GRF1 gene and its application in the regulation of skin prickles.

Abstract

This invention relates to a GRF1 gene and its application in regulating the prickle trait, belonging to the field of plant genetic engineering. This invention discloses the GRF1 gene and its application in regulating the prickle trait.

Description

Technical Field

[0001] This invention relates to a GRF1 gene and its application in the regulation of prickly traits, belonging to the field of plant genetic engineering. Background Technology

[0002] Many plants in the genus *Rosa* have high ornamental value; however, many species, such as roses, hybrid tea roses, and wild roses, have thorns on their stems, causing considerable inconvenience in field cultivation, flower harvesting, and transportation. Therefore, breeding new thornless or soft-thorned varieties of *Rosa* will effectively improve their quality, significantly increase production efficiency, and substantially reduce production costs.

[0003] Thorns are widely present in plants, formed by protrusions of epidermal cells and subcutaneous tissue on plant stems and branches. They share some similarities in origin with epidermal hairs. In recent years, significant research has been conducted on the molecular mechanisms of plant thorn formation, and several candidate genes related to thorn development have been isolated, such as CPC, WER, MYB5 (Yuan Xiaoyu. Research on the Discovery and Application of Genes Related to Rose Thorn Formation [D]. Yangzhou University, 2019.), and TTG1 (Luan Xiaofang. Cloning and Expression Analysis of the Transcription Factor RrTTG1 Related to Rose Thorn Formation [D]. Yangzhou University, 2014.). Therefore, identifying genes that regulate thorn formation is particularly important.

[0004] To address the aforementioned issues, this invention analyzed the transcriptomes of the prickle primordia and stem epidermis of both thorny and thornless wild roses, identifying differentially expressed genes in these tissues. Simultaneously, temporal transcriptome analysis of wild rose prickles identified characteristic genes for prickle formation. Integrating the data from both transcriptomes yielded 700 prickle-related genes. Expression pattern validation revealed that RmGRF1 is a predominantly expressed gene in the prickle primordia. Further cloning of this gene and transformation into Arabidopsis thaliana resulted in larger, unchanged leaf size and number, as well as larger, unchanged leaf epidermal and palisade cells. Interfering with the GRF1 gene in roses using RNAi technology inhibited prickle formation, resulting in roses with fewer prickles. Summary of the Invention

[0005] One of the objectives of this invention is to provide a Rosa gene RmGRF1.

[0006] The second objective of this invention is to disclose the application of the above-mentioned genes in regulating the skin prickles trait.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides the application of a gene in regulating the size of plant epidermal cells and / or palisade cells, characterized in that the gene is any one of the following:

[0009] 1) Genes with sequences as shown in SEQ ID NO:1 or SEQ ID NO:3;

[0010] 2) Genes encoding protein sequences as shown in SEQ ID NO:2 or SEQ ID NO:4.

[0011] The present invention also provides an application of an expression cassette in regulating the size of plant epidermal cells and / or palisade cells, characterized in that the expression cassette contains the aforementioned gene.

[0012] In some implementations, the expression cassette consists of the CaMV 35S promoter, the gene described above, and the nos terminator linked sequentially.

[0013] The present invention also provides an application of an expression vector in regulating the size of plant epidermal cells and / or palisade cells, characterized in that the expression vector contains the above-described expression cassette.

[0014] The present invention also provides the application of a host cell in regulating the size of plant epidermal cells and / or palisade cells, characterized in that the host cell contains the above-mentioned expression cassette or the above-mentioned expression vector; the host cell is a microbial cell or a non-regenerative plant cell.

[0015] The present invention also provides a method for reducing the number of thorns in Rosa species, characterized by reducing the expression of the above-mentioned genes in Rosa species and selecting plants with reduced thorn number.

[0016] In some implementations, the above-mentioned method for reducing gene expression is to use RNAi interference.

[0017] In some implementations, the interference fragment used in the above-described RNAi interference method is as shown in bases 1362-1577 of SEQ ID NO.1 or bases 1362-1577 of SEQ ID NO.3, or their reverse sequence.

[0018] Compared with existing technologies, the beneficial effects of this invention are: this invention obtains the thorn-dominantly expressed gene RmGRF1 and discovers that this gene has the function of regulating the size of plant epidermal cells and / or palisade cells. Inhibiting this gene can reduce the number of thorns in Rosa species. Attached Figure Description

[0019] Figure 1 Wild rose and its thornless germplasm were used to compare sampling sites for transcriptome sequencing.

