Genes regulating rice glutelin content and their use

Abstract

The application discloses a new gene GluA1-L1 which is a gene locus positively affecting rice quality, can regulate the content of glutelin in rice endosperm, and is used for creating high-quality rice varieties and providing a gene resource for cultivating high-quality rice and breeding new low-glutelin rice varieties.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural biotechnology, and particularly relates to a gene GluA1-L1 for regulating the content of rice glutelin and application of the gene GluA1-L1 in regulating the quality and yield of rice. BACKGROUND

[0002] The content of starch and storage protein is directly related to the yield and quality of rice. The storage protein accounts for about 10% of the content of rice and 90% of the total protein content. The storage protein in rice can be mainly divided into glutelin, globulin, prolamin and albumin, of which glutelin accounts for the largest proportion. The content of glutelin accounts for more than 60% of the storage protein and is the preferred target for improving the quality of rice protein. Rice glutelin is a protein family encoded by multiple genes and can be divided into four subfamilies: GluA, GluB, GluC and GluD. Properly increasing the synthesis and accumulation of storage protein is beneficial to improving the quality of rice, and excessive accumulation of storage protein will cause endoplasmic reticulum stress. Abnormal synthesis of storage protein will directly affect the quality of rice, resulting in problems such as increased chalkiness, soft endosperm and the like, and the abnormal synthesis of storage protein will be aggravated under high temperature. Higher content of glutelin will increase the burden of patients with kidney disease and accelerate the decline of kidney function. Therefore, mining the natural variation sites of storage protein and analyzing the molecular network for regulating the synthesis of storage protein can provide important theoretical basis and gene resources for breeding high-quality rice and selecting new varieties of low-gluten rice.

[0003] Developing a major gene for glutelin production is one of the ways to select new varieties of low-gluten rice. For example, the patent document with publication number CN115786367A discloses a gene LGC2 for controlling the content of rice glutelin. After knocking out the gene LGC2 in rice by gene editing technology, a gene editing rice material with low glutelin in grains can be obtained. SUMMARY

[0004] In the research of screening genes related to glutenin production in rice, we found a high expression gene in endosperm by RNA-seq, which is annotated as LOC_Os08g03410. The gene encodes a putative glutenin, and the amino acid sequence is shown as SEQ ID NO: 1. The function loss of the gene will cause the rice seeds to exhibit a chalky endosperm phenotype. Through multiple sequence alignment and phylogenetic tree analysis, we named it GluA1-L1. Compared with the wild type ZH11 strain, the thousand-grain weight and yield per plant of the strain knocking out GluA1-L1 decrease. Therefore, GluA1-L1 is a gene locus positively affecting rice quality, which suggests that the transcription level of GluA1-L1 can be improved by gene editing and the like to create high-quality rice. At the same time, the function of GluA1-L1 is also clear, which provides a new breeding locus for creating low-gluten rice. Subsequent CRISPR / Cas9 and the like can be used to knock out the glutenin coding gene to obtain multiple mutants to achieve rapid breeding of low-gluten rice. On the other hand, overexpression of the gene GluA1-L1 can also increase the thousand-grain weight and yield per plant. Based on the above research findings, the present application includes the following technical solutions.

[0005] The first aspect of the present application provides a polypeptide selected from the group consisting of:

[0006] (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, which is protein GluA1-L1;

[0007] (b) a conservatively modified variant of the polypeptide of (a) which has the function of the polypeptide of (a) and which comprises a substitution, deletion, or addition, and / or insertion of one or more amino acid residues of the polypeptide of (a) as shown in SEQ ID NO: 1;

[0008] (c) a polypeptide derived from the polypeptide of (a) and having more than 95% identity, preferably more than 96% identity, preferably more than 97% identity, preferably more than 98% identity, more preferably more than 99% identity, to the polypeptide sequence defined in (a), and having the function of the polypeptide of (a); or

[0009] (d) a polypeptide comprising a fragment of the polypeptide sequence of (a) or (b) or (c).

[0010] The above function refers to the function of positively regulating the content of glutenin in endosperm.

