Nucleic acid molecule ir340 for early flowering of rice and use thereof

Abstract

The present application belongs to the field of genetic engineering, and particularly relates to a nucleic acid molecule IR340 and application thereof. The present application creates a nucleic acid molecule IR340 with the function of enhancing expression and / or early flowering of rice.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a nucleic acid molecule IR340 and application thereof. BACKGROUND

[0002] Flowering is an important sign of the transition from vegetative growth to reproductive growth in plants. The timing of flowering not only determines the adaptability of plant materials in different regions, but also affects the accumulation of photosynthetic products, ultimately affecting biomass or yield. OsEhd1 is an important flowering regulation gene in rice, playing the role of a key convergence point for multiple signal transduction regulations. The 5' regulatory region of OsEhd1 contains a very rich response element of regulatory signals. Artificial modification of these elements using gene editing methods can affect the flowering date / heading date of rice. For example, CN108841855A and CN117625602A disclose a method of editing the promoter region of OsEhd1 to delay the flowering date / heading date of rice. However, it is not certain in the art whether editing the regulatory region of OsEhd1 can achieve early flowering date / heading date of rice. SUMMARY

[0003] The purpose of the present application is to provide a novel nucleic acid molecule.

[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0005] The present application provides a nucleic acid molecule, characterized in that the nucleic acid molecule sequence is shown in SEQ ID NO. 10.

[0006] The present application also provides an expression cassette, characterized in that the expression cassette is sequentially connected by the above-mentioned nucleic acid molecule, a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2, and a terminator sequence shown in SEQ ID NO. 3.

[0007] In some embodiments, the nucleotide sequence of the above-mentioned nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 4 or SEQ ID NO. 5.

[0008] The present application also provides a method for early flowering of rice, characterized in that it comprises any one of the following:

[0009] (1) using genetic engineering methods to target modify the sequence of the rice genomic region shown in SEQ ID NO. 1, and selecting plants with early flowering of rice;

[0010] (2) inserting the above-mentioned expression cassette into the rice genome, and selecting plants with early flowering of rice.

[0011] In some embodiments, the genetic engineering means described above employs a CRISPR / Cas9 gene editing method.

[0012] The present application also provides a kit for early flowering of rice, characterized in that it comprises any one of the following:

[0013] (1) an RNA molecule capable of simultaneously recognizing the target sequences shown in SEQ ID NO. 6 and SEQ ID NO. 7;

[0014] (2) a DNA molecule encoding the RNA of (1);

[0015] (3) a vector expressing the RNA of (1).

[0016] In some embodiments, the kit described above further comprises a Cas9 protein or a nucleic acid molecule encoding the Cas9 protein or a vector expressing the Cas9 protein.

[0017] In some embodiments, the RNA molecule described above is a combination of the sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9.

[0018] The present application also provides the use of the nucleic acid molecule described above in enhancing gene expression.

[0019] The present application also provides the use of the nucleic acid molecule, the expression cassette, the method or the kit described above in early flowering of rice.

[0020] The present application also provides a detection method for detecting whether the rice contains the nucleic acid molecule described above, characterized in that the genomic DNA sample of the rice to be detected is used as a template, and primers 5'-GACGGCTGGGAACATATGCAC-3' and 5'-TCCCAGCTTCAGTGTTTGCT-3' are used for PCR amplification; if the amplification product contains a 308 bp band, the rice to be detected contains the nucleic acid molecule.

[0021] The innovation and beneficial effects of the present application are as follows: in the background of the complexity and unpredictability of the OsEhd1 regulatory region, the present application obtains a new type of nucleic acid molecule IR340, which has the technical effects of improving gene expression and early flowering of rice. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Regulatory elements between T9 and T11 target sites.

[0023] Figure 2 Gene editing vector map.

[0024] Figure 3Picture of heading date of IR340 homozygous (middle) and heterozygous mutant plants (right) and wild type plants (left).

[0025] Figure 4 Schematic diagram of genotype structure of IR340.

[0026] Figure 5 Expression amount of OsEhd1, OsHd3a and OsRFT1 genes in IR340.

[0027] Figure 6 Detection of IR340. DETAILED DESCRIPTION

[0028] The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise defined, all terms used are in accordance with common usage of the same. All patents, patent applications, academic papers, industry standards and other published publications cited herein are incorporated by reference in their entirety.