[0020] Figure 2 Wild rose was used as a sampling site for time-series transcriptome sequencing of prickles.

[0021] Figure 3 Spatiotemporal representation of RmGRF1. Prickle represents spiculated tissue.

[0022] Figure 4 Map of the RmGRF1 overexpression vector used for heterologous transformation of Arabidopsis thaliana.

[0023] Figure 5 Phenotyping of transgenic Arabidopsis plants. a-b) Rosette leaf phenotype; cf) Leaf length, leaf width, petiole length, leaf area, and number of leaves of the 5th true leaf. n>18.

[0024] Figure 6 Epidermal cells of transgenic Arabidopsis leaves. a) Epidermal cell morphology of wild type and three RmGRF1 transgenic lines (1, 2, 11); b) Statistical analysis of epidermal cell area of ​​leaves; c) Statistical analysis of epidermal cell number of leaves.

[0025] Figure 7 Palisade cells in transgenic Arabidopsis leaves. a) Palisade cell morphology in wild-type and three RmGRF1 transgenic lines (1, 2, 11); b) Palisade cell area statistics in leaves; c) Palisade cell number statistics in leaves.

[0026] Figure 8 RrGRF1-RNAi vector map. Detailed Implementation

[0027] The following definitions and methods are provided to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terms are to be understood in accordance with their conventional usage by those skilled in the art. All patent literature, academic papers, industry standards, and other publicly available publications cited herein are incorporated herein by reference in their entirety.

[0028] Unless otherwise specified, nucleic acids are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino-to-carboxyl orientation. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include numbers that define the range. As used herein, “nucleic acid” includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides and hybridize with single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the terms “encoding” or “encoded” when used in the context of a particular nucleic acid mean that the nucleic acid contains the essential information to guide the translation of that nucleotide sequence into a particular protein. Codons are used to represent the information encoding the protein. As used herein, “full-length sequence” referring to a particular polynucleotide or the protein it encodes means the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence. Full-length polynucleotides encode the full-length, catalytically active form of the specific protein. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. This term is also used for naturally occurring amino acid polymers. The terms “residue,” “amino acid residue,” or “amino acid” are used interchangeably herein to refer to an amino acid incorporated into a protein, polypeptide, or peptide (collectively, “protein”). Amino acids can be naturally occurring amino acids and, unless otherwise limited, may include known analogs of natural amino acids that can function in a similar manner to naturally occurring amino acids.

[0029] "Plant" includes indexes for whole plants, plant organs, plant tissues, seeds, and plant cells, as well as their offspring. Plant cells include, but are not limited to, cells from seeds, suspension cultures, plumules, meristematic regions, callus, leaves, roots, seedlings, gametophytes, sporophytes, pollen, and microspores. "Offspring" includes any subsequent generations of a plant.

[0030] In this application, the terms "comprising," "including," or variations thereof should be understood to include other elements, numbers, or steps besides those described. "Test plant" or "test plant cell" refers to a plant or plant cell in which genetic modification has taken effect, or a progeny cell of such a modified plant or cell containing the modification. "Control," "control plant," or "control plant cell" provides a reference point for measuring phenotypic changes in the test plant or plant cell.

[0031] Negative or control plants may include, for example: (a) wild-type plants or cells, i.e., plants or cells with the same genotype as the genetic modification starting material, the genetic modification producing the test plants or cells; (b) plants or plant cells with the same genotype as the starting material but transformed with an empty construct (i.e., a construct with no known effect on the target trait, such as a construct containing the target gene); (c) plants or plant cells that are non-transformed isomers of the test plants or plant cells; (d) plants or plant cells that are genetically identical to the test plants or plant cells but not exposed to conditions or stimuli that would induce the expression of the target gene; or (e) the test plants or plant cells themselves, which are under conditions where the target gene is not expressed.

[0032] Those skilled in the art will readily recognize that advances in molecular biology, such as site-specific and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques, have provided a wide range of appropriate tools and procedures for modifying or engineering the amino acid sequences and potential gene sequences of proteins of interest in agriculture.