[0011] MKTASMAASLLIPLCLCILLLRGASAVSDQQEAGRRDSCDRIDRRIRALEPTRRVDSEAGHTELYDDRDGQLPCAGVAAARITIQRNGLLLPSYSNSPRLAYIVHGRGIVGVVIPGCPETYQETSSSSSQEQEDDERRRRGRRGDEERRRSSEGEEEEEEETSRRSFEQSIRDEHQRITTVRQGDVVAIPAGAPFWVHNDGDSPLVAISVHDVSNSANQLDQTSRRFRLAGGQARSEGRQRYGEGESSESETERGGEESYNILSGFDTELLAESMRVSPDIARKLQGRSDKRGNIVRVRRGGLRMLRPATERVTDEEMMRGANAAAAAGNGIDEAVCLMKLRENVADPMKADLYTPNGGRITVLNSQKLPVLKLIKMSVNRGVMRRNAILAPHWNINAHAAVYATSGSARLQVVSSEGRRVFDGELRRRQMVVVPQSFAVAGRAGDEGFAWVSFQTSDGAMNAPVVGKSSALRGMPADVLANAFGVSREEARMVKFGRGQELAIFSPKSGAAARRRRSPGHRDDGVLAAPA (SEQ ID NO: 1).

[0012] A second aspect of the present application provides a polynucleotide, which is selected from the group consisting of:

[0013] (A) a polynucleotide encoding the polypeptide of claim 1;

[0014] (B) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1;

[0015] (C) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 2, which is a CDS region sequence of the encoding gene of protein GluAl-Ll;

[0016] (D) a polynucleotide having a nucleotide sequence with a homology of ≥ 95%, preferably ≥ 96%, preferably ≥ 97%, preferably ≥ 98%, more preferably ≥ 99% to the nucleotide sequence as set forth in SEQ ID NO: 2;

[0017] (E) a nucleotide sequence complementary to the nucleotide sequence of any one of (A)-(D).

[0018] wherein the nucleotide sequence of SEQ ID NO: 2 is:

[0019]

[0020] A third aspect of the present application provides a DNA molecule comprising the polynucleotide as described above, for example an expression cassette / expression frame of the protein GluA1-L1.

[0021] In one embodiment, the DNA molecule as described above comprises a GluA1-L1 encoding gene with a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 and a promoter upstream, for example a Cauliflower Mosaic Virus (CAMV) 35S promoter, a Ubiquitin gene promoter from rice, i.e. a Ubi promoter, or a promoter from Huagengxian 74 (HGX74) with a nucleotide sequence as shown in SEQ ID NO: 4.

[0022] In the nucleotide sequence SEQ ID NO: 3, the GluA1-L1 full gene sequence is composed of a 5' UTR region, a 3' UTR region, an intron and an exon (coding region, i.e. CDS).

[0023] A fourth aspect of the present application provides a recombinant plasmid for expressing the protein GluA1-L1 as claimed in claim 1, comprising the DNA molecule as described above, which is formed by cloning the DNA molecule as described above in a plasmid vector suitable for expression in Agrobacterium, selected from binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, for example a plant transgenic vector or a modified vector such as pHB-YFP, pHB-FLAG, pBin19, pUN1301, a fluorescent reporter vector pGreenII0800-LUC, pCAMBIA3300, pCAMBIA1301, pCAMBIA2301, pBI121 or pTF102.

[0024] A fifth aspect of the present application provides a microbial engineering bacterium, which is a transformant comprising the recombinant plasmid as described above, for mediating the transfer of the recombinant plasmid as described above into a plant, for example a rice or a grass crop, etc., preferably the microorganism is Agrobacterium, for example Agrobacterium tumefaciens, Agrobacterium EHA105, Agrobacterium GV3101. For example, the recombinant plasmid as described above is transferred into an Agrobacterium strain by freeze-thaw method to form a microbial engineering bacterium.

[0025] Preferably, the microbial host of the microbial engineering bacterium as described above is Agrobacterium, for example Agrobacterium tumefaciens, Agrobacterium EHA105, Agrobacterium GV3101.

[0026] The sixth aspect of the present application provides the use of the polypeptide as described above, the polynucleotide as described above, the DNA molecule as described above, the recombinant plasmid as described above or the microbial engineering bacteria as described above in regulating the content of glutelin in grains, in cultivating glutelin-low plant varieties, in improving rice quality, or in cultivating glutelin-high and / or high-thousand-grain-weight and / or high-yield-per-plant plant varieties.

[0027] The plant is a cereal plant selected from the group consisting of rice, wheat, maize, barley, oat, rye, sorghum, millet, preferably the plant is rice.

