Method for producing lettuce with improved leaf traits

Abstract

The present disclosure belongs to the field of plant genetic engineering. Specifically, the present disclosure relates to a method for producing lettuce with improved leaf traits, particularly but not limited to a method for obtaining lettuce with improved leaf traits, a method for detecting lettuce plants with improved leaf traits, and related primers and kits, and also relates to lettuce plants with improved leaf traits and a method for regulating lettuce leaf traits. The method provided by the present disclosure obtains lettuce with improved leaf traits by expressing a gene editing tool in lettuce to perform gene editing on a target sequence of lettuce.

Description

Technical Field

[0001] The present disclosure belongs to the field of plant genetic engineering, and relates to a method for producing lettuce with improved leaf traits, particularly but not limited to a method for obtaining lettuce with improved leaf traits, a method for detecting lettuce plants with improved leaf traits, and related primers and kits, and also relates to lettuce plants with improved leaf traits and a method for regulating lettuce leaf traits. Background Art

[0002] Lettuce (Lactuca sativa L.) is a widely consumed vegetable, and its taste and quality are highly concerned in the market. Among the many characteristics of lettuce, the storability, softness (taste), and appearance of the leaves are one of the important factors affecting consumers' choices and eating experiences. However, traditional lettuce varieties have certain limitations in terms of storability, softness (taste), and appearance, and it is difficult to fully meet the diverse needs of the market. Summary of the Invention

[0003] To solve the above technical problems, the present application designs a site-directed gene editing tool construct, and uses the gene editing tool to achieve site-directed editing of the target gene of lettuce plants, and screens out lettuce varieties with improved leaf traits.

[0004] Specifically, the present disclosure provides a modified non-reproductive lettuce plant cell, and the lettuce plant cell contains a mutated LsCTi1-1 gene. The mutated LsCTi1-1 gene includes: there is a nucleotide insertion mutation between positions 967 and 968 in SEQ ID NO: 11;

[0005] Compared with the non-mutated LsCTi1-1 gene, the mutated LsCTi1-1 gene is weakened or knocked out.

[0006] In some exemplary embodiments provided by the present disclosure, the mutated LsCTi1-1 gene includes: there is a single nucleotide insertion between positions 967 and 968 in SEQ ID NO: 11.

[0007] In some exemplary embodiments provided by the present disclosure, the mutated LsCTi1-1 gene includes: an adenine (A) is inserted between positions 967 and 968 in SEQ ID NO: 11.

[0008] In some exemplary embodiments provided by the present disclosure, the lettuce plant cell includes a mutated LsCTi1-1 protein;

[0009] The amino acid sequence of the mutated LsCTi1-1 protein is as shown in SEQ ID NO: 4.

[0010] In some exemplary embodiments provided by the present disclosure, the lettuce plant cells comprise a nucleotide sequence that edits the mutant LsCTi1-1 protein.

[0011] In some exemplary embodiments provided by the present disclosure, the nucleotide sequence that edits the mutant LsCTi1-1 protein is as shown in SEQ ID NO:12; the CDS nucleotide sequence of the mutant LsCTi1-1 protein is as shown in SEQ ID NO:3.

[0012] In a second aspect, the present disclosure provides a nucleic acid molecule that encodes the mutant LsCTi1-1 protein shown in SEQ ID NO:4.

[0013] In some exemplary embodiments provided by the present disclosure, the nucleic acid molecule is as shown in SEQ ID NO:12; the CDS nucleotide sequence encoding the mutant LsCTi1-1 protein is as shown in SEQ ID NO:3.

[0014] In a third aspect, the present disclosure provides a method for obtaining a lettuce plant, wherein all or part of the cells of the lettuce plant are modified by gene editing, physical mutagenesis, and / or chemical mutagenesis to express the mutant LsCTi1-1 protein shown in SEQ ID NO:4 and / or the endogenous LsCTi1-1 gene mutation of the lettuce plant comprises the above nucleic acid molecule.

[0015] In some exemplary embodiments provided by the present disclosure, the gene editing tool is a CRISPR / Cas nuclease or its derivative technology, a zinc finger nuclease or its derivative technology (ZFN), or a transcription activator-like effector (TALE) or its derivative technology.

[0016] In some exemplary embodiments provided by the present disclosure, the CRISPR / Cas nuclease is a TraC effector protein, preferably an eTraC protein, and its amino acid sequence is as shown in the amino acid sequence of SEQ ID NO:10.

[0017] In a fourth aspect, the present disclosure provides a gRNA molecule that comprises a variable DNA recognition region and a Cas effector protein interaction region, and the DNA target sequence recognized by the DNA recognition region of the gRNA molecule comprises at least SEQ ID NO:5 or its complementary sequence.

[0018] In some exemplary embodiments provided by the present disclosure, the DNA target sequence is located in the LsCTi1-1 gene.

[0019] In a fifth aspect, the present disclosure provides a composition that comprises a Cas effector protein and the above gRNA.

[0020] In a sixth aspect, the present disclosure provides an expression vector that encodes the gRNA and Cas effector protein as described above.