[0033] In some embodiments, the nucleotide sequence of this application may be modified to perform conserved amino acid substitutions. Principles and examples of conserved amino acid substitutions are further described below. In some embodiments, the nucleotide sequence of this application may be substituted without altering the amino acid sequence according to disclosed monocotyledonous codon preferences; for example, a codon encoding the same amino acid sequence may be substituted with a codon preferred by monocotyledons without changing the amino acid sequence encoded by the nucleotide sequence. In some embodiments, a portion of the nucleotide sequence in this application may be substituted with a different codon encoding the same amino acid sequence, thereby changing the nucleotide sequence without altering the encoded amino acid sequence. Conserved variants include those sequences that encode an amino acid sequence of one of the proteins of the embodiments due to genetic codon degeneracy. In some embodiments, a portion of the nucleotide sequence in this application may be substituted according to a codon preferred by monocotyledons. Those skilled in the art will recognize that amino acid additions and / or substitutions are generally based on the relative similarity of amino acid side-chain substituents, such as the hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substituents having the various properties considered above are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC) (incorporated herein by reference). Conserved substitutions, such as replacing one amino acid with another amino acid having similar properties, can be performed. Sequence identity verification includes hybridization techniques. For example, a known nucleotide sequence, in whole or in part, can be used as a probe for selective hybridization with other corresponding nucleotide sequences present in cloned genomic DNA fragments or cDNA fragment groups (i.e., genomic libraries or cDNA libraries) from a selected organism. The hybridization probe may be a genomic DNA fragment, cDNA fragment, RNA fragment, or other oligonucleotide, and may be labeled with a detectable group such as 32P or other detectable markers. Thus, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides based on sequences from the embodiment. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are generally known in the art. Hybridization of the sequences can be performed under stringent conditions. As used herein, the terms "stringent conditions" or "stringent hybridization conditions" refer to conditions under which the probe will hybridize with its target sequence to a detectable extent (e.g., at least 2, 5, or 10 times the background) relative to hybridization with other sequences.Harsh conditions are sequence-dependent and vary across different environments. By controlling hybridization harshness and / or washing conditions, target sequences 100% complementary to the probe can be identified (homologous probe method). Alternatively, harsh conditions can be adjusted to allow for some sequence mismatches in order to detect lower similarities (heterologous probe method). Typically, probe lengths are less than about 1000 or 500 nucleotides. Typically, harsh conditions are those where the salt concentration is less than about 1.5 M Na ions at pH 7.0 to 8.3, typically about 0.01 M to 1.0 M Na ion concentration (or other salts), and the temperature conditions are: at least about 30 °C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides). Harsh conditions can also be achieved by adding a destabilizing agent such as formamide. Exemplary low-threshold conditions include hybridization at 37°C using 30% to 35% formamide buffer, 1M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing at 50°C to 55°C in 1× to 2× SSC (20× SSC = 3.0M NaCl / 0.3M trisodium citrate). Exemplary medium-threshold conditions include hybridization at 37°C using 40% to 45% formamide, 1.0M NaCl, and 1% SDS, followed by washing at 55°C to 60°C in 0.5× to 1× SSC. Exemplary high-threshold conditions include hybridization at 37°C using 50% formamide, 1M NaCl, and 1% SDS, followed by a final wash at 60°C to 65°C in 0.1× SSC for at least about 20 minutes. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Hybridization duration is typically less than about 24 hours, typically from about 4 hours to about 12 hours. Specificity typically depends on post-hybridization washing, with key factors being the ionic strength and temperature of the final washing solution. The Tm (thermodynamic melting point) of DNA-DNA hybrids can be approximated by the formula from Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5℃ + 16.6 (logM) + 0.41 (%GC) - 0.61 (%formamide) - 500 / L; where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, "formamide%" is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. Tm is the temperature at which 50% of the complementary target sequence hybridizes with a perfectly matched probe (at a given ionic strength and pH). Washing is typically performed at least until equilibration is reached and a low hybridization background level is achieved, such as for 2 hours, 1 hour, or 30 minutes. Each 1% mispairing should lower Tm by approximately 1°C; therefore, Tm, hybridization, and / or washing conditions can be adjusted to hybridize with the desired sequence of homology. For example, if a sequence with ≥90% homology is required, Tm can be lowered by 10°C.Typically, the stringency conditions are selected to be approximately 5°C lower than the Tm of the specific sequence and its complementary sequence at the defined ionic strength and pH. However, under very stringent conditions, hybridization and / or washing can be performed at 4°C lower than the Tm; under moderately stringent conditions, hybridization and / or washing can be performed at 6°C lower than the Tm; and under low stringency conditions, hybridization and / or washing can be performed at 11°C lower than the Tm.