[0028] In an application embodiment, the content of glutelin in grains is increased, glutelin-high and / or high-thousand-grain-weight and / or high-yield-per-plant plant varieties such as new rice varieties are cultivated by overexpressing the polypeptide as described above, such as GluA1-L1, in plants such as rice by the following method:

[0029] (i) cloning the coding gene of the polynucleotide as described above, such as protein GluA1-L1, on a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, i.e. GluA1-L1 overexpression vector, transforming plants by Agrobacterium-mediated method to obtain transgenic plants overexpressing the polypeptide as described in claim 1, such as GluA1-L1; and / or

[0030] (ii) cloning the coding gene of the polynucleotide as described above, such as protein GluA1-L1, on a plant chromosome by gene editing technology to obtain transgenic plants overexpressing the coding gene of the polypeptide as described above, such as GluA1-L1; and / or

[0031] (iii) placing the existing coding gene of GluA1-L1 in the plant genome under the regulation of a functionally enhanced promoter, such as 35S promoter or Ubi promoter.

[0032] In an embodiment, the expression of the gene GluA1-L1 in the chromosome of wild-type rice is down-regulated, inactivated, functionally weakened or knocked out by the following method to reduce the content of glutelin in rice, improve the quality of rice, cultivate glutelin-low rice new varieties:

[0033] (1) knocking out the GluA1-L1 gene in the chromosome of wild-type rice by gene editing technology;

[0034] (2) down-regulating the expression level of the GluA1-L1 gene in the chromosome of wild-type rice;

[0035] (3) replacing the GluA1-L1 gene in the chromosome of wild-type rice with a mutant of the GluA1-L1 gene encoding a loss-of-function or down-regulated gene; and / or

[0036] (4) blocking, inhibiting or interfering with the expression of the gene GluA1-L1 in the chromosome of wild-type rice.

[0037] Further, the above-mentioned mode (2) can be selected from the group consisting of:

[0038] (2-1) mutating the promoter region and / or coding region of the gene GluA1-L1 to down-regulate the expression level of the gene GluA1-L1;

[0039] (2-2) mutating the upstream regulatory factor of the gene GluA1-L1 to down-regulate the expression level of the gene GluA1-L1; or

[0040] (2-3) introducing the interacting protein of GluA1-L1 in wild-type rice to change the function of the gene GluA1-L1.

[0041] Alternatively, the gene editing technology in the above-mentioned step (ii) or step (1) can be selected from the group consisting of homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system and MuGENT.

[0042] The present application first discovers a new gene GluA1-L1 in rice, and preliminarily reveals its function of regulating the content of glutelin in rice endosperm and regulating the quality of rice. The new gene GluA1-L1 encodes a putative glutelin precursor and is highly expressed in rice endosperm. Down-regulation of the expression level of GluA1-L1 leads to a significant decrease in the thousand-grain weight, insufficient grain filling, and a clear chalky endosperm phenotype. This function enables it to become a gene locus for positively regulating the quality of rice, for improving agronomic traits such as single plant yield and grain width, and has application prospects for improving the quality of rice and creating low-gluten rice, and provides important genetic resources for improving the quality of rice. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The expression level of the gene GluA1-L1 and the phenotype of the replacement line parent grain are shown. Among them, (a) detection of the expression level of GluA1-L1 in the grain of the parent near-isogenic line 7 days after fertilization, the data is mean±SEM, n=3, using two-tailed t-test for significant difference test, ****P<0.0001; (b) phenotype of mature grain of the parent near-isogenic line; (c) expression level of GluA1-L1 in each tissue, wherein YP, young panicle; DAF, grain at different days after fertilization, the data is mean±SEM, n=3; (d) expression level of GluA1-L1 in each tissue site in the public database mbkbase.

[0044] Figure 2 A multiple sequence alignment of glutens in rice is shown. In the figure, the GluA1-L1 protein sequence is aligned with other glutens in rice, GluA1, GluA2, GluA3, GluB1a, etc. The sequence alignment was performed using DNAMAN 8.0 software.

[0045] Figure 3 A phylogenetic tree of the glutens gene family in rice is shown. In the figure, the phylogenetic tree of the glutens family, GluA1-L1, etc. was constructed using MEGA11 software, using the Neighbor-Joining method and based on the JTT matrix to infer the evolutionary history, with 1,000 step tests. The phylogenetic tree is drawn to scale.

[0046] Figure 4 The grain development phenotype of wild type and knockout gene GluA1-L1 glua1-l1 mutant is shown. Among them, (a) glua1-l1 mutant grain and wild type ZH11 grain development performance, scale is 2 cm; (b) genotype of glua1-l1 mutant. After PCR amplification using specific primers, sequencing and comparing with the wild type ZH11 reference sequence to determine the genotype of glua1-l1 mutant.

[0047] Figure 5 The cross-sectional morphology of wild type and glua1-l1 mutant grains under scanning electron microscope is shown. In the figure, the comparison of starch granule morphology of glua1-l1 mutant and wild type ZH11 grain cross-section under scanning electron microscope, scale is 20 μm.

