Lettuce LsNP1 gene and application thereof in preparation of male sterile plant

Abstract

The invention discloses a lettuce LsNP1 gene and application of the lettuce LsNP1 gene in preparation of male sterile plants. Specifically, the invention discloses a protein LsNP1 with an amino acid sequence of SEQ ID NO: 1 and application of a coding gene of the protein LsNP1 in forming a male sterility phenotype of lettuce. Experiments show that the number of pollen grains of the mutant obtained after the LsNP1 gene is knocked out is obviously reduced, the pollen activity is obviously reduced, the pollen grains are abnormal in shape, and anther cannot normally scatter and inseminate during flowering, so that lettuce forms a male sterility phenotype. The male sterile lettuce is successfully created, valuable gene resources are provided for germplasm resource innovation and genetic improvement of the lettuce, a new field is developed for application of the LsNP1 gene, and a new material is provided for application of a lettuce male sterile line. By utilizing the gene and the method, the lettuce male sterile line germplasm can be rapidly created, the cultivation process of a new lettuce variety is accelerated, and the gene and the method have a wide application prospect in the field of agricultural production.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a lettuce LsNP1 gene and its application in the preparation of male sterile plants. Background Art

[0002] Nuclear genetic male sterility is a common phenomenon in flowering plants, caused by mutations in nuclear male sterility genes. Studies have found that these genes are involved in multiple key processes of anther development, including transcriptional regulation, lipid metabolism, sugar metabolism, etc. The development of plant anthers depends on the precise regulation of hundreds of transcription factors, and some transcription factors and their homologs form conserved regulatory pathways for anther development. In addition, lipid metabolism genes are involved in the formation of anther cuticle and pollen wall, and sugar metabolism genes affect the normal development of anthers and pollen. Mutations in these genes can lead to male sterility in plants, providing potential application value for hybrid breeding and seed production.

[0003] Although nuclear male sterile mutants provide excellent genetic materials for emasculation in hybrid seed production. However, natural mutant germplasms may harm other growth processes of plants while affecting pollen development, thus limiting their direct application in hybrid breeding. In traditional methods, the recessive nuclear male sterility trait can be transferred to other genetic backgrounds through backcrossing, but it takes a long time through multiple generations of screening. Therefore, although nuclear male sterile mutants have important theoretical and application values, they have not been widely used in hybrid seed production.

[0004] The CRISPR / Cas9 technology has been widely applied in plant genetics and crop improvement. By targeting mutagenesis of genes required for pollen development, new male sterile line germplasms can be generated in different crops. It is reported that researchers have rapidly created male sterile lines in various crops such as maize (ZmMS26, ZmMYB84), rice (OsCYP703A3, OsOPR7), soybean (GmMS1), rapeseed (BnOPR3, BnCYP704B1) using gene editing technology, which can be applied to commercial hybrid seed production. However, currently, the male sterile gene resources available for lettuce ( Lactuca sativa ) are relatively limited, which restricts the development of lettuce hybrid breeding.

[0005] The use of male sterility genes can better utilize heterosis. Through cross-breeding, the excellent traits of different varieties can be combined to cultivate new varieties with high yield, good quality, and strong stress resistance. Lettuce contains rich inorganic salts, vitamins, niacin and other nutrients beneficial to the human body. It is not only delicious but also has medicinal value. Niacin in lettuce is considered an insulin activator and is beneficial to diabetic patients; it is rich in potassium ions, which helps maintain the body's water-salt balance and has the effects of reducing blood pressure and preventing arrhythmia for patients with hypertension, heart disease, etc.; Lettuce has good economic benefits due to its rich nutritional and medicinal values. Therefore, by deeply exploring male sterility genes in lettuce and using genetic transformation technology to conduct research and utilization of functional genes, it can provide valuable gene resources for the innovation of lettuce germplasm resources and genetic improvement, quickly create male sterile line germplasm, and thus accelerate the breeding process of new varieties, showing broad application prospects in the field of agricultural production. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to make lettuce exhibit a male sterile phenotype. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0007] To solve the above technical problems, the present invention first provides the use of a protein, and the use can be any of the following: A1) Use in making lettuce exhibit a male sterile phenotype; A2) Use in preparing male sterile lettuce; The name of the protein can be LsNP1, and can be any of the following: B1) A protein with the amino acid sequence of SEQ ID NO:1; B2) A protein obtained by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO:1, having more than 80% identity with the protein shown in B1) and having the same function; B3) A fusion protein with the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of B1) or B2).

