctgttcaacacgctcgggttcaagaccgacgcgtcgctctactccgcggtgatcacgggggccgtgaacgtgctgtccacgctggtgtccgt

[0264] ctactcggtggacagggtggggaggcggatgctgctgctggaggccggcgtgcagatgttcctgtcgcaggtggccatcgccgtcgtgctg

[0265] gggatcaaggtgacggaccggtcggacaacctcggccacgggtgggcgatcatggtggtggtgatggtgtgcacgttcgtgtcctccttcgc

[0266] gtggtcgtggggcccgctcgggtggctcatccccagcgagacgttcccgctggagacgcggtcggcggggcagagcgtcaccgtgtgcg

[0267] tcaacctgctcttcaccttcgtcatcgcgcaggcgttcctctccatgctctgccacctcaagtacgccatcttcgccttcttctccgcgtgggtcgt

[0268] cgtcatgtcgctcttcgtgctcttcttcctgcccgagaccaagaacatccccatcgaggagatgaccgagagggtgtggaagcagcactggtt

[0269] ctggaagcgcttcatggacgacgccgacaaacaccacgtcgtccccaatggcgggaagagcaacggcgccaccgtttaa

[0270] SEQ ID No.5DUP3的氨基酸序列(来源于HJX74)

[0271] MAGGFSVSGSGVEFEAKITPIVIISCIMAATGGLMFGYDVGISGGVTSMDDFLREFFPTVLK

[0272] KKHEDKESNYCKYDNQGLQLFTSSLYLAGLTATFFASYTTRRLGRRLTMLIAGVFFIVGVIF

[0273] NGAAQNLAMLIVGRILLGCGVGFANQAVPLFLSEIAPTRIRGGLNILFQLNVTIGILFANLV

[0274] NYGTAKIHPWGWRLSLSLAGIPAALLTLGALFVVDTPNSLIERGRLEEGKAVLRKIRGTDN

[0275] VEPEFNEIVEASRVAQEVKHPFRNLLQRRNRPQLVIAVLLQIFQQFTGINAIMFYAPVLFNTL

[0276] GFKTDASLYSAVITGAVNVLSTLVSVYSVDRVGRRMLLLEAGVQMFLSQVAIAVVLGIKVT

[0277] DRSDNLGHGWAIMVVVMVCTFVSSFAWSWGPLGWLIPSETFPLETRSAGQSVTVCVNLL

[0278] FTFVIAQAFLSMLCHLKYAIFAFFSAWVVVMSLFVLFFLPETKNIPIEEM

[0279] TERVWKQHWFWKRFMDGADKHHVVPNGGKSNGATVSEQ ID No. 6DUP3 Japonica Amino acid sequence of (derived from japonica)