[0021] In a seventh aspect, the present disclosure provides a ribonucleoprotein complex that includes the gRNA and Cas effector protein as described above.

[0022] In an eighth aspect, the present disclosure provides a method for reducing the expression of the LsCTi1-1 gene, the method including directly or indirectly delivering the above-described vector or the above-described ribonucleoprotein complex to a lettuce target cell or a lettuce target structure.

[0023] In some exemplary embodiments provided by the present disclosure, compared with lettuce plants in which the expression of the LsCTi1-1 gene is not reduced, the leaves of lettuce plants containing lettuce plant cells with reduced expression of the LsCTi1-1 gene are softer (better taste), have a lower weight loss rate, and have a higher glossiness.

[0024] In a ninth aspect, for the Cas effector protein in the above-described gRNA molecule, the above-described composition, the above-described vector, the above-described ribonucleoprotein complex, or the above-described method for reducing the expression of the LsCTi1-1 gene, wherein the Cas effector protein is a transposon and CRISPR-Cas12 intermediate (eTraC) effector protein.

[0025] In a tenth aspect, the present disclosure provides a genetically modified non-reproductive lettuce plant cell that includes: the above-described gRNA molecule, the above-described composition, the above-described vector, and / or the above-described ribonucleoprotein complex;

[0026] The lettuce plant cell further includes: reduced expression of the LsCTi1-1 gene.

[0027] In some exemplary embodiments provided by the present disclosure, reducing the expression of the LsCTi1-1 gene results in the expression of the protein shown in SEQ ID NO: 4.

[0028] In some exemplary embodiments provided by the present disclosure, compared with lettuce plants in which the expression of the LsCTi1-1 gene is not reduced, the leaves of lettuce plants containing the lettuce plant cell are softer (better taste), have a lower weight loss rate, and have a higher glossiness.

[0029] In an eleventh aspect, the present disclosure provides a method for detecting lettuce with a mutation in the LsCTi1-1 gene, the method including: contacting a sample to be detected with at least two primers for amplifying a target amplification product in a nucleic acid amplification reaction;

[0030] Performing a nucleic acid amplification reaction;

[0031] Detect the presence of the target amplification product;

[0032] The target amplification product contains the nucleic acid sequence set forth in SEQ NO ID:9.

[0033] In some exemplary embodiments provided by the present disclosure, the two primers include the primers shown in SEQ ID NO:7 and SEQ ID NO:8.

[0034] In a twelfth aspect, the present disclosure provides a kit for detecting lettuce with reduced LsCTi1-1 gene expression, and the kit includes the primers shown in SEQ ID NO:7 and SEQ ID NO:8.

[0035] Technical effects

[0036] The present disclosure provides a mutant lettuce LsCTi1-1 protein and its encoding gene. Lettuce plants with this mutation can significantly improve the smoothness and softness of leaves while maintaining the single-plant yield and nutrients of lettuce plants basically unchanged.

[0037] Definitions

[0038] As used herein, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation procedures used herein are all terms and conventional procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present disclosure are well known to those skilled in the art and are more comprehensively described in the following literature: Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided below.

[0039] As used herein, the term "and / or" encompasses all combinations of the items connected by this term, and should be regarded as each combination having been separately listed herein. For example, "A and / or B" encompasses "A", "A and B", and "B". For example, "A, B and / or C" encompasses "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0040] As used herein, the terms "comprising", "comprises" and "comprised of" shall be understood to have at least the recited feature, while not excluding any other unspecified feature. The term "plant" as used herein includes whole lettuce plants, whether immature or mature, including plants from which seeds, grains or anthers have been removed. Any seed or embryo capable of producing a plant is also considered a lettuce plant.

[0041] As used herein, the terms "gene", "genome" cover not only chromosomal DNA present in the cell nucleus, but also organelle DNA present in subcellular components of the cell, such as mitochondria, plastids.

[0042] "Genetically modified organism" or "genetically modified cell" means an organism or cell that contains within its genome an exogenous polynucleotide or a modified gene or an expression regulatory sequence. For example, the exogenous polynucleotide can be stably integrated into the genome of the organism or cell and be inherited through successive generations. The exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. The modified gene or expression regulatory sequence means that the sequence in the genome of the organism or cell contains single or multiple deoxynucleotide substitutions, deletions and additions.

[0043] As used herein, "polynucleotide", "nucleic acid sequence", "nucleotide sequence", "DNA sequence" or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural or altered nucleobases. Nucleotides are referred to by their single letter designations: "A" is adenosine or deoxyadenosine (corresponding to RNA or DNA respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0044] As used herein, a polynucleotide is an isolated polynucleotide. Also provided are biological samples and processed products comprising any of the foregoing polynucleotides. In certain embodiments, the biological sample can be a whole lettuce plant or any plant part comprising a mutated LsCTi1-1 gene. In certain embodiments, the processed product is a processed product of a whole lettuce plant or any plant part (such as seeds, leaves, and / or stems or other parts) of a mutated LsCTi1-1 gene, including: cut, sliced, grated, pureed, dried, canned, jarred, washed, packaged, dried, dehydrated, frozen, and / or heated leaves, stems, and / or seeds, or any other part thereof. The processed product includes sugars or other carbohydrates, fibers, proteins, and / or aromatic compounds extracted, purified, or isolated from a lettuce plant of one or more of the examples. In further embodiments, the processed product includes washed and packaged lettuce leaves, stems, and / or seeds (or parts thereof).