[0048] Figure 6 The agronomic traits of wild type and glua1-l1 mutant are shown. Among them, (a-b) plant type and ear type of ZH11 and glua1-l1 mutant, scale is 10 cm, 2 cm respectively; (c-f) comparison of single plant yield, thousand seed weight, grain length and grain width of ZH11 and glua1-l1 mutant, data is mean±SD, n=12, using two-tailed t-test for significant difference test, ns, no significant difference, *P<0.05, **P<0.01.

[0049] Figure 7The haplotype analysis of the gene GluA1-L1 is shown. Among them, (a) there is a G-T SNP change in the coding region of GluA1-L1 gene, which leads to early termination of CDS, and this type of variation mainly exists in Indica III; (b-c) haplotype analysis and phylogenetic tree of the promoter region of GluA1-L1, japonica rice mainly uses haplotype I and II, indica rice mainly uses haplotype III and IV. DETAILED DESCRIPTION

[0050] In the process of exploring the molecular mechanism of regulating the content of storage proteins in rice, the research group identified a new gene locus GluA1-L1 highly expressed in endosperm. Compared with ZH11 wild type strain, the grain of GluA1-L1 knockout showed chalky endosperm, and the thousand-grain weight, single plant yield and grain width were significantly reduced, and the grain length had no obvious difference, confirming that GluA1-L1 is a gene locus that positively affects rice quality, suggesting that the in situ expression and improvement of the transcription level of GluA1-L1 through gene editing and other methods to create high-quality rice. At the same time, GluA1-L1 has become a new breeding locus for creating low glutelin rice. The new mechanism of GluA1-L1 affecting rice quality revealed in this study provides a new theoretical guidance for crop genetic improvement and molecular design breeding.

[0051] The above functions of the gene GluA1-L1 enable the overexpression or inactivation of GluA1-L1 in wild-type plants, especially in rice, to be applied to create new varieties of transgenic plants with expected agronomic traits such as high glutelin content, low glutelin content, high thousand-grain weight, and / or high single-plant yield.

[0052] As used herein, the term "wild type" represents the original plant that has not been genetically modified, has not been subjected to mutagenic treatment, such as rice ZH11, Huajiang indica 74, etc.

[0053] Correspondingly, the terms "transgenic plant", "(plant) mutant" and "genetically engineered plant" and the like in this paper represent the same meaning, which refers to the plant after genetic engineering modification of the wild type plant.

[0054] In the description of the technical solutions of the present application, the term "and / or" used in terms such as "A and / or B", "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used in phrases such as "A, B, and / or C", the term "and / or" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0055] As used herein, the term "(glutelin content / 1,000-grain weight / single plant yield) increase," "enhance" or "augment" can mean an increase of at least 10% compared to a reference level (such as wild-type rice), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any increase between 10% and 100% compared to a reference level.

[0056] Similarly, the term "(glutelin content) decrease" or "decrease" can mean a decrease of at least 10% compared to a reference level (such as wild-type rice), for example, a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any decrease between 10% and 100% compared to a reference level.

[0057] In this document, for the sake of brevity, certain protein such as GluA1-L1 is sometimes used interchangeably with its encoding gene (DNA) name GluA1-L1, and one of skill in the art would understand that they refer to different types of substances in different contexts. The meaning is readily understood by one of skill in the art in light of the context and the context. For example, for GluA1-L1, when used to describe a function or a class that regulates glutelin content in endosperm, it refers to the protein; when used as a gene, it refers to the encoding gene of the protein.

[0058] One of skill in the art can anticipate that a conservative variant polypeptide of GluA1-L1 having high homology (identity) to the amino acid sequence of SEQ ID NO: 1, for example, 90% or more, also has the same or similar function is also reasonable.

[0059] As used herein, the term "conservatively modified polypeptide" refers to a polypeptide that substantially retains the same biological function or activity as the polypeptide. The variations refer, inter alia, to small amino acid mutations, which "mutations" include, but are not limited to, substitutions, deletions, insertions, chemical modifications of amino acid residues, preferably positive mutations, i.e., mutations that improve the function of GluAl-Ll. The substitutions can be non-conservative substitutions, conservative substitutions, or a combination of non-conservative and conservative substitutions. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and thus typically includes the substitution of an amino acid in a polypeptide with an amino acid of the same or similar class. However, as used herein, a conservative mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitutions if the conservative mutation can alternatively be an aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or a restricted residue to restricted residue substitution. It is well known in the art that common instances of conservative substitutions include: interchanges among the aromatic amino acids F, W, Y; interchanges among the hydrophobic amino acids L, I, V; interchanges among the polar amino acids Q, N; interchanges among the basic amino acids K, R, H; interchanges among the acidic amino acids D, E; and interchanges among the hydroxyl amino acids S, T. In addition, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions can be made, for example, according to the following table.

