Gene for regulating and controlling glutelin content of rice and application thereof

Abstract

The invention discloses a novel gene GluA1-L1, which is a gene locus positively influencing rice quality, can regulate and control the glutelin content in rice endosperm, is used for creating high-quality rice varieties, and provides gene resources for cultivating high-quality rice and breeding new low-glutelin rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a gene GluA1-L1 for regulating rice glutenin content and an application of the gene in regulating rice quality and yield. Background Art

[0002] The content of starch and storage protein is directly related to the yield and quality of rice. Storage protein accounts for about 10% of the rice content and 90% of the total protein content. Storage protein in rice can be mainly divided into glutenin, globulin, alcohol-soluble protein and albumin, among which glutenin accounts for the largest proportion. Gluten content accounts for more than 60% of storage protein and is the preferred target for improving rice protein quality. Rice glutenin is a family of proteins 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 rice quality, while excessive accumulation of storage protein can cause endoplasmic reticulum stress. Abnormal synthesis of storage protein will directly affect rice quality, leading to problems such as increased chalkiness and powdery endosperm. High temperature will aggravate the abnormal synthesis of storage protein. Higher content of gluten will increase the burden on patients with kidney disease and accelerate the decline of kidney function. Therefore, exploring the natural variation sites of storage proteins and analyzing the molecular networks that regulate the synthesis of storage proteins can provide important theoretical basis and genetic resources for breeding high-quality rice and selecting new low-gluten rice varieties.

[0003] Developing major genes for gluten production is one of the ways to breed new low-gluten rice varieties. For example, patent document with publication number CN115786367A discloses a gene LGC2 that controls the gluten content of rice. By knocking out the LGC2 gene in rice through gene editing technology, gene-edited rice materials with low gluten content in grains can be produced. Summary of the invention

[0004] In the study of screening genes related to gluten production in rice, we found a gene highly expressed in the endosperm through RNA-seq, which was annotated as LOC_Os08g03410. This gene encodes a putative glutenin, and the amino acid sequence is shown in SEQ ID NO:1. The loss of function of this gene will cause rice seeds to show a powdery 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 single-plant yield of the strain knocked out GluA1-L1 decreased. Therefore, GluA1-L1 is a gene locus that positively affects rice quality, suggesting that the transcription level of GluA1-L1 can be increased by gene editing and other methods to create high-quality rice. At the same time, the clear function of GluA1-L1 also provides a new breeding locus for the creation of low-gluten rice. In the future, multiple mutants can be obtained by knocking out gluten-encoding genes through CRISPR / Cas9 and other technologies to achieve rapid breeding of low-gluten rice. On the other hand, overexpression of the gene GluA1-L1 may also increase thousand-grain weight and yield per plant. Based on the above research findings, the present invention includes the following technical solutions.

[0005] The first aspect of the present invention provides a polypeptide, wherein the polypeptide is selected from the following group:

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

[0007] (b) a conservative variant polypeptide derived from (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence SEQ ID NO:1 and having the function of the polypeptide of (a);

[0008] (c) a conservative variant polypeptide derived from (a) that has 95% or more homology, preferably 96% or more homology, preferably 97% or more homology, preferably 98% or more homology, and more preferably 99% or more homology with the polypeptide sequence defined in (a), and has the function of the polypeptide of (a); or

[0009] (d) A derivative polypeptide having a sequence containing the polypeptide sequence described in (a) or (b) or (c).

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

[0011] (SEQ ID NO: 1).

[0012] The second aspect of the present invention provides a polynucleotide, wherein the polynucleotide is selected from the group consisting of:

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

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

[0015] (C) a polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 2, which is the CDS region sequence of the gene encoding protein GluA1-L1;

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

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

[0018] Wherein, the nucleotide sequence SEQ ID NO:2 is:

[0019]

[0020] The third aspect of the present invention 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 above-mentioned DNA molecule comprises a GluA1-L1 encoding gene with a nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3 and a promoter located upstream, wherein the promoter is, for example, a cauliflower mosaic virus (CAMV) 35S promoter, a rice-derived ubiquitin gene promoter, i.e., an Ubi promoter, or a promoter derived from Huajingxian 74 (Huagengxian74, HGX74) with a nucleotide sequence as shown in SEQ ID NO:4.