[0045] As used herein, "lettuce" and "Lettuce" are used interchangeably and refer to the vegetable Lactuca sativa L.

[0046] As used herein, "sample" or "specimen" refers to whole or non-whole lettuce plant tissue (such as broken lettuce leaves or lettuce plant tissue, shredded lettuce plant tissue, or freeze-dried tissue, etc.). It can also be an extract containing whole or non-whole seeds or lettuce plant tissue. The biological sample can include all or part of a processed product of a lettuce plant, such as dehydrated lettuce. In certain embodiments, the biological sample is "non-renewable" (i.e., cannot be regenerated into a lettuce plant or a lettuce plant part).

[0047] As used herein, "polypeptide", "peptide", "amino acid sequence", and "protein" are used interchangeably in the present disclosure and refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence", and "protein" also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0048] The term "identity" as used in the context of sequences has its recognized meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. (See, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are numerous methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0049] In peptides or proteins, suitable conservative amino acid substitutions are known to those of skill in the art and generally can be made without altering the biological activity of the resulting molecule. In general, those of skill in the art recognize that single amino acid substitutions in the non-essential regions of a polypeptide generally do not alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p. 224).

[0050] As used herein, "promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present disclosure, the promoter is a promoter capable of controlling gene transcription in a cell, whether or not it is derived from said cell. The promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

[0051] As used herein, "reducing the expression of the LsCTi1-1 gene" refers to a mutation in which the gene function is lost or reduced compared to the unmutated or wild-type allele.

[0052] As used herein, "mutation" can be an increase of 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 15 bp, 20 bp, 25 bp or more at the DNA target sequence in the genome of LsCTi1-1 compared to the wild type; the "mutation" can also be a decrease of 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 15 bp, 20 bp, 25 bp or more at the DNA double-strand break position in the genome of LsCTi1-1 compared to the wild type.

[0053] As used herein, "mutated" or "edited" refers to comprising knocking down or knocking out the endogenous lettuce LsCTi1-1 gene, i.e., reducing the expression of the LsCTi1-1 gene. In certain embodiments, such knockdown or knockout mutations result in the LsCTi1-1 gene expressing a protein as shown in SEQ ID NO: 4. "Mutated" can also refer to mutating the Cas enzyme to obtain beneficial effects related to the Cas enzyme such as higher editing efficiency for the target sequence.

[0054] As used herein, "plant" or "plantlet" includes the entire lettuce plant and any of its descendants, cells, tissues, parts or components. Thus, the terms "plant" or "plantlet" include the entire immature or mature lettuce plant, including plants or plantlets from which seeds, fruits or anthers have been removed.

[0055] As used herein, "plant part" includes any part of a plant, such as but not limited to: seeds (including mature and immature seeds); fruits; plant sections; plant cells; plant cell cultures; or plant organs (such as pollen, embryos, flowers, fruits, buds, leaves, roots, stems, and explants). A plant tissue or plant organ can be a seed, protoplast, callus, or any other group of plant cells organized into a structural or functional unit. A plant cell or tissue culture can regenerate a plant with the physiological and morphological characteristics of the plant from which the cells or tissue were obtained, and can regenerate a plant that is substantially identical in genotype to that plant. The renewable cells in a plant cell or tissue culture can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, or stems. In contrast, some plant cells are unable to regenerate into plants and are herein referred to as "non-reproductive" plant cells.

[0056] As used herein, "trait" refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism.

[0057] As used herein, "trait" particularly refers to measurable indicator parameters of crop plants, including but not limited to: leaf greenness, yield, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, plant vegetative tissue nitrogen content, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, plant vegetative tissue free amino acid content, total plant protein content, fruit protein content, seed protein content, plant vegetative tissue protein content, herbicide resistance, drought resistance, nitrogen uptake, root lodging, harvest index, stem lodging, plant height, disease resistance, cold resistance, and salt tolerance, etc.

[0058] As used herein, the time in "lettuce fruit harvest time" or "over time" refers to the number of days post-harvest (DPH).

[0059] As used herein, "nuclease" refers to a nuclease that has completely or partially lost its cleavage activity herein. In some embodiments, the nucleases of the present disclosure can be selected from TraC effector proteins, preferably the eTraC protein, the amino acid sequence of which is shown in SEQ ID NO: 10, and the structure of the gRNA corresponding to the TraC effector protein includes a scaffold. The TraC effector protein and its corresponding gRNA can be described and selected in more detail with reference to Chinese Patent CN117187213A. If any of the foregoing definitions are inconsistent with the definitions in any patent or non-patent reference cited herein, any patent or non-patent reference cited herein, or any patent or non-patent reference elsewhere, it should be understood that the foregoing definitions will be used herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a schematic diagram of the vector map of the p010-0072 vector in Example 1.