[0060] Original residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0061] A "non-conservative substitution" refers to a substitution or mutation of an amino acid in a polypeptide with an amino acid having substantially different side chain properties. A non-conservative substitution can use amino acids from groups listed above, but outside of the groups, rather than within. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the region of the substitution (e.g., a proline in place of glycine), (b) the charge or hydrophobicity, or (c) the side chain volume.

[0062] A "deletion" refers to a modification made to a polypeptide by removing one or more amino acids from a reference polypeptide. A deletion can include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids making up the sequence of GluAl-Ll (SEQ ID NO: 1), while retaining improved properties of GluAl-Ll function. Deletions can be to the interior and / or the terminal portions of a polypeptide. In various embodiments, deletions can comprise contiguous segments or can be non-contiguous.

[0063] "Insertion" refers to a modification made to a polypeptide by adding one or more amino acids from the reference polypeptide. In some embodiments, the improved engineered GluA1-L1 includes one or more amino acids inserted into a naturally occurring GluA1-L1 and one or more amino acids inserted into other improved GluA1-L1 polypeptides. The insertion can be internal, or carboxy terminal or amino terminal. Insertions, as used herein, include fusion proteins as known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more amino acids in the naturally occurring polypeptide.

[0064] By overexpressing a protein GluA1-L1 of an amino acid sequence as shown in SEQ ID NO: 1 in a plant, such as rice, it is possible to breed a transgenic plant variety with expected agronomic traits such as high gluten content, high thousand seed weight, high single plant yield, etc.

[0065] For the construction of such overexpressing GluA1-L1 genetically engineered plants, a conventional recombinant plasmid transformation method such as Agrobacterium-mediated transformation or a gene editing technology can be used to introduce a GluA1-L1-encoding gene such as SEQ ID NO: 2 or SEQ ID NO: 3 into a plant.

[0066] Preferably, the above "introducing a GluA1-L1-encoding gene into a plant" can be achieved by introducing a recombinant expression vector containing the GluA1-L1-encoding gene into a recipient plant.

[0067] A recombinant expression vector containing the coding gene can be constructed using an existing plant expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. The plant expression vector can also contain a 3' untranslated region of the foreign gene, i.e., a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the untranslated region of the 3' end of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nopaline synthase gene Nos), the 3' end of the plant gene (such as the soybean storage protein gene) transcription.

[0068] When the above-mentioned coding gene is used to construct a recombinant expression vector, any one of the enhanced promoters or constitutive promoters (such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of maize, or a tissue-specific expression promoter (such as a seed-specific expression promoter) such as SEQ ID NO: 4) can be added before the transcription initiation nucleotide, which can be used alone or in combination with other plant promoters. In addition, when the coding gene is used to construct a recombinant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent regions of start codons, but must be in the reading frame of the coding sequence to ensure correct translation of the entire sequence. The source of the enhancer is broad and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0069] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a gene that can express an enzyme or a luminescent compound that can produce a color change in plants (GUS gene, luciferase gene, etc.), a resistant antibiotic marker (gentamicin marker, kanamycin marker, etc.), or an anti-chemical reagent marker gene (such as an anti-herbicide gene).

[0070] In the above method, the recombinant expression vector carrying the above-mentioned coding gene is introduced into the recipient plant, which can be specifically: transforming plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc. Conventional biological methods, and cultivating the transformed plant tissue into a plant.

[0071] The transformed cells, tissues or plants are understood to include not only the final product of the transformation process, but also its transgenic progeny.

[0072] Taking the construction of a transgenic plant overexpressing GluA1-L1 using a conventional Agrobacterium transformation method as an example, the construction method of the transgenic plant includes:

[0073] 1) Providing Agrobacterium such as EHA105 carrying an expression vector containing the coding sequence of the polypeptide GluA1-L1;

[0074] 2) Contacting plant cells or tissues or organs with the Agrobacterium in step 1) to transfer the coding sequence into plant cells and integrate it into the chromosomes of the plant cells;

[0075] 3) Selecting plant cells or tissues into which the coding sequence is transferred; and

[0076] 4) Regenerating the plant cells or tissues in step 3) into a plant.