[0029] As used herein, "rice" is any rice plant and includes all plant varieties that can be bred with rice, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, roots, root tips, anthers, and the like. Nucleic acids are written left to right in 5' to 3' orientation, unless otherwise indicated; amino acid sequences are written left to right in amino to carboxyl orientation, unless otherwise indicated. The amino acids can be represented by either their commonly accepted single-letter codes or IUPAC-IUB Biochemical Nomenclature Commission recommended single letter codes. Likewise, nucleotides can be represented by the commonly accepted single letter codes. Numerical ranges are inclusive of the numbers defining the range. As used herein, "nucleic acid" includes polynucleosides of deoxyribonucleotides or ribonucleotides in either single- or double-stranded form, and unless otherwise limited, includes known analogues of natural nucleotides that have similar binding properties as the naturally occurring nucleotides (e.g., peptide nucleic acids) that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the term "encoding" or "encoded" with reference to a specified nucleic acid indicates that the nucleic acid includes the information necessary to direct the synthesis of a specified protein. The information is in the form of codons that indicate the presence of a specific amino acid or a truncation of the protein. As used herein, "full-length sequence" with reference to a particular polynucleotide or its encoded protein refers to the entire nucleic acid sequence or the entire amino acid sequence having the native (non-synthetic) endogenous sequence. A full-length polynucleotide encodes a full-length, catalytically active form of the particular protein. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term is used to refer to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids. The term is also used to refer to naturally occurring amino acid polymers. The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively "protein"). The amino acid can be a naturally occurring amino acid and, unless otherwise limited, can include known analogues of natural amino acids that can function in a manner similar to the naturally occurring amino acids.

[0030] As used herein, the terms "isolated" and "purified" are used interchangeably to refer to a nucleic acid or polypeptide, or biologically active portion thereof, that is substantially or essentially free from components, which naturally accompany or potentially accompany the nucleic acid or polypeptide as found in its natural environment. Thus, an isolated or purified nucleic acid or polypeptide produced by recombinant techniques is substantially free of other cellular material or culture medium when produced by recombinant techniques, or is substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" nucleic acid is generally free from sequences (such as protein encoding sequences) that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid can comprise less than about 0.5 kb of nucleotide sequences that naturally flank the nucleic acid in the genomic DNA of the cell from which the nucleic acid is derived.

[0031] In this application, the words "comprise," "comprises," or "comprising" are used in their inclusive, open-ended sense, that is, meaning "including, but not limited to," and allow the inclusion of additional elements, numbers, or steps to those that are described. A "subject plant" or "subject plant cell" refers to a plant or plant cell in which a genetic modification has taken effect, or a progeny cell of such a modified plant or cell that comprises the modification. A "control" or "control plant" or "control plant cell" provides a reference point against which a change in phenotype of a subject plant or plant cell is measured. A control plant or plant cell can include, for example: (a) a wild-type plant or cell, i.e., a plant or cell of the same genotype as the starting material for a genetic modification that produced the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but that has been transformed with an empty construct, i.e., a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene; (c) a plant or plant cell that is a non-transformed segregant of the subject plant or plant cell; (d) a plant or plant cell that is genetically identical to the subject plant or plant cell but that has not been exposed to a condition or stimulus that induces expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

[0032] Those of skill in the art will readily recognize, for example, that advances in the field of molecular biology, such as site-specific mutagenesis and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques, provide a wide range of appropriate tools and procedural steps for modifying or engineering the amino acid sequence and potentially the genetic sequence of a protein of interest in agriculture.

[0033] In some embodiments, alterations can be made to the nucleotide sequences of the application to make conservative amino acid substitutions. Principles and examples of conservative amino acid substitutions are further described below. In certain embodiments, alterations can be made to the nucleotide sequences of the application that do not change the amino acid sequence, e.g., the codons encoding the same amino acid sequence can be replaced with monocot plant preferred codons, without changing the amino acid sequence encoded by the nucleotide sequence. In some embodiments, portions of the nucleotide sequences in the application are replaced with different codons that encode the same amino acid sequence, thereby changing the nucleotide sequence while not changing the amino acid sequence it encodes. Conservative variants include those sequences that encode the same amino acid sequence of a protein of the embodiments due to the degeneracy of the genetic code. In some embodiments, portions of the nucleotide sequences in the application are replaced according to monocot plant preferred codons. Those of skill in the art will recognize that amino acid additions and / or substitutions generally are based on the relative similarity of the amino acid side chains, for example, as is shown by the hydrophobic, charged, size, and other properties of the amino acids. Exemplary amino acid substitution groups that take various of the foregoing properties into consideration are well-known in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Guidance in appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C), incorporated herein by reference. Conservative substitutions can be made, such as replacing one amino acid with another having similar properties. Identification of sequence identity includes hybridization techniques. For example, all or a portion of a known nucleotide sequence is used as a probe to selectively hybridize to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments from a selected organism (i.e., a genomic or cDNA library).