[0022] The nucleotide sequence SEQ ID NO: 3 is the complete gene sequence of GluA1-L1 consisting of the 5'UTR region, the 3'UTR region, introns and exons (coding region, ie, CDS).

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

[0024] The fifth aspect of the present invention provides a microbial engineering bacterium, which is a transformant containing the recombinant plasmid as described above, and is used to mediate the transfer of the recombinant plasmid as described above into plants such as rice or gramineous crops, and preferably the microorganism is Agrobacterium such as Agrobacterium tumefaciens, Agrobacterium EHA105, Agrobacterium GV3101. For example, the recombinant plasmid is transferred into the Agrobacterium strain by freeze-thaw method to form a microbial engineering bacterium.

[0025] Preferably, the microbial host of the above-mentioned microbial engineering bacteria is Agrobacterium, such as Agrobacterium tumefaciens, Agrobacterium EHA105, and Agrobacterium GV3101.

[0026] The sixth aspect of the present invention 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 gluten content in grains, in cultivating low-gluten plant varieties, in improving rice quality, or in cultivating high-gluten and / or high 1,000-grain weight and / or high single-plant yield plant varieties.

[0027] The plant is a grass crop selected from the group consisting of rice, wheat, corn, barley, oats, rye, sorghum, and millet, preferably the plant is rice.

[0028] In one application embodiment, the polypeptide such as GluA1-L1 as described above is overexpressed in plants such as rice by the following method, thereby increasing the gluten content in the grains, cultivating high gluten and / or high thousand-grain weight and / or high single-plant yield plant varieties such as new rice varieties:

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

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

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

[0032] In one embodiment, the expression of the gene GluA1-L1 in the chromosome of wild-type rice is downregulated, inactivated, weakened or knocked out by the following method, thereby reducing the gluten content in rice, improving the quality of rice, and cultivating new low-gluten rice varieties:

[0033] (1) Knock out the GluA1-L1 gene in the chromosomes of wild-type rice using gene editing technology;

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

[0035] (3) replacing the gene GluA1-L1 in the chromosome of wild-type rice with a mutant of the gene GluA1-L1 that encodes a gene with lost or downregulated function; and / or

[0036] (4) Block, inhibit or interfere with the expression of gene GluA1-L1 in chromosomes of wild-type rice.

[0037] Furthermore, the above method (2) can be selected from the following group:

[0038] (2-1) mutation of the promoter region and / or coding region of the GluA1-L1 gene leads to downregulation of the expression level of the GluA1-L1 gene;

[0039] (2-2) mutation of the upstream regulatory factor of the GluA1-L1 gene leads to downregulation of the expression level of the GluA1-L1 gene; or

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

[0041] Optionally, the gene editing technology described in the above step (ii) or step (1) can be selected from the following group: homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT.

[0042] The present invention discovered a new gene GluA1-L1 in rice for the first time, and preliminarily revealed its function of regulating the gluten content in rice endosperm and regulating the quality of rice. The new gene GluA1-L1 encodes a putative gluten precursor and is highly expressed in rice endosperm. The downregulation of the expression of GluA1-L1 leads to a significant decrease in its thousand-grain weight, insufficient grain filling, and an obvious powdery endosperm phenotype. This function enables it to become a gene locus that positively regulates rice quality, and is used to improve agronomic traits such as rice yield per plant and grain width. It has the application prospect of improving rice quality and creating low-gluten rice, and provides an important gene resource for rice quality improvement. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0044] Figure 2 Multiple sequence alignment of rice glutelins is shown. In the figure, the multiple sequence alignment of GluA1-L1 protein sequence with other rice glutelins such as GluA1, GluA2, GluA3, and GluB1a was performed using DNAMAN 8.0 software.