[0280] MAGGFSVSGSGVEFEAKITPIVIISCIMAATGGLMFGYDVGISGGVTSMDDFLREFFPTVLK

[0281] KKHEDKESNYCKYDNQGLQLFTSSLYLAGLTATFFASYTTRRLGRRLTMLIAGVFFIVGVIF

[0282] NGAAQNLAMLIVGRILLGCGVGFANQAVPLFLSEIAPTRIRGGLNILFQLNVTIGILFANLV

[0283] NYGTAKIHPWGWRLSLSLAGIPAALLTLGALFVVDTPNSLIERGRLEEGKAVLRKIRGTDN

[0284] VEPEFNEIVEASRVAQEVKHPFRNLLQRRNRPQLVIAVLLQIFQQFTGINAIMFYAPVLFNTL

[0285] GFKTDASLYSAVITGAVNVLSTLVSVYSVDRVGRRMLLLEAGVQMFLSQVAIAVVLGIKVT

[0286] DRSDNLGHGWAIMVVVMVCTFVSSFAWSWGPLGWLIPSETFPLETRSAGQSVTVCVNLL

[0287] FTFVIAQAFLSMLCHLKYAIFAFFSAWVVVMSLFVLFFLPETKNIPIEEMTERVWKQHWFW

[0288] KRFMDDADKHHVVPNGGKSNGATV

[0289] SEQ ID NO. 7 DUP3-CRISPR-F1

[0290] CATCATCTCCTGCATCATGGGTTTTAGAGCTAGAAAT

[0291] SEQ ID NO. 8 DUP3-CRISPR-R1

[0292] CCATGATGCAGGAGATGATGCGGCAGCCAAGCCAGCA

[0293] SEQ ID NO. 9 DUP3-F1

[0294] ACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGGAGGGTTCTCTGTG

[0295] SEQ ID NO. 10 DUP3-R1

[0296] ACCACTTTGTACAAGAAAGCTGGGTCTTAAACGGTGGCGCCGTTGC

[0297] SEQ ID NO. 11 DUP3-CSSL-F

[0298] GAACACTTACGTTTGTCACTC

[0299] SEQ ID NO. 12 DUP3-CSSL-R

[0300] AGGTGTCTAATCCGTTGCTTG

[0301] SEQ ID NO. 13 DUP3 Promoter

[0302] tgcatgaaaccgacaccgattacgtgcgcgcgcggacttggactgcatacggctggtattcgagtgttccaagtccggggaatagtactgctc

[0303] cacgcgaaggatttctcgcgaaatcgtcacgtaccttacggtggcaactcgtgtatgcagcgcccaaggatcgagctgtgatcaatcgtgcac

[0304] gcgtcaagcgtcagtgaaagaaattccatggaaattttcaccatgttaattcgtgaggcttgagtacatttgtcggtgtgtacacaatcagttcatg

[0305] gtcgatcaagtggagtagttccatccggtaagcgtcaaacatcgtatcacagcacgattccgacgacaaggagttcacccagttcgatcggctt

[0306] acgtgaaaattcccataggcacggagagtacgtacgtgcagattgtaagttgcaaaacggtgccaaaattcaaatcatctaccgaacgtgacg

[0307] tgatgtttgcgcaacacgtgtgcgtggatattaccagatttttggctttgaatattagttgatcgatctgacatgaccacttgactatgatacggttgt

[0308] atgaactcaacaagagtgagagcgtgcgaatatttttcgatgttcaattcgttgatatgcagagaagaacgaagcaaaattaaatttgtgtagaca

[0309] taggtacggaggttatttggagagtgactggccgttagaccacacgtttgttaattcgtgttcacggtttttttttttgttctttctcatgaactacacat

[0310] ggctgattaacggatacctattaattattactagcaactgtgtccggtactacgtacgtatatcttatacaatcatatcttaatgagaatcacgagta

[0311] gatcaaatccaataacaatacaaagatgactaaactagcagctcttgggcaagaatagcggcggctaatcaccactgcttgtgttctgattaaac

[0312] gccgtcatctacactagctactactaatatagtactatgggaatgtttaggtgcagatgtgtaaagttttagcgtgtcgcatcgggtattatatagag

[0313] tgttatatggggtgttcgatcactaataaaaaaactaattacagtatccgtcagtaaactgcgagatgaatttactaagcctaattgatcagtcatta

[0314] gtacatgtttactgtagcaccatattgtcaaaccatgaagcaattaggtttaaaagatccgtctcgcaaagtagtcgaaatctatgcaattagttatt

[0315] tttttctatatttaatacttcatgcaagtgtttaaacgtttcatgtgatatggtgtaaaattttggggtgtgatctaaacagaccctatgataccacgttta

[0316] ctagaaggaaaatattacaagctaggtaagccgactagccgaactagaagtcattttctttagctcgcattttccacagaaccacaatttcccaag

[0317] taatagtactacactactactccaccatgtccatcgcacatgttgcttaattttccttagcaaatattcaagtcaaggtcctccattcacatgatcagc

[0318] tcgccaaagattcaagtgagcatcaccgtacagtaccaagacagcaaccagcgtcaaggcactgacatgcgggcccgcctaacgccagga

[0319] atcaatactgtagtaccgtacatccagcgtcaaaaaggccggcgttcaaacttcaatatatccaactatatatccaaatttccaaactgctgctcct

[0320] acttgctctctccctcttttccaaaaggagctcaaaaagctttaaaaaaaggttttaatttggataagcaaaaaaaattaaagtgggatttgaaaag

[0321] tcttgtgctgcttatgcaacagtcgcagacttgcggcacggccagcctccggcccgtgggccgccgggaaagccgtcgctgaccggccgg

[0322] ctagccagcccgggaggcgacccggcggtgggtcccacctatgccacgtggccgcatcctgcctctccgatgcgtttgaaatgtctcccccc

[0323] gtgtgatcgggctcccactccctctctatatatagcgagcggcttggagccctgtga

[0324] SEQ ID NO.14DUP3 Japonica 启动子

[0325] tgcatgaaaccgacaccgattacgtgcgcgcgcggacttggactgcatacggctggtattcgagtggctggtattcgagtgttccaagtccgg