[0034] In some embodiments, a fragment of a nucleotide sequence and the amino acid sequence it encodes is also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide of an embodiment or a portion of the amino acid sequence of a polypeptide. A fragment of the nucleotide sequence may encode a protein fragment that retains the biological activity of the native or corresponding full-length protein and thus has protein activity. Mutant proteins include biologically active fragments of native proteins containing consecutive amino acid residues that retain the biological activity of the native protein. Some embodiments also include transformed plant cells or transgenic plants containing a nucleotide sequence of at least one embodiment. In some embodiments, plants are transformed using an expression vector containing a nucleotide sequence of at least one embodiment and a promoter operatively linked thereto that drives expression in plant cells. Transformed plant cells and transgenic plants represent plant cells or plants whose genome contains a heteropolynucleotide. Generally, the heteropolynucleotide is stably integrated into the genome of the transformed plant cell or transgenic plant to pass the polynucleotide to offspring. The heteropolynucleotide may be integrated into the genome alone or as part of an expression vector. In some embodiments, the plants involved in this application include plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant masses, and plant cells that are whole plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, rachis, husks, straw, roots, root tips, anthers, etc. This application also includes plant cells, protoplasts, tissues, callus, embryos, flowers, stems, fruits, leaves, and roots derived from transgenic plants of this application or their progeny, and thus at least partially containing the nucleotide sequences of this application.

[0035] In the context of nucleic acid amplification, the term "amplification" refers to any process in which an additional copy of a selected nucleic acid (or its transcribed form) is produced. Common amplification methods include replication methods based on various polymerases, including polymerase chain reaction (PCR), ligase-mediated methods such as ligase chain reaction (LCR), and RNA polymerase-based amplification methods (e.g., via transcription).

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance thereof are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0037] Example 1: Discovery and Expression Detection of Splinter Development Genes

[0038] Wild rose (Rosa multiflora) is a deciduous shrub belonging to the genus Rosa. It is diploid, with thorns on its stems and petioles, and is one of the main parents of modern roses. Thornless wild rose (Rosa multiflora Thunb. var. inermis) is a variety of wild rose with smooth stems and no thorns. Both the wild rose and thornless wild rose used in this invention are common species cultivated at Huazhong Agricultural University, and are also widely cultivated elsewhere and are available to the public.

[0039] Total RNA was extracted from the prickle primordium of *R. multiflora* stem (PP), the immature bark of *R. multiflora* stem (IB), and the immature bark of *R. multiflora* 'inermis' No. 1 stem (IBI) (sampling sites are shown in the table below). Figure 1 Total RNA was extracted from the thorns of wild rose at five developmental stages (I–V) (sampling sites are shown in [reference]). Figure 2After total RNA extraction and DNase I treatment, mRNA was isolated using Oligo(dT) magnetic beads to construct cDNA libraries. The quality of the cDNA libraries was assessed using an ABI StepOnePlus Real-Time PCR system and an Agilent 2100 bioanalyzer, and sequencing was performed on an Illumina HiSeq™ 2000 sequencing platform. In the comparative transcriptome experiments of wild rose and thornless wild rose, three biological replicates were set up for each sample, resulting in a total of nine samples. After sequencing, the data were analyzed to identify differentially expressed genes in different samples. In the time-series transcriptome experiments of wild rose thorns, four biological replicates were set up for each sample, resulting in a total of 20 samples. After sequencing, the data were analyzed to identify characteristic genes in different samples.

[0040] A total of 6,728 differentially expressed genes were obtained in the three comparison groups of PP vs IB, PP vs IBI, and IB vs IBI, and 11,976 characteristic genes were obtained in the temporal transcriptome analysis of wild rose thorns. Combined analysis of the two sets of RNA-seq data yielded a total of 700 thorn-related genes.

[0041] Among the aforementioned thorn-related genes, we sought genes that exhibited high expression levels in PP tissues but low or no expression in IB and IBI tissues. Furthermore, these genes also needed to meet the characteristic of having the highest expression level in thorns during stage I, followed by a continuous decline. Based on these screening criteria, a gene homologous to the Arabidopsis thaliana GROWTH-REGULATING FACTOR 1 (GRF1) gene was found to simultaneously meet the above conditions. This gene from *Rosa rugosa* was named RmGRF1. The RmGRF1 gene sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0042] The inventors conducted further functional tests on RmGRF1. The spatiotemporal expression pattern of the RmGRF1 gene was detected using qRT-PCR. The results showed that the gene was most highly expressed in the prickle primordia, with relatively high expression in terminal buds, lateral buds, and leaves, while expression was low or extremely low in other tissues. Samples from the prickle development stages I–V of *Rosa rugosa* were collected, and the expression level in these samples was detected by qRT-PCR. The results showed that the gene expression was highest in stage I prickles, very low in stage II prickles, and continued to decline thereafter until stage V. Figure 3 These results indicate that RmGRF1 exhibits a spiculation-dominant expression characteristic, and its expression pattern is closely related to spiculation initiation.