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

[0046] Figure 4 The grain development phenotypes of the wild type and the glua1-l1 mutant with the knockout gene GluA1-L1 are shown. (a) Grain development performance of the glua1-l1 mutant and the wild type ZH11, scale bar is 2 cm; (b) Genotype of the glua1-l1 mutant. The genotype of the glua1-l1 mutant was determined by PCR amplification using specific primers and sequencing, and then aligning it with the wild type ZH11 reference sequence.

[0047] Figure 5 The cross-sectional morphology of wild-type and glua1-l1 mutant grains under scanning electron microscopy is shown. In the figure, the cross-sectional morphology of starch granules in the grains of glua1-l1 mutant and wild-type ZH11 under scanning electron microscopy is compared. The scale bar is 20 μm.

[0048] Figure 6 The agronomic traits of the wild type and glua1-l1 mutant are shown. Among them, (ab) plant type and ear type of ZH11 and glua1-l1 mutant, the scale bar is 10cm and 2cm respectively; (cf) comparison of single plant yield, 1000-grain weight, grain length and grain width between ZH11 and glua1-l1 mutant, data are mean±SD, n=12, two-tailed t-test was used 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. (a) There is a GT SNP change in the coding region of the GluA1-L1 gene, which leads to premature termination of the CDS. This variation type mainly exists in Indica III; (bc) Haplotype analysis and phylogenetic tree of the promoter region of GluA1-L1, japonica rice is mainly dominated by haplotypes I and II, and indica rice is mainly dominated by haplotypes III and IV. DETAILED DESCRIPTION

[0050] While exploring the molecular mechanism of regulating storage protein content in rice, our research group identified a new gene locus GluA1-L1 that is highly expressed in the endosperm. Compared with the wild-type strain ZH11, the grains with GluA1-L1 knockout showed floury endosperm, and the thousand-grain weight, single-plant yield and grain width were significantly reduced, while there was no significant difference in grain length. GluA1-L1 was confirmed to be a gene locus that positively affects rice quality, suggesting that high-quality rice can be created by achieving in situ expression and increasing the transcription level of GluA1-L1 through gene editing and other methods. At the same time, GluA1-L1 has become a new breeding locus for creating low-gluten rice. The new mechanism of GluA1-L1 affecting rice quality revealed in this study provides new theoretical guidance for crop genetic improvement and molecular design breeding.

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

[0052] As used herein, the term "wild type" represents an original plant that has not been genetically modified or mutagenized, such as rice ZH11, Huajingxian 74, and the like.

[0053] Correspondingly, the terms "transgenic plants", "(plant) mutants" and "genetically engineered plants" in this article have the same meaning, and all refer to plants that have been genetically engineered from wild-type plants.

[0054] In the description of the technical solutions of the present invention, 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, the term "and / or" used in phrases such as "A, B and / or C" 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 terms "(gluten content / 1000-grain weight / yield per plant) increase", "enhance" or "increase" may 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 an increase of 100%, or any increase between 10%-100%, compared to a reference level.

[0056] Similarly, the term "reduction (in gluten content)" or "decreased" can mean a reduction of at least 10% compared to a reference level (such as wild-type rice), for example, a reduction 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 a 100% reduction, or any reduction between 10%-100%, compared to a reference level.

[0057] In this article, for the sake of simplicity of description, a certain protein such as GluA1-L1 is sometimes used interchangeably with the name of its coding gene (DNA) GluA1-L1. Those skilled in the art should understand that they represent different types of substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context. For example, for GluA1-L1, when used to describe the function or category of regulating the gluten content in the endosperm, it refers to the protein; when described as a gene, it refers to the coding gene of the protein.