[0077] In another embodiment, when constructing a transgenic rice variety with low prolamin content, there are various technical means for inactivating, deactivating and / or not expressing the gene GluA1-L1 in rice, which can be used alone or in combination. For example, one inactivation method is to perform a frameshift mutation on the GluA1-L1 coding gene to change the amino acid sequence of the polypeptide GluA1-L1 and / or terminate translation.

[0078] The application will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the application and not used to limit the scope of the application.

[0079] Examples

[0080] In the examples, the amount, content and concentration of various substances are involved, wherein the percentage content refers to the mass percentage content unless otherwise specified.

[0081] In the examples herein, if no specific description is made for the reaction temperature or operating temperature, the temperature generally refers to room temperature (15-30°C).

[0082] In the examples, the molecular biology experiments including plasmid construction, enzyme digestion, preparation of competent cells, transformation, etc. are mainly performed according to the Molecular Cloning Experiments Guide (3rd edition), J. Sambrook, D. W. Russell (USA) edited, Huang Peitang et al. translated, Science Press, Beijing, 2002. For example, the competent cell transformation method and the preparation method of competent cells are both performed according to the Molecular Cloning Experiments Guide (3rd edition) Chapter 1 page 96. If necessary, the specific experimental conditions can be determined by simple tests.

[0083] The PCR amplification experiments are performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. If necessary, it can be adjusted by simple tests.

[0084] In the examples, the primer synthesis and gene sequencing are commissioned to Shengong Bioengineering (Shanghai) Co., Ltd. and Huada Gene.

[0085] The molecular biology methods and transgenic plant construction methods including CRISPR-Cas9 system plasmid construction for knocking out the gene GluA1-L1 and gene editing technology are operated by using the technical means commonly used in the art.

[0086] Plant materials

[0087] The wild type (WT) used in this study is ZH11 (preserved in the laboratory). The GluA1-L1 gene deletion mutants glua1-l1#1 and glua1-l1#2 are obtained by using CRISPR / cas9 technology and are used for further experiments.

[0088] Genetic transformation

[0089] 1. Callus induction: Mature and plump seeds of rice were selected and dehulled; 75% alcohol was used to sterilize the seeds for 1-2 min and the alcohol was poured out; the seeds were washed with sterilized distilled water for 2 times; 0.15% mercuric chloride (containing 0.1% Tween-20) was added to soak the seeds for 15-18 min, and the seeds were shaken several times during the soaking; the mercuric chloride was poured out and the seeds were washed with sterilized distilled water for 5 times. The sterilized seeds were inoculated into callus induction medium and cultured at 32°C under illumination for 5-10 days.

[0090] 2. Agrobacterium streak activation: 2 days before infection, Agrobacterium was streaked on LB medium containing 50 mg / L kanamycin and incubated at 28°C.

[0091] 3. Suspension, infection and co-culture of Agrobacterium: before infection, the activated Agrobacterium was scraped into suspension medium and incubated at 28°C with 180 rpm shaking for 3-3.5 h, and then the concentration of the bacterial solution was adjusted to OD600=0.1-0.2 with the suspension medium. The callus induced for 5-10 days was put into the Agrobacterium suspension, and infected for 1.5 min. The bacterial solution was poured out and the callus surface was dried with sterilized filter paper. The callus surface was covered with a layer of sterilized filter paper and dried in a clean bench for 30 min. After drying, the callus was transferred into co-culture medium with a layer of sterilized filter paper on the surface, and first incubated at 20°C in the dark overnight, and then incubated at 25°C in the dark for 2 days.

[0092] 4. Bacteria removal: after co-culture, the callus was transferred to an empty sterilized container with a pair of tweezers. The callus was repeatedly washed with sterilized distilled water for 7-8 times, and the first 3 times were fast washing, and the last 3-4 times were soaking for 3-5 min each time. Finally, the callus was soaked in sterilized distilled water containing 500 mg / L Cn for 30 min. The solution was poured out, and the water on the callus surface was dried with sterilized filter paper as much as possible. A layer of sterilized filter paper was covered on the callus surface, and dried in a clean bench for 1 h.

[0093] 5. Selection: the callus after bacteria removal was placed on selection medium and cultured at 32°C under illumination for 14 days.

[0094] 6. Differentiation: after selection for 14 days, the resistant callus was transferred into differentiation medium and cultured at 28°C (light cycle of 14 h illumination / 10 h darkness).

[0095] 7. Rooting: when the resistant callus formed regenerated seedlings with a height of 3-4 cm on the differentiation medium, the seedlings were transferred into rooting medium for culture until complete plants were formed.