[0008] In the above use, the protein LsNP1 can be derived from lettuce ( Lactuca sativa ).

[0009] The connection in B3) can be directly connected through a peptide bond or connected through a linker.

[0010] The substitution of amino acid residues in B2) can be a conservative substitution of amino acid residues.

[0011] To facilitate the separation, purification, detection, and / or localization of the protein described in B1) or B2), a tag may be attached to the amino terminus or carboxyl terminus of the protein described in B1) or B2). The tags include, but are not limited to: GST (glutathione S-transferase) tag protein, Trx (thioredoxin) tag protein, nitrogen utilization substrate A (NusA) tag protein, His tag protein (His-tag), Strep tag protein, MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO (small ubiquitin-like modifier) tag protein, HA (hemagglutinin) tag protein, Myc tag protein, LacZ tag protein, CBD (cellulose-binding domain) tag protein, bacteriophage T7 protein kinase (T7PK) tag protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), mCherry (monomeric red fluorescent protein), or AviTag tag protein, or a combination of the above tag proteins. Those skilled in the art know how to select a suitable tag protein according to the desired purpose. The use of the tag does not change the function of the target protein, and its purpose is for separation, purification, detection, or tracing. Therefore, the tag proteins applicable to this application are not limited to specific types. The tag can be separated from the target protein by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site and using TEV protease to cleave and remove the tag).

[0012] The application can be achieved by downregulating the content and / or activity of the protein LsNP1.

[0013] Furthermore, the application includes preparing male-sterile lettuce or causing lettuce to form a male-sterile phenotype by reducing the expression level of the coding gene of the protein LsNP1 ( LsNP1 gene) in lettuce (such as knocking out or silencing LsNP1 the gene).

[0014] The present invention also provides an application of a biological material, and the application can be any one of the following: C1) Application in causing lettuce to form a male-sterile phenotype; C2) Application in preparing male-sterile lettuce; The biological material can be any one of the following: D1) A nucleic acid molecule encoding the protein LsNP1; D2) An expression cassette containing the nucleic acid molecule described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) A recombinant host cell containing the nucleic acid molecule described in D1), or a recombinant host cell containing the expression cassette described in D2), or a recombinant host cell containing the recombinant vector described in D3); D6) A transgenic plant tissue containing the nucleic acid molecule described in D1), or a transgenic plant tissue containing the expression cassette described in D2); D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2).

[0015] All of the above biological materials can express the nucleic acid molecule described in D1).

[0016] In the above applications, the nucleic acid molecule described in D1) can be a coding sequence or a DNA molecule with a nucleotide sequence of SEQ ID NO:2.

[0017] The present invention also provides the use of a substance for reducing the activity and / or content of the protein LsNP1 in any of the following: E1) Use in making lettuce form a male sterile phenotype; E2) Use in preparing male sterile lettuce.

[0018] The substance can be any substance that reduces the activity and / or content of the protein LsNP1 through regulation of gene expression at the gene level or regulation at the protein level.

[0019] The regulation of gene expression at the gene level can include regulation of expression at the chromatin level (such as histone modification, chromatin remodeling), transcriptional level (such as regulation of promoters, transcription factors, co-regulators), post-transcriptional level (such as RNA splicing, microRNA regulation), and post-translational level (such as ubiquitination, SUMOylation, acetylation, glycosylation, phosphorylation, methylation, NEDD8 modification, etc.). The regulation at the protein level can include regulation of the activity and / or content of the protein by protein degradation, protein interaction, or other methods capable of regulating protein activity.

[0020] In the above applications, the substance includes a substance that inhibits the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the coding gene of the protein LsNP1.

[0021] Furthermore, the substance includes a substance that causes deletion or inactivation of the coding gene of the protein LsNP1 through site-directed mutagenesis technology, gene knockdown technology, gene editing technology, and / or gene knockout technology, or a substance that targets and binds to the protein to reduce its content or inactivate its function.