[0058] Those skilled in the art can expect that it is reasonable that conservative variant polypeptides of GluA1-L1 having a high homology (identity) with the amino acid sequence of SEQ ID NO: 1, for example, more than 90%, have the same or similar functions.

[0059] As used herein, the term "conservative variant polypeptide" refers to a polypeptide that substantially maintains the same biological function or activity of the polypeptide. The variation refers in particular to a small amount of amino acid mutations, wherein the "mutation" includes, but is not limited to, replacement, deletion, insertion, chemical modification of amino acid residues, preferably a forward mutation, i.e., a mutation that improves the function of GluA1-L1. The substitution may be a non-conservative substitution, a conservative substitution, or a combination of a non-conservative substitution and a conservative substitution. "Conservative" amino acid substitutions or mutations refer to the interchangeability of residues with similar side chains, and therefore generally include substitutions of amino acids in the same or similar amino acid definition categories in the polypeptide. However, as used herein, if a conservative mutation may instead be 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 a restricted residue substitution, then the conservative mutation does not include a substitution of hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or a small residue to a small residue. It is well known in the art that common cases of conservative substitutions include: mutual substitutions between aromatic amino acids F, W, and Y; mutual substitutions between hydrophobic amino acids L, I, and V; mutual substitutions between polar amino acids Q and N; mutual substitutions between basic amino acids K, R, and H; mutual substitutions between acidic amino acids D and E; and mutual substitutions between hydroxyl amino acids S and 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 performed, for example, according to the following table.

[0060] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; 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) Ile; Leu; Met; Phe; Ala Leu

[0061] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups listed above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk.

[0062] "Deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. 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 reference GluA1-L1 (SEQ ID NO: 1) sequence, while retaining the improved properties of GluA1-L1 function. Deletion can be directed to the interior and / or ends of the polypeptide. In various embodiments, deletions can comprise continuous segments or can be discontinuous.

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

[0064] By making plants such as rice overexpress the protein GluA1-L1 with an amino acid sequence as shown in SEQ ID NO: 1, it is possible to breed transgenic plant varieties with desired agronomic traits such as high gluten content / high thousand-grain weight / high single plant yield.

[0065] The construction of such a genetically engineered plant overexpressing GluA1-L1 can be achieved by introducing a GluA1-L1 encoding gene such as SEQ ID NO: 2 or SEQ ID NO: 3 into the plant using conventional recombinant plasmid transformation methods such as Agrobacterium-mediated transformation or gene editing technology.

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

[0067] The existing plant expression vector can be used to construct a recombinant expression vector containing the coding gene. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment. The plant expression vector can also contain the 3' non-translated region of the foreign gene, that is, a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos), the non-translated region transcribed at the 3' end of the plant gene (such as the soybean storage protein gene) all have similar functions.

[0068] When the above coding gene is used to construct a recombinant expression vector, any enhanced promoter or constitutive promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (Ubiquitin), or tissue-specific expression promoter (such as seed-specific expression promoter) such as SEQ ID NO: 4) can be added before the transcription start 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 can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the enhancer is wide, and can be natural or synthetic. The translation start region can come from the transcription start 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 genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.

[0070] In the above method, a recombinant expression vector carrying the above coding gene is introduced into the recipient plant, specifically by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant tissue is cultivated into a plant.

[0071] Transformed cells, tissues or plants are understood to include not only the end product of the transformation process, but also their transgenic progeny.

[0072] Taking the construction of a transgenic plant overexpressing GluA1-L1 using the traditional 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, wherein the expression vector contains a coding sequence for the polypeptide GluA1-L1;

[0074] 2) contacting the plant cells, tissues or organs with the Agrobacterium in step 1) so that the coding sequence is transferred into the plant cells and integrated into the chromosomes of the plant cells;

[0075] 3) selecting plant cells or tissues into which the coding sequence has been transferred; and

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

[0077] In another embodiment, when constructing a transgenic rice variety with low gluten content, there are multiple technical means to inactivate, reduce and / or not express the GluA1-L1 gene in rice, and these technical means can be used alone or in combination. For example, one inactivation method is to perform a frameshift mutation on the GluA1-L1 encoding gene, so that the amino acid sequence of the polypeptide GluA1-L1 is changed and / or the translation is terminated.