[0326] ggaatagtactgctccacgcgaaggatttctcgcgaaatcgtcacgtaccttacggtggcaactcgtgtatgcagcgcccaaggatcgagctg

[0327] tgatcaatcgtgcacgcgtcaagcgtcagcgaaagaaattccatggaaattttcaccatgttaattcgtgaggcttgagtacatttgtcggtgtgt

[0328] acacaatcagttcatggtcgatcaagtggagtagttccatccggtaagcgtcaaacatcgtatcacagcacgattccgacgacaaggagttca

[0329] cccagttcgatcggcttacgtgaaaattcccataggcacggagagtacgtacgtgcagattgtaagttgcaaaacggtgccaaatattagttgat

[0330] cgatctgacatgaccacttgactatgatacggttgtatgaactcaacaagagtgagagcgtgcgaatatttttcgatgttcaattcgttgatatgca

[0331] gagaagaacgaagcaaaattaaatttgtgtagacataggtacggaggttatttggagagtgactggccgttagaccacacgtttgttaattcgtg

[0332] ttcacggttttttttttttgttctttctcatgaactacacatggctgattaacggatacctattaattattactagcaactgtgtccggtactacgtacgtat

[0333] atcttatacaatcatatcttaatgagaatcacgagtagatcaaatccaataacaatacaaagatgactaaactagcagctcttgggcaagaatag

[0334] cggcggctaatcaccactgcttgtgttctgattaaacgccgtcatctacactagctactactaatatagtactatgggaatgtttagatgcagaagt

[0335] gtaaagttttagcgtgtcgtatcgggtattatatagggtgttatatggggtgttcgatcactaataaaaaaactaattacagtatccgttagtaaact

[0336] gcgagacgaatttactaagactaattgatcagtcattagcacatgtttactgtagcaccatattgtcaaaccatgaaacaattaggtttaaaagatc

[0337] tgtctcgcaaagtagtcgaaatctatgcaattagttattttttttctatatttagtactttatgcaagtgtttaaacgtttcatgtgatatggtgtaaaatttt

[0338] ggggtgtgccctatgataccacgtttactagaaggaaaatattacaagctaggtaagccgactagccgaactagaagtcattttctttagctcgc

[0339] attttccacagaaccacaatttcccaagtaatagtactacactactactccaccatgtccatcgcacatgttgcttaattttccttagcaaatattcaa

[0340] gtcaaggtcctccattcacatgatcagctcgccaaagattcaagtgagcatcaccgtacagtaccaagacagcaaccagcgtcaaggcactg

[0341] acatgcgggcccgcctaacgccaggaatcaatactgtagtaccgtacatccagcgtcaaaaaggccggcgttcaaacttcaatatatccaact

[0342] atatatccaaatttccaaactgctgctcctacttgctctctccctcttttccaaaaggagctcaaaaagctttaaaaaaaggttttaatttggataagc

[0343] aaaaaaaattaaagtgggatttgaaagtcttgtgctgcttatgcaacagtcgcagacttgcggcacggccagcctccggcccgtgggccgcc

[0344] gggaaagccgtcgctgaccggccggctagccagcccgggaggcgacccggcggtgggtcccacctatgccacgtggccgcatcctgcct

[0345] ctccgatgcgtttgaaatgtctccccccgtgtgatcgggctcccactccctctctatatatagcgagcggcttggagccctgtga

Claims

1. DUP3 and / or its alleles DUP3 Japonica and the use of the protein encoded by the DUP3 in reducing ineffective tillering of crops, increasing crop yield and / or improving nitrogen fertilizer utilization efficiency of crops, wherein the amino acid sequence of the protein encoded by the DUP3 or its allele is shown in any one of the following: 1) the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6; 2) an amino acid sequence that differs from the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6 due to substitution, deletion and / or insertion of one or more amino acid residues; 3) an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and especially at least 95% or 98% identical to the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6; 4) An active fragment of the amino acid sequence described in (1) or (2) or (3).