[0043] Example 2: Gene Function Study

[0044] The inventors heterologously transformed RmGRF1 into Arabidopsis thaliana to study its function. The vector used CaMV 35S as the promoter and NOS as the terminator. For ease of study, the gene was preceded by a GFP reporter gene and a His tag (see vector diagram). Figure 4 The Arabidopsis transformation method employed was the conventional Agrobacterium-mediated transformation technique. Phenotypic observation and recording were performed on the transformed Arabidopsis T1 and T2 generations throughout their entire growth period. Results showed that when the plants reached 4 weeks of age, the leaf size of the RmGRF1 transgenic line was significantly larger than that of the wild type. Figure 5 ab). Leaf length, leaf width, petiole length, and leaf area were measured in the three transgenic lines, and the fifth true leaf was compared with the true leaves of the wild type at the same stage for statistical analysis. The results showed that, compared with the wild type, the leaf length, petiole length, and leaf area of ​​the fifth true leaf of the three transgenic lines were significantly larger than those of the wild type. Figure 5 (cf). However, the total number of blades remains unchanged. Figure 5 Therefore, RmGRF1 affects leaf size, not leaf number.

[0045] The inventors further investigated the effects of RmGRF1 on leaf development in transgenic Arabidopsis thaliana at the cellular level, observing the epidermal and palisade cells of the fifth true leaf from the three transgenic Arabidopsis thaliana lines. The results showed that, under the same proportion of field of view, the cell area of ​​both epidermal and palisade cells in the three transgenic lines was significantly larger than that in the wild-type control, while the number of epidermal and palisade cells remained unchanged. Figure 6 and Figure 7 Therefore, RmGRF1 affects leaf development by promoting the expansion of leaf epidermal and palisade cells, rather than cell proliferation, leading to larger leaves.

[0046] Since the RmGRF1 gene promotes the expansion of leaf epidermal cells and palisade cells, inhibiting the RmGRF1 gene can inhibit the expansion of prickle cells, thereby reducing the number of prickles. To verify the effectiveness of this technology, the inventors amplified the homologous gene RrGRF1 of RmGRF1 in the wild rose 'Baobai', a closely related species, and determined its gene sequence (as shown in SEQ ID NO.3). The sequences of RrGRF1 and RmGRF1 are highly similar, with only a few base differences. A fragment from SEQ ID NO.1 to SEQ ID NO.3, at bases 1362-1577, was selected as the interference fragment for RrGRF1, and this fragment, along with its reverse sequence, was used to construct an RNAi vector. Using 'Baobai' as the transformation recipient, an Agrobacterium-mediated transformation was performed using an RNAi vector containing RrGRF1 (vector diagram shown). Figure 8 Agrobacterium was used to infect somatic embryos of rose 'Baobai', eventually yielding transformed seedlings. Transformed seedlings were selected based on expression detection, and the prickly condition of these plants was investigated. It was found that GRF1 gene interference significantly reduced the number of prickles compared to the control, indicating that inhibiting the GRF1 gene can effectively reduce prickles.

[0047] Table 1. Statistics on the number of thorns on the 'whitening' stem segments of roses after GRF1 gene interference.

[0048]

[0049] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for reducing the number of thorns on Rosa species, characterized in that, To reduce gene expression in Rosa species, select plants with fewer thorns. The gene can be any one of the following: 1) Genes with sequences as shown in SEQ ID NO:1 or SEQ ID NO:3; 2) Genes encoding protein sequences as shown in SEQ ID NO:2 or SEQ ID NO:

4.

2. The method according to claim 1, characterized in that, The method for reducing gene expression is to use RNAi interference.

3. The method according to claim 2, characterized in that, The interference fragment used in the RNAi interference method is the sequence shown in SEQ ID NO.1, bases 1362-1577, or SEQ ID NO.3, bases 1362-1577.

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