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

[0079] Example

[0080] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

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

[0082] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., mainly with reference to "Molecular Cloning Experiment Guide" (3rd edition), compiled by J. Sambrook, DW Russell (USA), translated by Huang Peitang, etc., Science Press, Beijing, 2002). For example, the competent cell transformation method and the competent preparation method are all carried out with reference to "Molecular Cloning Experiment Guide" (3rd edition), Chapter 1, page 96. If necessary, the specific experimental conditions can be determined by simple experiments.

[0083] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0084] The primer synthesis and gene sequencing in the examples were commissioned to Sangon Biotech (Shanghai) Co., Ltd. and BGI.

[0085] Molecular biological methods and transgenic plant construction methods, including CRISPR-Cas9 system plasmid construction for gene GluA1-L1 knockout and gene editing technology, are performed using technical means commonly used in the field.

[0086] Plant materials

[0087] The wild type (WT) used in this study was ZH11 (kept in this laboratory). The mutants glua1-l1#1 and glua1-l1#2 due to GluA1-L1 gene deletion were obtained using CRISPR / cas9 technology and used for further experiments.

[0088] Genetic transformation

[0089] 1. Callus induction: Select mature and full rice seeds, remove the shells; disinfect with 75% alcohol for 1-2 minutes, pour off the alcohol; rinse twice with sterile distilled water; add 0.15% mercuric chloride (containing 0.1% Tween-20) and soak for 15-18 minutes, shaking several times; pour off the mercuric chloride and rinse 5 times with sterile distilled water. Inoculate the sterilized seeds into the callus induction medium and culture at 32℃ for 5-10 days.

[0090] 2. Activation by streaking of Agrobacterium: 2 days before infection, streak Agrobacterium on LB medium containing 50 mg / L kanamycin and culture at 28°C.

[0091] 3. Suspension, infection and co-cultivation of Agrobacterium: Before infection, scrape the activated Agrobacterium into the suspension medium, shake and culture at 28℃, 180rpm for 3-3.5h, and then adjust the bacterial solution concentration to OD600=0.1-0.2 with the suspension medium. Put the callus tissue induced for 5-10 days into the Agrobacterium suspension and infect for 1.5min. Pour out the bacterial solution and use sterile filter paper to absorb the bacterial solution on the surface of the callus. Cover the surface of the callus with sterile filter paper and blow dry it in the clean bench for 30min. After drying, transfer the callus to the co-cultivation medium covered with a layer of sterile filter paper on the surface, first culture it in the dark at 20℃ overnight, and then transfer it to a 25℃ incubator to continue dark culture for 2d.

[0092] 4. Cleaning bacteria: After the co-culture is completed, transfer the callus tissue to an empty sterilized container with tweezers. Wash the callus repeatedly with sterile distilled water for 7 to 8 times, of which the first 3 times can be washed quickly, and the next 3 to 4 times can be washed for 3-5 minutes each time. Finally, soak the callus in sterile distilled water containing 500 mg / L Cn for 30 minutes. Pour out the solution, use sterile filter paper to absorb as much water as possible on the surface of the callus, cover the surface of the callus with a layer of sterile filter paper, and blow dry it on a clean bench for 1 hour.

[0093] 5. Screening: Place the callus after sterilization on the screening medium and culture at 32℃ under light for 14 days.

[0094] 6. Differentiation: After 14 days of screening, the resistant calli were transferred to differentiation medium and cultured at 28°C (with a photoperiod of 14 h light / 10 h dark).