[0061] Figure 2 It is a schematic diagram of the T-DNA insertion region of the vector map of the p010-0072 vector in Example 1.

[0062] Figure 3A and Figure 3B It is a comparison diagram of the leaf phenotypes of the T1 generation genomic editing homozygous lettuce plants with the target mutation and wild-type lettuce plants ( Figure 3A and Figure 3B from left to right are wild-type, edited T1-1, edited T1-2, edited T1-3), it can be seen that compared with the wild-type, the surface of the edited lettuce leaves is soft and smooth, the leaves are light green with a slightly purple-red color, and have a higher gloss. In terms of plant type, the edited lettuce has more leaf branches.

[0063] Figure 4 It is a peak map (excerpt) of the detection results of the homozygous lettuce plant (T1 generation) numbered edited T1-1 with the target mutation and a schematic diagram of comparative analysis.

[0064] Figure 5 It is a peak map (excerpt) of the detection results of the homozygous lettuce plants (T2 generation) numbered edited T2-1 to edited T2-3 with the target mutation and a schematic diagram of comparative analysis.

[0065] Figure 6 It is a peak map (excerpt) of the detection results of the homozygous lettuce plants (T3 generation) numbered edited T3-1 to edited T3-3 with the target mutation and a schematic diagram of comparative analysis. It can be seen that 1 bp is inserted into the target region 1 (target sequence, SEQ ID NO:5) of the LsCTi1-1 gene, ultimately resulting in premature termination of protein translation, and the amino acid sequence is truncated from 166 aa to 99 aa. DETAILED DESCRIPTION OF THE INVENTION

[0066] The present disclosure will now be described with reference to the following examples which are intended to illustrate, but not limit, the present disclosure.

[0067] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0068] In the experimental methods of the following examples, unless otherwise specified, for each numbered T1 generation and its offspring, 10 plants are randomly selected and mixed into 1 sample for detection or analysis. If the number is less than 10, the largest non - 1 number is taken.

[0069] In the following examples, the selection marker gene used is the NptII resistance gene. Lettuce tolerant to aminoglycoside antibiotics shows normal green germination similar to that of wild - type lettuce that has not been treated with aminoglycoside antibiotics during germination, while wild - type lettuce germinated with the same amount of aminoglycoside antibiotics will not show normal green but yellow. This indicates that the presence of the NptII resistance gene makes lettuce germinated resistant to aminoglycoside antibiotics. Exemplarily, aminoglycoside antibiotics can be selected from neomycin, kanamycin, paromomycin, etc.

[0070] In the following examples, the lettuce variety used is Red leaf lettuce (Lactucasativa L. cv. ‘Jeokchima’).

[0071] In the following examples, the host cells (recombinant cells) containing the vector are Agrobacterium rhizogenes and / or Agrobacterium tumefaciens. The Agrobacterium rhizogenes is preferably a wild - type or a derivative strain selected from one or more of A4, Arqua1, MSU440, C58C1, Ar1193, K599, ATCC15834, and NCPPB1855; the Agrobacterium tumefaciens is preferably a wild - type or a derivative or an unarmed strain C58 selected from one or more of EHA101, EHA105, LBA4404, GV3101, and AGL1.

[0072] In the following examples, the identification test steps for gene editing are conventional experimental steps in the art. When detecting genomic editing sites, primers are designed with cultivated lettuce as the reference genome. The sequence shown in SEQ ID NO:11 is the LsCTi1 - 1 genomic sequence. A pair of primers (i.e., SEQ ID NO:7 and SEQ ID NO:8) are designed on both sides of the editing site as shown in SEQ ID NO:9, and SEQ ID NO:9 is the amplified DNA fragment to show the editing of the target site. The primers for the site - specific PCR test for genomic editing are shown in Table 1:

[0073] Table 1: Primers for site - specific PCR test for genomic editing

[0074]

[0075] Example 1: Construction of gene - editing vector

[0076] Construction of vector: The binary vector used in the plant gene editing process in this example is derived from Escherichia coli, and there have been no reports of pathogenicity or potential evolution into pathogenicity. The vector in this example (numbered p010-0072-g1 / 2, the schematic diagram of vector construction is as shown in Figure 1 ) has an origin of replication ori derived from the natural plasmid of Escherichia coli. The vector contains the eTraC nuclease coding sequence, which guides the eTraC protein to anchor to the gRNA of the target gene, and the selection marker NptII gene. The vector map is as shown in Figure 1 and Figure 2 . Among them, eTraC is selected from its functional mutants, and the amino acid sequence. The target sequence of TraC-gRNA is shown in Table 2 below, and the targeting sequence of the spacer of its gRNA is used to edit the gene at the target site shown in Table 2 of the target site to obtain the mutant genotype of lettuce LsCTi1-1:

[0077] Table 2: Statistical table of gene editing target sequences

[0078]