[0096] Field investigation of rice

[0097] Wild type ZH11 and mutant rice gluai-l1#1, gluai-l1#2 were planted in the Shanghai Songjiang Test Base of the Institute of Molecular Plant Sciences, Chinese Academy of Sciences, with 18 plants for each strain, to perform phenotype investigation and gene identification.

[0098] Example 1: Discovery and identification of gene GluA1-L1

[0099] We used a set of chromosome fragment replacement line materials with Oryza Sativa L. ssp. Indica variety Huagengxian 74 (HGX74) as the receptor parent and Oryza rufipogon as the donor parent to construct the materials for locating and cloning QTLs related to rice heat tolerance. In the preliminary location, we identified that the Y261 replacement line material strain from wild rice fragments exists a temperature-dependent QTL that regulates rice yield and rice quality. In order to fully explore the gene regulatory network that regulates rice quality, we selected near-isogenic lines (NIL-TT5 Y261 , NIL-TT5 HGX74 ) for transcriptome sequencing of grains in the grain filling stage. There is a significantly down-regulated gene GluA1-L1 in the differential genes in RNA-seq, which encodes a hypothetical glutelin gene and is specifically expressed in endosperm, and is not expressed in tissues such as leaves and stems. We verified the expression difference of GluA1-L1 in near-isogenic lines by qRT-PCR, which is consistent with the transcriptome results. The expression of GluA1-L1 in NIL-TT5 Y261 grains is weak.

[0100] Primers used for qRT-PCR:

[0101] The 5' end oligonucleotide primer sequence of qGluA1-L1 is:

[0102] 5'-GACCGACGAGGAGATGATGAG-3';

[0103] The 3' end primer sequence is:

[0104] 5'-ATCTTGATGAGCTTGAGGACGG-3'.

[0105] Example 2: Knockout of gene GluA1-L1 using CRISPR / Cas9 gene editing technology

[0106] To further verify the function of LOC_Os08g03410, we created a knockout mutant of GluA1-L1 gene, glua1-l1, using CRISPR / Cas9 technology. We designed a knockout target in the first exon region and constructed it into a CRISPR / Cas9 vector. We performed genetic transformation in the japonica rice ZH11 background by Agrobacterium tumefaciens EHA105-mediated mature embryo transformation, screened positive plants by hygromycin B, and planted T2 and above in Songjiang, Shanghai, and investigated the phenotype.

[0107] The 5' end oligonucleotide primer sequence for the construction of the CRISPR / Cas9 knockout vector is:

[0108] 5'-ggcACCAGCTCGACCAAACCTCC-3'.

[0109] The 3' end primer sequence is:

[0110] 5'-aaacGGAGGTTTGGTCGAGCTGGT-3'.

[0111] Through the above CRISPR / Cas9 gene editing technology, we obtained two GluA1-L1 gene knockout mutants, glua1-l1#1 and glua1-l1#2.

[0112] Results and Discussion

[0113] 1. We screened a high expression gene GluA1-L1 in endosperm through transcriptome data analysis, which encodes a putative glutelin and affects rice quality.

[0114] In our previous work, we used a set of chromosome substitution lines with the indica rice (Oryza Sativa L. ssp. Indica) variety Huagengxian 74 (HGX74) as the recipient parent and the common wild rice (Oryza rufipogon) as the donor parent to construct the chromosome substitution line material for the work of locating and cloning QTLs related to heat tolerance in rice. We identified Y261 substitution line material from a wild rice fragment, which showed a significant chalky endosperm phenotype in grains, and carried out subsequent research.

[0115] To explore the regulatory network of high temperature affecting rice grain quality, we sequenced the transcriptome of the 7-day post-fertilization grain of near-isogenic line (carrying wild rice chromosomal fragment and genetic background of Huajingxian 74). Through the analysis of RNA-seq, we noticed that there was a significantly down-regulated gene LOC_Os08g03410 in the differential genes, which encoded a putative glutelin gene and was specifically expressed in endosperm (as shown in Figure 1 ). To further characterize the function of the gene, we analyzed its sequence similarity and named it GluA1-L1, and meanwhile generated a homozygous knockout line glua1-l1 in Zhonghua 11 (ZH11) background through CRISPR / Cas9 technology (as shown in Figure 2 and Figure 3 ). In the loss-of-function mutant glua1-l1, its grain had a clear chalky endosperm phenotype (as shown in Figure 4 ).