[0095] 7. Rooting: When the resistant callus forms 3-4 cm high regenerated seedlings on the differentiation medium, transfer them to the rooting medium and culture them until a complete plant is formed.

[0096] Rice field inspection

[0097] The wild-type ZH11 and mutant rice glua1-l1#1 and glua1-l1#2 were planted in the Shanghai Songjiang Experimental Base of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, with 18 plants of each line for phenotypic investigation and genetic identification.

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

[0099] We used a set of chromosome segment replacement lines constructed with the Asian cultivated rice indica (Oryza Sativa L.ssp.Indica) variety Huagengxian74 (HGX74) as the recipient parent and the common wild rice (Oryza rufipogon) as the donor parent to locate and clone QTLs related to heat tolerance in rice. In the initial positioning, we identified a Y261 replacement line material strain from a wild rice segment that had a temperature-dependent QTL that regulated rice yield and rice quality. In order to fully explore the gene regulatory network that regulates rice quality, we selected a near-isogenic line pair (NIL-TT5 Y261 、NIL-TT5 HGX74 ) Transcriptome sequencing was performed on grains in the filling stage. Among the differentially expressed genes in RNA-seq, there was a significantly downregulated gene, GluA1-L1, which encodes a putative glutenin gene and is specifically expressed in the endosperm, but not in leaves, stems and other tissues. We verified the difference in the expression level of GluA1-L1 in the near-isogenic lines by qRT-PCR, and consistent with the transcriptome results, the expression level of GluA1-L1 was significantly higher in NIL-TT5. Y261 The expression in kernels was weak.

[0100] Primers used for qRT-PCR:

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

[0102] 5'-GACCGACGAGGAGATGATGAG-3';

[0103] The 3' primer sequence is:

[0104] 5'-ATCTTGATGAGCTTGAGGACGG-3'.

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

[0106] To further verify the function of LOC_Os08g03410, we used CRISPR / Cas9 technology to create a knockout mutant of the GluA1-L1 gene - glua1-l1, selected the first exon region to design the knockout target, and constructed it into the CRISPR / Cas9 vector. Genetic transformation was carried out in the Japonica rice ZH11 background by the rice mature embryo transformation method mediated by Agrobacterium tumefaciens EHA105, positive plants were screened by hygromycin B, and the T2 generation and above were planted in Songjiang, Shanghai for phenotypic observation.

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

[0108] 5'-ggcACCAGCTCGACCAAACCTCC-3'.

[0109] The 3' primer sequence is:

[0110] 5'-aaacGGAGGTTTGGTCGAGCTGGT-3'.

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

[0112] Results and Discussion

[0113] 1. Through transcriptome data analysis, we screened out a highly expressed gene in the endosperm, GluA1-L1, which encodes a putative glutenin that affects rice grain quality.

[0114] In the previous work of this study, we used a set of chromosome replacement line materials constructed with the Asian cultivated rice indica variety Huagengxian74 (HGX74) as the recipient parent and the common wild rice (Oryza sativa L.ssp. Indica) as the donor parent to positionally clone QTLs regulating rice heat tolerance. We identified a Y261 replacement line material from a wild rice fragment, whose grains showed obvious floury endosperm phenotype, and subsequent research was carried out.

[0115] To explore the regulatory network of high temperature affecting rice quality, we selected grains of near-isogenic lines (carrying wild rice chromosome fragments and the genetic background of Huajingxian 74) 7 days after fertilization for transcriptome sequencing. Through RNA-seq analysis, we noticed that there was a significantly down-regulated gene among the differentially expressed genes, LOC_Os08g03410, which encodes a putative glutenin gene and is specifically expressed in the endosperm (such as Figure 1 To further characterize the function of this gene, its sequence similarity was analyzed and named GluA1-L1. At the same time, CRISPR / Cas9 technology was used to generate a homozygous knockout line glua1-l1 (as shown in Figure 2) in the background of the japonica rice variety Zhonghua11 (ZH11). Figure 2 and Figure 3 In the loss-of-function mutant glua1-l1, its grains have an obvious floury endosperm phenotype (as shown in Figure 4 shown).