[0079] The specific method of genetic transformation of the vector is as follows: The seeds of red leaf lettuce (Red leaf lettuce (Lactuca sativa L.cv. ‘Jeokchima’)) are surface sterilized with 70% ethanol for 1 minute, then immersed in 1.0% sodium hypochlorite solution for 15 minutes, and then sown on MS medium (BD, Sparks, USA) solidified with 0.8% Bacto agar and supplemented with 3% sucrose. The culture dish is incubated at 25°C for 7 days under a photoperiod of 16 hours of light and 8 hours of darkness. The cotyledon explants of the germinated seedlings are excised and inverted on the MS co-culture medium (supplemented with 30 g / L sucrose, 0.8% plant agar, 0.1 mg / L α-naphthaleneacetic acid and 0.5 mg / L 6-benzylaminopurine) for two days. Then the explants are incubated with the Agrobacterium suspension carrying the required construct (p010-0072-g1, p010-0072-g2 or the empty vector pKSE401) for 10 minutes. After co-culture, the excess Agrobacterium cells in the explants are removed with sterile filter paper. The treated explants are inverted on the MS co-culture medium again and incubated in the dark at 25°C for 48 hours.

[0080] Example 2: Obtaining gene-edited plants

[0081] Kanamycin is used as a selection marker to identify the explants with adventitious buds grown in Example 1, that is, the tender buds. Observe the appearance of the tender buds and record the tolerance of the adventitious buds. Obtain kanamycin-tolerant lettuce tender buds.

[0082] The kanamycin-tolerant lettuce shoots were subjected to elongation culture and then transferred to a rooting medium, and the rooted lettuce plants were transferred to a greenhouse to obtain rooted seedlings. It was identified whether the vector described in Example 1 was integrated into the lettuce genome, and the integrated seedlings were designated as transgenic seedlings.

[0083] The genomes of the transgenic seedlings were extracted, and the method of targeted PCR sequencing was used to identify whether the transgenic materials were edited. Plants with gene editing and plants without gene editing were screened, and several plants were selected for phenotypic, off-target and genetic stability analysis.

[0084] Example 3: Phenotypic analysis, off-target and genetic stability analysis

[0085] The transgenic seedlings obtained by gene editing of the target sequence were transferred to a greenhouse to grow until the T1 generation of lettuce was obtained. The T1 generation was selected for qualitative analysis of lettuce leaf traits. Several T1 generation lettuce plants with a weight loss rate lower than that of the wild type, thinner leaves than the wild type and better gloss were selected for targeted PCR sequencing and found to be the editing target of SEQ ID NO: 5, and there were mutations Figure 4 as shown in the edited T1-1 exemplary plant in

[0086] Genomic DNA was extracted from individual plants of multiple T1 generation plants with the Figure 4 shown target mutations respectively, and multiple pairs of primers were used to detect whether there was vector residue in the T1 generation plants. The target gene editing type and potential off-target sites were verified by targeted PCR and sequencing. Finally, lines with homozygous mutations at the target site, no residual exogenous vector sequence, and no editing at potential target sites and other sites were selected to continue self-crossing, T2 generation lettuce seeds were harvested, and self-crossing was continued for propagation, T3 generation lettuce seeds were harvested, and T3 generation plants were cultured.

[0087] Since the genomic editing site is located inside the target gene. According to the primers described in Table 1, the above T1, T2 and T3 generations were identified and tracked. The results showed that in the edited materials of the exemplary selected T1, T2 and T3 generations in this example, the genomic editing sites could be stably inherited, and there were no editing elements, no off-targets, and the editing sites were homozygous. As Figure 4 , Figure 5 and Figure 6 shown in the peak maps, Figure 4 Exemplary description of the selected T1 generation plant numbered edited T1-1; Figure 5 Exemplary description of the selected T2 generation plants numbered edited T2-1, edited T2-2, edited T2-3; Figure 6 Exemplary description of the selected T3 generation plants numbered edited T3-1, edited T3-2, edited T3-3.

[0088] Figure 4 and Figure 5 and Figure 6 Among them, the sequences marked in dark color are the gene editing target sequence regions bound by gRNA. WT is the wild-type control, and the T1 generation is detected for each individual plant; 30 plants are detected for each of the T2 and T3 generations. After mixing 10 plants into 1 sample, they are divided into 3 samples each (i.e., edited T2-1, edited T2-2, edited T2-3, edited T3-1, edited T3-2, edited T3-3) for detection. The control for all generations is the corresponding wild-type lettuce, and the experimental treatment is gene-edited lettuce.

[0089] After identification, 1 bp (adenine, A) was inserted between the 967th and 968th base pairs of the target sequence of the LsCTi1-1 gene (SEQ ID NO: 11) corresponding to the T1, T2, and T3 generation plants of the gene-edited lettuce, truncating the expressed amino acid sequence from 166 aa (SEQ ID NO: 2) to 99 aa (SEQ ID NO: 4). Through phenotypic analysis and genomic analysis, it can be concluded that compared with the wild type, the edited lettuce has soft and smooth leaf surfaces, the leaves are light green with a slight purplish-red color, and have a higher gloss. In terms of plant type, the edited lettuce has more leaf branches, and the number of branches increases by 9.1% - 18.2%. (As shown in Figure 3). In addition, there are basically no differences in the yield, nutrition, etc. of the harvested lettuce plants compared with the wild-type lettuce plants.