2. DUP3 and / or its alleles DUP3 Japonica Application in reducing ineffective tillering of crops, increasing crop yield and / or improving nitrogen fertilizer utilization efficiency of crops, The nucleotide sequence of the DUP3 gene is shown in any one of the following: 1) the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.3; 2) A nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3 due to substitution, deletion and / or insertion of one or more nucleotide sequences, but the activity of the protein encoded by it is the same as that of the protein encoded by the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3; 3) a nucleotide sequence that is at least 70%, preferably at least 80%, and more preferably at least 90% identical to the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3, and the activity of the protein encoded by it is the same as the activity of the protein encoded by the nucleotide sequence shown in SEQ ID NO. 1 or SEQ ID NO. 3; 4) Nucleotide sequences that differ in sequence from SEQ ID NO. 1 or SEQ ID NO. 3 due to the degeneracy of the genetic code; 5) An active fragment comprising the nucleotide sequence of any one of 1) to 4); 6) comprising a nucleotide sequence that hybridizes to a complementary sequence of the nucleotide sequence of any one of 1) to 5) under moderate stringency conditions, preferably high stringency hybridization conditions; or 7) comprising a nucleotide sequence complementary to the nucleotide sequence of any one of 1) to 5); The DUP3 Japonica The nucleotide sequence of the gene is shown as any of the following: a) the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4; b) a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4 due to substitution, deletion and / or insertion of one or more nucleotide sequences, but the activity of the protein encoded by it is the same as that of the protein encoded by the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4; c) a nucleotide sequence that is at least 70%, preferably at least 80%, more preferably at least 90% identical to the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4, and the activity of the protein encoded by it is the same as the activity of the protein encoded by the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 4; d) a nucleotide sequence that differs in sequence from SEQ ID NO. 2 or SEQ ID NO. 4 due to the degeneracy of the genetic code; e) an active fragment comprising the nucleotide sequence of any one of a) to d); f) comprising a nucleotide sequence that hybridizes to the complementary sequence of the nucleotide sequence of any one of a) to e) under moderate stringency conditions, preferably high stringency hybridization conditions; or g) comprises a nucleotide sequence complementary to the nucleotide sequence of any one of a) to e).

3. A recombinant construct comprising DUP3 and / or its allele DUP3 as defined in claim 1 or 2 Japonica .

4. The recombinant construct of claim 3, comprising a DUP3 promoter, a DUP3 Japonica promoter, Actin promoter or 35s promoter.

5. The recombinant construct of claim 4, wherein the nucleotide sequence of the DUP3 promoter is as shown in any one of the following: (1) the nucleotide sequence shown in SEQ ID NO. 13; (2) a nucleotide sequence that hybridizes to the complementary sequence of SEQ ID NO. 13 under moderately stringent conditions, preferably high stringent hybridization conditions; (3) a nucleotide sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and especially at least 95% or 98% identical to SEQ ID NO. 13; (4) An active fragment of the nucleotide sequence of any one of (1)-(3); or (5) A nucleotide sequence complementary to the nucleotide sequence of any one of (1) to (4).

6. The recombinant construct of claim 4, wherein the DUP3 Japonica The nucleotide sequence of the promoter is shown in any of the following: (1) the nucleotide sequence shown in SEQ ID NO. 14; (2) a nucleotide sequence that hybridizes to the complementary sequence of SEQ ID NO. 14 under moderately stringent conditions, preferably high stringent hybridization conditions; (3) a nucleotide sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and especially at least 95% or 98% identical to SEQ ID NO. 14; (4) An active fragment of the nucleotide sequence of any one of (1)-(3); or (5) A nucleotide sequence complementary to the nucleotide sequence of any one of (1) to (4).

7. A method comprising a DUP3 gene or an allele thereof as defined in claim 1 or 2. Japonica Or the host cell of the recombinant construct according to claim 3-6, which comprises animal cells, microbial cells and plant cells.

8. A method for cultivating crops with reduced ineffective tillers, increased yield and / or improved nitrogen fertilizer utilization efficiency, the method comprising increasing the expression of the DUP3 gene or its allele DUP3 as defined in claim 1 or 2. Japonica expression level.

9. The method according to claim 8, comprising transfecting the recombinant construct according to claim 3 or the host cell according to claim 7 into plant cells or tissues to obtain transgenic crop plants.

10. The method according to claim 8, comprising the step of: introducing a gene comprising the DUP3 gene or an allele thereof as defined in claim 1 or 2 into a cell line; Japonica A hybrid crop plant is obtained by crossing a crop plant with another plant of the crop.

11. The method according to claim 8, comprising but not limited to gene editing methods, preferably CRISPR / Cas9 technology methods.

12. The use according to claim 1 or 2, wherein the crop is a monocotyledonous plant, preferably rice.

13. The method of claims 8-11, wherein the crop is a monocotyledonous plant, preferably rice.

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