[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 (e.g. Figure 3 Based on the above results, we believe that GluA1-L1 may be an atypical glutenin gene, and the protein it encodes has a large amino acid sequence difference from the existing GluA, GluB, GluC and GluD subfamily genes. GluA1-L1 may be a new type of glutenin subfamily gene, and the clear function of GluA1-L1 provides a new breeding site for creating low-glutenin rice and improving rice quality.

[0117] 2. The starch granules of the glua1-l1 mutant are spherical, irregularly and loosely arranged.

[0118] To further characterize the microstructure of the glua1-l1 mutant grains, we observed the starch granule morphology of the cross-sections of mature grains of wild-type ZH11 and glua1-l1 mutants by scanning electron microscopy. Compared with the neat and smooth cross-sections of ZH11 grains, the cross-sections of glua1-l1 mutants were irregular. The starch granules of the glua1-l1 mutant were mostly spherical, irregularly and loosely arranged, while the wild-type showed a uniform polyhedral structure, with regular and tight arrangement of starch granules and sharp edges (such as Figure 5This structural abnormality of starch granules may be the main reason for the poor quality of rice in the glua1-l1 mutant, and the functional loss of GluA1-L1 leads to powdery endosperm in the grains.

[0119] 3. The glua1-l1 mutant showed significant decreases in agronomic traits such as thousand-grain weight and yield per plant.

[0120] As a new glutenin gene, GluA1-L1 positively regulates rice quality. In order to further investigate the effect of GluA1-L1 on other agronomic traits, we investigated agronomic traits such as thousand-grain weight and yield per plant of the glua1-l1 mutant. Wild-type ZH11 and glua1-l1 mutant plants were planted in Songjiang, Shanghai, and there was no significant difference in plant height between the two. Compared with ZH11, the yield per plant and thousand-grain weight of the glua1-l1 mutant were significantly reduced, the grain width was reduced, but there was no significant difference in grain length (such as Figure 6 This indicates that GluA1-L1 affects the accumulation of grain dry matter, resulting in a decrease in 1000-grain weight and yield per plant. This also suggests that overexpression of the GluA1-L1 gene may 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 breeding high-quality rice.

[0122] Given that GluA1-L1 positively regulates rice quality, in order to fully explore its application value in cultivating high-quality rice. http: / / ricevarmap.ncpgr.cn / Haplotype analysis of SNP sites in the entire GluA1-L1 gene sequence (including the 2kb upstream promoter region and the 1kb downstream region of the 3'UTR) detected a total of about 415 SNP variations. Among them, there were about 40 variations in the gene coding region, about 200 in the promoter region, and about 110 SNP variations in the 3'UTR and the 1kb region downstream. There was a GT SNP change in its coding region (physical position 1597644), which caused GluA1-L1 to terminate prematurely. It was only retrieved in Indica III, accounting for about 1.10% (such as Figure 7 The discovery of this natural variation can provide a new genetic resource for creating low-gluten rice, and new rice varieties with lower gluten can be further cultivated by hybridization with low-gluten rice varieties. Considering its application in positively regulating rice quality, we conducted haplotype analysis on the 100 SNP sites before its start codon ATG and constructed a phylogenetic tree (as shown in Figure 2). Figure 7A total of 14 haplotypes were divided, among which haplotypes I and II were mainly japonica rice, and haplotypes III and IV were mainly indica rice. There was obvious differentiation between indica and japonica rice. The abundant SNP changes in the promoter and 3'UTR provided rich genetic resources for the exploration of high-expression level of GluA1-L1 and the cultivation of high-quality rice.