[0090] Since various changes can be made to the above compositions and methods without departing from the scope of the present disclosure, all matters contained in the above description and shown in the drawings should be construed as illustrative rather than restrictive.

[0091] Sequence Listing:

[0092] >SEQ ID NO:1 LsCTi1-1 CDS original (501 bp):

[0093] ATGACTGCACTATCAAACTCCCTTGTTTTACCCATGAACAAACCAAATCATTTATCATCAGATTTGAAGTCGCTTGACCAGAGTACCAAATTGTTGTTTGGACAAACCCATGTGAGTAAAGTACAATTACACACATCTAAAAGAACACTCTCCATTCAAGCAAGATATAGTGATGATGGAAAATCCAACAATGGGAATGCCTTTGTTGGTGGCTTTGTTCTTGGTGGGTTAATTATTGGCACACTTGGTTGTGTATATGCA CC TCAGATAAGCAAGGCGTTATCTGTAGCTGGAACTGACAAAAAGGAGTTGCTGAAGAAGCTCCCTACATTCATTTATGATGAAGAAAAAGCTTTGGAGAAAACACGGAAAAAGCTAGCAGAGAAGATAGCACAGCTGAATGATGCCATTGATGACGTATCTTTGCAATTGAAATCAGATGATGAGGAGTCAATTGGAAATGGTGCTGTTGTTCCTGAAAAGACTCAATCTGTTGCTTGA

[0094] >SEQ ID NO:2 LsCTi1-1 amino acid original(166 aa):

[0095] MTALSNSLVLPMNKPNHLSSDLKSLDQSTKLLFGQTHVSKVQLHTSKRTLSIQARYSDDGKSNNGNAFVGGFVLGGLIIGTLGCVYAPQISKALSVAGTDKKELLKKLPTFIYDEEKALEKTRKKLAEKIAQLNDAIDDVSLQLKSDDEESIGNGAVVPEKTQSVA*

[0096] >SEQ ID NO:3 LsCTi1-1 CDS edited (502 bp):

[0097] ATGACTGCACTATCAAACTCCCTTGTTTTACCCATGAACAAACCAAATCATTTATCATCAGATTTGAAGTCGCTTGACCAGAGTACCAAATTGTTGTTTGGACAAACCCATGTGAGTAAAGTACAATTACACACATCTAAAAGAACACTCTCCATTCAAGCAAGATATAGTGATGATGGAAAATCCAACAATGGGAATGCCTTTGTTGGTGGCTTTGTTCTTGGTGGGTTAATTATTGGCACACTTGGTTGTGTATATGCACACTCAGATAAGCAAGGCGTTATCTGTAGCTGGAACTGACAAAAAGGAGTTGCTGAAGAAGCTCCCTACATTCATTTATGATGAAGAAAAAGCTTTGGAGAAAACACGGAAAAAGCTAGCAGAGAAGATAGCACAGCTGAATGATGCCATTGATGACGTATCTTTGCAATTGAAATCAGATGATGAGGAGTCAATTGGAAATGGTGCTGTTGTTCCTGAAAAGACTCAATCTGTTGCTTGA

[0098] >SEQ ID NO:4 LsCTi1-1 amino acid edited(99 aa)

[0099] MTALSNSLVLPMNKPNHLSSDLKSLDQSTKLLFGQTHVSKVQLHTSKRTLSIQARYSDDGKSNNGNAFVGGFVLGGLIIGTLGCVYAHSDKQGVICSWN*

[0100] >SEQ ID NO:5 target sequence target 1

[0101] ATAATCTAACCTGAGGTGCA

[0102] >SEQ ID NO:6 target sequence target 2

[0103] gttgtgtatatgcacctcag

[0104] >SEQ ID NO:7 D-LsCTi1-1-F123

[0105] gcttgaccagagtaccaaattgttg

[0106] >SEQ ID NO:8 D-LsCTi1-1-R123

[0107] cagttccagctacagataacgcc

[0108] >SEQ ID NO:9 part of Ls-CTi1-1

[0109] gcttgaccagagtaccaaattgttg tttggacaaacccatgtgagtaaagtacaattacacacatctaaaagaacactctccattcaagcaagatataggtacaaatttgccctcgtttcctttttctttccatttcttcatttcttccattcctaagttactgttttattcatctccagtgatgatggaaaatccaacaatgggaatgcctttgttggtggctttgttcttggtgggttaattattggcacacttggttgtgtatatgca cc tcaggttagattattaaaaacttagcataatcctatgatttgtttaaactttgcactaaatcaaaagcataatcttgatttaaaccagataagcaa ggc gttatctgtagctggaactg