[0034] In some embodiments, fragments of the nucleotide sequences and the amino acid sequences they encode are also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide or a portion of the amino acid sequence of a polypeptide of the embodiments. Fragments of the nucleotide sequence can encode protein fragments that retain the biological activity of the native or corresponding full-length protein and thus have the protein activity. Mutant proteins include biologically active fragments of the native protein that comprise contiguous amino acid residues that retain the biological activity of the native protein. Some embodiments also include transformed plant cells or transgenic plants comprising at least one nucleotide sequence of the embodiments. In some embodiments, plants are transformed using an expression vector comprising at least one nucleotide sequence of the embodiments operably linked to a promoter that drives expression in plant cells. Transformed plant cells and transgenic plants represent plant cells or plants that comprise a heterologous polynucleotide within the genome. Generally, the heterologous polynucleotide is stably integrated within the genome of the transformed plant cell or transgenic plant such that the polynucleotide is passed to the progeny. The heterologous polynucleotide can be integrated into the genome either alone or as part of an expression vector. In some embodiments, plants contemplated by the present application include plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, fruits, ears, kernels, husks, cobs, roots, root tips, anthers, and the like. The present application also includes plant cells, protoplasts, tissues, calli, embryos, and flowers, tassels, ears, grain, and other plant reproductive materials that are produced by, or are derived from, a transgenic plant or its progeny, and thus contain the nucleotide sequences of the present application in at least one of their cells.

[0035] The following examples are intended to illustrate the present application and are not intended to limit the scope of the application. Modifications or substitutions of the methods, steps or conditions of the present application are considered to be within the scope of the present application without departing from the spirit and scope of the present application. Unless otherwise specified, the examples were performed according to conventional experimental procedures, such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2001) or according to the conditions suggested by the manufacturer. Unless otherwise specified, the chemical reagents used in the examples were conventional commercially available reagents and the techniques used in the examples were conventional techniques known to those skilled in the art.

[0036] Example

[0037] Example 1 Analysis of Ehd1 Promoter Elements

[0038] Ehd1 is a positive regulator of flowering time in rice. Overexpression of Ehd1 can promote early flowering in rice, thereby shortening the growth period and reducing planting costs. However, the strategy of creating early-maturing rice by transgenically overexpressing Ehd1 is too costly, and knocking out Ehd1 using gene editing technology cannot achieve the technical effect of early flowering in rice. Editing the promoter can regulate gene expression, but so far, the field technicians have edited the Ehd1 promoter, which has shown a technical effect of delaying flowering time.

[0039] The inventors aim to achieve the technical effect of early flowering in rice by editing the Ehd1 promoter. Through online software CRISPR-GE (http: / / skl.scau.edu.cn / ), the target site analysis of the expression regulatory region of Ehd1 was performed. According to the GC content of the target site, the off-target possibility, etc., 15 target sites were selected, and a plurality of editing vectors were constructed by grouping these target sites two by two. Using online plant 5' cis-regulatory element analysis software PLACE (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace), the sequence between the two target sites was predicted for cis-regulatory elements, and it was found that there were rich regulatory elements between T9 and T11 target sites (sequences as shown in SEQ ID NO. 6 and SEQ ID NO. 7), including 3 CAAT-boxes, 2 TATA-boxes, 2 A-boxes, 1 Box4, dehydration stress response element (DRE), 1 ABA response element (ABRE) and other cis-acting elements. Figure 1 ) Some of these cis-acting elements may bind or interact with the upstream or downstream genes of Ehd1 to directly regulate the heading date of rice.

[0040] Example 2 Targeted editing of the Ehd1 regulatory region

[0041] According to the target sequence, a nucleic acid molecule encoding an sgRNA molecule was synthesized, which could recognize the designed target sequence. A CRISPR / Cas9 editing vector was constructed, which was a commonly used vector in the field of rice gene editing, and could express sgRNA recognizing the target sequence and editing enzyme Cas9 in rice, while carrying the antibiotic resistance gene required for genetic transformation (vector map see Figure 2 ).