[0123] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by a person skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. A polypeptide, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, which is protein GluA1-L1; (b) a conservative variant polypeptide derived from (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence SEQ ID NO:1 and having the function of the polypeptide of (a); (c) a conservative variant polypeptide derived from (a) that has a homology of more than 95% with the polypeptide sequence defined in (a) and has the function of the polypeptide of (a); or (d) A derivative polypeptide having a sequence containing the polypeptide sequence described in (a) or (b) or (c).

2. A polynucleotide selected from the group consisting of: (A) a polynucleotide encoding the polypeptide of claim 1; (B) a polynucleotide encoding a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; (C) a polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 2, which is the CDS region sequence of the gene encoding protein GluA1-L1; (D) a polynucleotide having a nucleotide sequence with a homology of ≥ 95% to the nucleotide sequence shown in SEQ ID NO: 2; (E) A nucleotide sequence complementary to the nucleotide sequence described in any one of (A) to (D).

3. A DNA molecule, characterized in that Comprising the polynucleotide as claimed in claim 2.

4. The DNA molecule according to claim 3, characterized in that The present invention comprises a GluA1-L1 coding gene with a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 and a promoter located upstream, which is an expression box / frame of the protein GluA1-L1.

5. A recombinant plasmid, characterized in that: Used for expressing the protein GluA1-L1 as claimed in claim 1, comprising the DNA molecule as claimed in claim 3.

6. A microbial engineering bacterium, characterized in that: The transformant comprises the recombinant plasmid as claimed in claim 5.

7. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, the DNA molecule according to claim 3, the recombinant plasmid according to claim 4, or the microbial engineering bacteria according to claim 6 in regulating the gluten content in grains, in cultivating low-gluten plant varieties, in improving rice quality, or in cultivating high-gluten and / or high 1,000-grain weight and / or high single-plant yield plant varieties.

8. The use according to claim 7, characterized in that The polypeptide as claimed in claim 1, such as GluA1-L1, is overexpressed in plants such as rice by the following method, thereby increasing the gluten content in grains, cultivating plants with high gluten and / or high thousand-grain weight and / or high single-plant yield, such as new rice varieties: (i) cloning the polynucleotide as claimed in claim 2, such as the gene encoding the protein GluA1-L1, into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, i.e., a GluA1-L1 overexpression vector, and transforming the plant by Agrobacterium-mediated method to obtain a transgenic plant overexpressing the polypeptide as claimed in claim 1, such as GluA1-L1; and / or (ii) cloning the polynucleotide as claimed in claim 2, such as the gene encoding the protein GluA1-L1, into a plant chromosome by gene editing technology to obtain a transgenic plant that overexpresses the gene encoding the polypeptide as claimed in claim 1, such as GluA1-L1; and / or (iii) Placing the existing GluA1-L1 encoding gene in the plant genome under the regulation of a promoter with enhanced function.

9. The use according to claim 7, characterized in that The expression of the gene GluA1-L1 in the chromosome of wild-type rice is down-regulated, inactivated, weakened or knocked out by the following methods, thereby reducing the gluten content in rice and cultivating new low-gluten rice varieties: (1) Knock out the GluA1-L1 gene in the chromosomes of wild-type rice using gene editing technology; (2) down-regulating the expression level of the gene GluA1-L1 in the chromosomes of wild-type rice; (3) replacing the gene GluA1-L1 in the chromosome of wild-type rice with a mutant of the gene GluA1-L1 that encodes a gene with lost or downregulated function; and / or (4) Block, inhibit or interfere with the expression of gene GluA1-L1 in chromosomes of wild-type rice.

10. The use according to claim 9, characterized in that Method (2) is selected from the following group: (2-1) mutation of the promoter region and / or coding region of the GluA1-L1 gene leads to downregulation of the expression level of the GluA1-L1 gene; (2-2) mutation of the upstream regulatory factor of the GluA1-L1 gene leads to downregulation of the expression level of the GluA1-L1 gene; or (2-3) Introducing the interacting protein of GluA1-L1 into wild-type rice to change the function of the gene GluA1-L1.

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