[0110] >SEQ ID NO:10 eTraC

[0111] MSVSFSLNAKKIRLENYAMKMRLYPSPTQAEQMDKMFLALRLAYNMTFHEVFQQNPAVCGDPDEDGNVWPSYKKMANKTWRKALIDQNPAIAEAPAAAITTNNGLFLSNGQKAWKTGMHNLPANKADRKDFRFYSLRKPRRSFAVQIRPRCIIPSDTNQKVARIKLPKIDGAIKARGFNRKIWFGPDGKHTYEEALAAHELSNNLTVRVSKDTCGDYFICITFSQGKVKGDKPTWEFYQEVRVSPIPEPIGLDVGIKDIAILNTGTKYENKQFKRDRAATLKKMSRQLSRRWGPANSAFRDYNKNIRAENRALERAQQDPGSSGVGPEAPVLKSVAQPSRRYLTIQKNRAKLERKIARRRDTYYHQVTREVAGKSSLLAVETLRVKNMLQNHRLAFALSDAAMSDFISKLKYKARRIQVPLVAIGTFQPSSQTCSVCGSINPAVKNLSIRVWTCPNCGTRHNRDINAAKNILAIAQNMLEKKVPFADEALPDEKPPAAPVKKAARKPRDAVFPDHPDLVIRFSKELTQLNDPRYVIVNKATNQIVDNAQGAGYRSAAKAKNCYKAKLAWSSKTNK

[0112] >SEQ ID NO:11 LsCTi1-1 pre-editing genomic sequence

[0113] TTGTACAGGAACTGTTGGACGTCAGCAATCAATTTTGGGTCGGTTTGACAATCTGATTTCCACCGTTGTTAACAACGCCGCTAAGCATTGGTATCATCGAACAGGGAAAATTAATAACTCGCATAATCGCCACCACCTTCTCCTCGATTTCTTTGTCTCTCCATTCTCTCTCTCTCTTCCCTCCACACCCGGTGATCTCTATTGCAAATCTGAAAACATGACTGCACTATCAAACTCCCTTGTTTTACCCATGAACAAACCAAATCATTTATCATCAGGTATCGTTACTCATTCGCTAGCATTACTGTTATTGCTCTTTCAGTTGAATCGTTGTTGTTTCACTTTTCCTTTTATTGATTCATTGCGACAAGTTTATGATTACGGATTGATTTTTGTGGGGAGTCCTTTACTCCTTTTCTATTTCCTCAATTTATCTCGTTTTCATGCATTGCTAGAGATTTCAAAACCCTAACATCTGCATTGCTCATAGCTCCTGTATCACTTCCCAATTTGCAAACAAAAAAATGTTTGTTTTTACCTAAAATTTAGGAATTTAGGATCTCTGAGGAGCTTTTTTATACCAATAAATCATTACATGTGTTTGGATGTGCTTATTCAAAGTGATTATAGCGACTTCAAGAATCAAATCAGATAATCATTTTCTACAATTGTGATCTCATGCTAGATTTGAAGTCGCTTGACCAGAGTACCAAATTGTTGTTTGGACAAACCCATGTGAGTAAAGTACAATTACACACATCTAAAAGAACACTCTCCATTCAAGCAAGATATAGGTACAAATTTGCCCTCGTTTCCTTTTTCTTTCCATTTCTTCATTTCTTCCATTCCTAAGTTACTGTTTTATTCATCTCCAGTGATGATGGAAAATCCAACAATGGGAATGCCTTTGTTGGTGGCTTTGTTCTTGGTGGGTTAATTATTGGCACACTTGGTTGTGTATATGCA CCTCAGGTTAGATTATTAAAAACTTAGCATAATCCTATGATTTGTTTAAACTTTGCACTAAATCAAAAGCATAATCTTGATTTAAACCAGATAAGCAAGGCGTTATCTGTAGCTGGAACTGACAAAAAGGAGTTGCTGAAGAAGCTCCCTACATTCATTTATGATGAAGAAAAAGCTTTGGAGGTATACATACCAATACAATCTGTTTTCAAAGCTTTTGTATGATATTGTAGTAAACATGTATAATCTCTTTGAGTTTGTTATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGAAAACACGGAAAAAGCTAGCAGAGAAGATAGCACAGCTGAATGATGCCATTGATGACGTATCTTTGCAATTGAAATCAGATGATGAGGAGTCAATTGGAAATGGTGCTGTTGTTCCTGAAAAGACTCAATCTGTTGCTTGAGTATATACTATTTTTGTGTTTTTGTCTTCTTCACAGATGTTTGATGCTTGTTTTAAGAAATAATAGTGGACGTATGTTAATTGGGTATAAAAGATATAAGTACTGGATATATAAATTTGTGGATGCAAATGTTGCTTTGATCATCTTGTAATACATTATACATGTAATGTTTTGGGTGTTTTTTGTGGTAAAA