[0042] The editing vector was transferred into Agrobacterium strain EHA105, and infected the embryonic callus induced from the seed of the restorer line R1607. After conventional infection, co-culture, selection, differentiation and rooting, the transformed plants were obtained.

[0043] Example 3 Identification of genotype and phenotype of edited plants

[0044] R1607 was transformed with the editing vector pGGM567, and 67 T0 plants were obtained. DNA fragment PCR amplification and sequencing found that 8 plants had large fragment deletions at the target position of the Ehd1 gene, and these plants had significantly delayed heading time than wild type. In addition, a very special plant material was identified: the PCR detection and sequencing results of T0 plants were a genotype similar to the homozygous deletion of 340bp mutant, and the forward primers flanking the two targets of the editing vector could only amplify a band smaller than that of the wild type plant, with a 340bp deletion, but its heading date phenotype was completely opposite to that of the homozygous deletion mutant, with a heading date significantly advanced by about 15 days Figure 3 ). More strangely, the heading date of the T1 population of this mutant also showed obvious early and late separation, with a ratio of about 3:1. Further identification results showed that the Ehd1 promoter position of these early heading plants produced an inverted repeat sequence, that is, during the CRISPR / Cas9-mediated gene editing process, a 340bp DNA fragment was removed from one sister chromatid, and this fragment was inserted into the other sister chromatid in the opposite direction, producing a 340bp inverted repeat sequence. The inventors named the inverted repeat insertion homozygous mutant IR340. From the phenotype of T0 and T1 plants of IR340, the mutant has very strong dominant characteristics and is a rare gain-of-function mutant. Crossing homozygous IR340 with wild type R1607, the heterozygous F1 individuals also showed the same heading date as the homozygous mutant, about 17 and 14 days earlier than wild type R1607, respectively. Detecting the expression of OsEhd1 gene in plants 45 days after sowing, it was found that the expression of OsEhd1 gene in homozygous and heterozygous plants of IR340 was significantly increased, reaching 3.0-3.7 times of that in wild type plants (Table 1), further proving that the early maturity characteristics of IR340 have obvious dominant characteristics.

[0045] Table 1 Heading date performance of edited rice IR340

[0046]

[0047] In summary, the data analysis results of the genotype of the IR340 early heading mutant showed that IR340 produced a 340bp sequence inverted repeat near the target position shown in SEQ ID NO. 7 (schematic diagram see Figure 4 ). The promoter sequence in IR340 is shown in SEQ ID NO. 10.

[0048] The IR340 pure-bred strain is transplanted into a greenhouse, and leaves are taken from the plants 45 days after sowing, with unedited R1607 as a control (CK). Through qRT-PCR detection, the expression of three heading-related genes OsEhd1, OsHd3a and OsRFT1 is significantly improved compared with the control. Figure 5

[0049] Detection method of IR340

[0050] The application further develops a detection method of IR340, specifically as follows:

[0051] The detection primers 5'-GACGGCTGGGAACATATGCAC-3' and 5'-TCCCAGCTTCAGTGTTTGCT-3' are used to detect plants carrying the IR340 site through PCR amplification and agarose gel electrophoresis, and plants carrying the IR340 site can amplify a DNA fragment of 308 bp in size, while plants not carrying the IR340 site cannot amplify a DNA fragment of 308 bp in size. Figure 6

[0052] Although the application has been described in detail with general description and specific embodiments, some modifications or improvements can be made on the basis of the application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the application, all belong to the scope of protection required by the application.​​

Claims

1. A nucleic acid molecule, characterized by: The nucleic acid molecule sequence is shown in SEQ ID NO.

10.

2. An expression box, characterized in that: It is formed by sequentially linking the nucleic acid molecule as described in claim 1, the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2, and the terminator of the sequence shown in SEQ ID NO.

3.

3. The expression box according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO.

5.

4. The application of the nucleic acid molecule according to claim 1 in enhancing the expression of the OsEhd1 gene.

5. The application of the expression cassette according to any one of claims 2-3 in advancing the flowering period of rice.

6. A method for detecting whether rice contains the nucleic acid molecule described in claim 1, characterized in that: Using a genomic DNA sample of the rice to be tested as a template, and primer pairs 5'-GACGGCTGGGAACATATGCAC-3' and 5'-TCCCAGCTTCAGTGTTTGCT-3' were used for PCR amplification; if the amplification product contains a 308 bp band, then the rice to be tested contains the nucleic acid molecule described in claim 1.

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