[0116] Through blast and multiple sequence alignment, it was found that GluA1-L1 had 40.08%, 40.46% and 41.21% sequence similarity with GluA1, GluA2 and GluA3 respectively, 37.67% and 39.58% similarity with GluB1a / b and GluB7, 41.79% and 38% similarity with GluC-1 and GluD-1 (as shown in Figure 3 ). Based on the above results, we believe that GluA1-L1 may be a non-typical glutelin gene, and the protein encoded by GluA1-L1 has a large amino acid sequence difference with the existing GluA, GluB, GluC and GluD subfamily genes. GluA1-L1 may be a new glutelin subfamily gene, and the function of GluA1-L1 provides a new breeding site for creating low glutelin rice and improving rice quality.

[0117] 2. The starch granule structure of glua1-l1 mutant is spherical, irregular and loose.

[0118] To further characterize the microstructure of glua1-l1 mutant grain, we observed the starch granule morphology of the cross section of mature grain of wild type ZH11 and glua1-l1 mutant by scanning electron microscope. Compared with the neat and smooth section of ZH11 grain, the section of glua1-l1 mutant presented irregular. The starch granules of glua1-l1 mutant were mostly spherical, irregular and loose, while the wild type showed uniform polyhedral structure, and the starch granules were arranged regularly and closely, with clear edges and corners (as shown in Figure 5The starch granules of glul-l1 mutant rice are abnormal in structure (Fig. 1). The abnormal structure of starch granules might be the main reason for the poor quality of glul-l1 mutant rice. The loss of function of Glul-L1 leads to the powderization of endosperm.

[0119] 3. The agronomic traits of glul-l1 mutant, such as 1000-grain weight and yield per plant, are significantly decreased.

[0120] GluA1-L1, as a new glutelin gene, positively regulates the quality of rice. To further investigate the effect of GluA1-L1 on other agronomic traits, we investigated the agronomic traits of glul-l1 mutant, such as 1000-grain weight and yield per plant. Wild-type ZH11 and glul-l1 mutant plants were planted in Songjiang, Shanghai. There was no significant difference in plant height between the two. Compared with ZH11, the yield per plant and 1000-grain weight of glul-l1 mutant were significantly decreased, and the grain width was reduced, while there was no significant difference in grain length (Fig. 2). Figure 6 This indicates that GluA1-L1 affects the accumulation of dry matter in grains, leading to a decrease in 1000-grain weight and yield per plant. This also implies that overexpression of GluA1-L1 might also increase 1000-grain weight and yield per plant.

[0121] 4. GluA1-L1 has obvious differentiation differences between Indica and Japonica subspecies, and has application potential in the cultivation of high-quality rice.

[0122] Since GluA1-L1 positively regulates the quality of rice, in order to fully exploit its application value in the cultivation of high-quality rice, we used the rice comprehensive database http: / / ricevarmap.ncpgr.cn / Haplotype analysis of SNP sites in the full gene sequence of GluA1-L1 (including the upstream 2 kb promoter region and the downstream 1 kb region of 3'UTR) detected about 415 SNP variations. Among them, there are about 40 variations in the coding region, about 200 in the promoter region, and about 110 SNP variations in the 3'UTR and its downstream 1 kb region. There is a G-T SNP change in its coding region (physical position 1597644), which makes GluA1-L1 terminate early, and is only detected in Indica III, accounting for about 1.10% (Fig. 3). Figure 7 The discovery of this natural variation provides a new genetic resource for creating low-gluten rice, which can be further bred into new varieties of low-gluten rice by crossing with low-gluten rice varieties. Considering its application in positively regulating the quality of rice, we performed haplotype analysis on the first 100 SNP sites of ATG, and constructed a phylogenetic tree (Fig. 4). Figure 7The 14 haplotypes were divided into two groups, with haplotype I and II mainly in japonica and haplotype III and IV mainly in indica. There was an obvious differentiation between japonica and indica, and the abundant SNP variations in the promoter and 3'UTR of GluA1-L1 provided rich genetic resources for mining GluA1-L1 with high expression level and cultivating high-quality rice.

[0123] The above embodiments are only preferred embodiments of the present application for fully illustrating the present application, and the protection scope of the present application is not limited to this. Any equivalent substitutions or changes made by those skilled in the art based on the present application are within the protection scope of the present application.

Claims

1. Use of the down-regulation or knockout of a gene encoding a polypeptide whose amino acid sequence is shown in SEQ ID NO: 1, said polypeptide being the protein GluA1-L1, in breeding low glutelin rice varieties.

2. Use according to claim 1, wherein Knocking out a wild-type rice chromosome in the genome editing technology GluA1-L1 gene.

3. Use according to claim 2, wherein the compound is ###0002### said gene editing technology is selected from the group consisting of homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT.

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