[0114] >SEQ ID NO:12 Edited genomic sequence of LsCTi1-1

[0115] TTGTACAGGAACTGTTGGACGTCAGCAATCAATTTTGGGTCGGTTTGACAATCTGATTTCCACCGTTGTTAACAACGCCGCTAAGCATTGGTATCATCGAACAGGGAAAATTAATAACTCGCATAATCGCCACCACCTTCTCCTCGATTTCTTTGTCTCTCCATTCTCTCTCTCTCTTCCCTCCACACCCGGTGATCTCTATTGCAAATCTGAAAACATGACTGCACTATCAAACTCCCTTGTTTTACCCATGAACAAACCAAATCATTTATCATCAGGTATCGTTACTCATTCGCTAGCATTACTGTTATTGCTCTTTCAGTTGAATCGTTGTTGTTTCACTTTTCCTTTTATTGATTCATTGCGACAAGTTTATGATTACGGATTGATTTTTGTGGGGAGTCCTTTACTCCTTTTCTATTTCCTCAATTTATCTCGTTTTCATGCATTGCTAGAGATTTCAAAACCCTAACATCTGCATTGCTCATAGCTCCTGTATCACTTCCCAATTTGCAAACAAAAAAATGTTTGTTTTTACCTAAAATTTAGGAATTTAGGATCTCTGAGGAGCTTTTTTATACCAATAAATCATTACATGTGTTTGGATGTGCTTATTCAAAGTGATTATAGCGACTTCAAGAATCAAATCAGATAATCATTTTCTACAATTGTGATCTCATGCTAGATTTGAAGTCGCTTGACCAGAGTACCAAATTGTTGTTTGGACAAACCCATGTGAGTAAAGTACAATTACACACATCTAAAAGAACACTCTCCATTCAAGCAAGATATAGGTACAAATTTGCCCTCGTTTCCTTTTTCTTTCCATTTCTTCATTTCTTCCATTCCTAAGTTACTGTTTTATTCATCTCCAGTGATGATGGAAAATCCAACAATGGGAATGCCTTTGTTGGTGGCTTTGTTCTTGGTGGGTTAATTATTGGCACACTTGGTTGTGTATATGCA CACTCAGGTTAGATTATTAAAAACTTAGCATAATCCTATGATTTGTTTAAACTTTGCACTAAATCAAAAGCATAATCTTGATTTAAACCAGATAAGCAAGGCGTTATCTGTAGCTGGAACTGACAAAAAGGAGTTGCTGAAGAAGCTCCCTACATTCATTTATGATGAAGAAAAAGCTTTGGAGGTATACATACCAATACAATCTGTTTTCAAAGCTTTTGTATGATATTGTAGTAAACATGTATAATCTCTTTGAGTTTGTTATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGAAAACACGGAAAAAGCTAGCAGAGAAGATAGCACAGCTGAATGATGCCATTGATGACGTATCTTTGCAATTGAAATCAGATGATGAGGAGTCAATTGGAAATGGTGCTGTTGTTCCTGAAAAGACTCAATCTGTTGCTTGAGTATATACTATTTTTGTGTTTTTGTCTTCTTCACAGATGTTTGATGCTTGTTTTAAGAAATAATAGTGGACGTATGTTAATTGGGTATAAAAGATATAAGTACTGGATATATAAATTTGTGGATGCAAATGTTGCTTTGATCATCTTGTAATACATTATACATGTAATGTTTTGGGTGTTTTTTGTGGTAAAA

[0116] >SEQ ID NO:13 sgRNA scaffold

[0117] GGUGGGAGGUCUGUCCCCACCAUGGGGUGCGAACCUUGUGUGCUCAUCAUUGCCGUGAGCGUUCGCACGUCCAAACGACCAUAUCAUCGUUGCCCUGCGACCAUUCAGCGGCAAUCAAGACGCAGGCAUGAUAUGUAACCAUGCAUUGGAAAGUGCAUGGAGCAGAAG。

Claims

1. A mutant LsCTi1-1 nucleic acid, characterized in that: The mutant LsCTi1-1 nucleic acid inserts a single nucleotide adenine base A between positions 967 and 968 of the wild-type LsCTi1-1 nucleic acid, and the wild-type LsCTi1-1 nucleic acid is shown in SEQ ID NO:

11.

2. A method for obtaining lettuce plants, characterized in that All or part of the cells of the parent lettuce plant are transformed by gene editing methods to mutate the endogenous LsCTi1-1 gene of the lettuce plant and obtain the mutant LsCTi1-1 nucleic acid as described in claim 1.

3. The method according to claim 2, characterized in that The method comprises the steps of: a) Using gene editing tools to perform gene editing in lettuce cells, lettuce seeds, lettuce tissues or lettuce parts, thereby obtaining gene-edited lettuce cells, lettuce seeds, lettuce tissues or lettuce parts; b) regenerating the gene-edited lettuce cells, lettuce seeds, lettuce tissues or lettuce parts described in step a) to obtain lettuce plants; The gene editing tool is CRISPR / Cas nuclease, zinc finger nuclease ZFN or transcription activator-like effector TALE.

4. The method according to claim 3, characterized in that The CRISPR / Cas nuclease is a TraC effector protein.

5. The method according to claim 4, characterized in that The TraC effector protein is eTraC protein, and its amino acid sequence is shown in SEQ ID NO:10.

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