[0022] It is well-known to those skilled in the art to inhibit gene expression, silence or knockout genes using site-directed mutagenesis techniques (including oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, cassette mutagenesis, etc.), gene knockdown techniques (including RNA interference technique, Morpholino interference technique, antisense nucleic acid technique, ribozyme technique, etc.), gene editing techniques (including zinc finger nuclease gene editing technique, TALEN gene editing technique, CRISPR gene editing technique, etc.) or gene knockout techniques (including complete gene knockout and conditional gene knockout). For example: shRNA, siRNA or miRNA targeting the gene encoding the protein LsNP1 can be used to inactivate gene expression or silence the gene at the post-transcriptional level or translational level. The CRISPR-Cas system containing sgRNA and Cas protein can also be used to LsNP1 knock out the gene. Or use site-directed mutagenesis technique to LsNP1 mutate the gene to generate a frameshift mutation or premature translation termination, thereby LsNP1 inactivating the gene or weakening its function. In some embodiments of the present invention, the CRISPR / Cas9 gene editing technique is used to knock out the LsNP1 gene in lettuce.

[0023] Those skilled in the art know that target sequences can be selected according to the LsNP1 sequence of the gene or the mRNA transcribed therefrom to design nucleic acid molecules such as siRNA, miRNA, shRNA or dsRNA. Such nucleic acid molecules can inhibit or interfere with gene transcription, translation or post-transcriptional and post-translational modifications, thereby affecting protein expression.

[0024] Furthermore, the substance may include nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, recombinant vectors (such as gene editing vectors), recombinant cells and viral vectors (such as lentiviruses and adeno-associated viruses).

[0025] Furthermore, the nucleic acid molecules may include (1) double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), etc. used in RNA interference technique; (2) antisense RNA (asRNA) and antisense oligonucleotide (AON), etc. used in antisense nucleic acid technique; (3) gRNA and sgRNA, etc. used in gene editing technique; (4) aptamers and ribozymes, etc.

[0026] In the above applications, the substance may be sgRNA or a CRISPR / Cas9 system containing the sgRNA, and the sgRNA targets the gene encoding the protein LsNP1.

[0027] Furthermore, the target sequence of the sgRNA may be as shown in SEQ ID NO:3.

[0028] The present invention also provides a method for preparing male sterile lettuce, which comprises reducing the content and / or activity of the protein LsNP1 in lettuce to obtain the male sterile lettuce.

[0029] In the above method, reducing the content and / or activity of the protein LsNP1 in lettuce is achieved by reducing the expression level of the gene encoding the protein LsNP1 in lettuce.

[0030] The nucleotide sequence of the gene encoding the protein LsNP1 described herein may be shown as SEQ ID NO:2.

[0031] In the above method, the reduction of the expression level of the gene encoding the protein LsNP1 in lettuce is performed using the CRISPR / Cas9 system, and the CRISPR / Cas9 system includes the sgRNA described herein.

[0032] The CRISPR / Cas9 system described herein also includes a Cas9 protein.

[0033] Furthermore, the Cas9 protein described herein is not limited to a specific protein, as long as it can be used in conjunction with the sgRNA of the present invention.

[0034] Further, the Cas9 protein described herein includes Streptococcus pyogenes ( Streptococcus pyogenes )Cas9 (spCas9, II-A subtype), spCas9 HF (high fidelity), nickase Cas9 (nCas9), Staphylococcus aureus ( Staphylococcus aureus )Cas9 (saCas9, subtype II-A), Neisseria meningitidis ( Neisseria meningitidis )Cas9 (NmCas9, subtype II-C), Francisella novicida ( Francisella novicida )Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus ( Streptococcus thermophilus )Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni ( Campylobacter jejuni )Cas9 (CjCas9) and Treponema dendriticum ( Treponema sp.) Cas9, and Cas9 orthologs of other organisms but not limited thereto. The Cas9 protein may also include high-fidelity Cas9 mutants (such as SpCas9-HF1, eSpCas9-1.1, and TrueCut™ HiFi Cas9 protein).

[0035] The method of the present invention can be implemented using any Cas9 protein known in the art. Those skilled in the art can make appropriate selections for the coding sequence of the Cas9 protein without departing from the principles of the embodiments of the present invention.

[0036] Further, the reduction of the expression level of the gene encoding the protein LsNP1 in lettuce by using the CRISPR / Cas9 system can be achieved by contacting the gene in the lettuce recipient cells with LsNP1 the sgRNA described herein and the Cas9 protein.

[0037] Further, the contacting step can be carried out as follows (1) and (2): (1) Directly introduce the sgRNA described herein into the lettuce recipient cells, or first construct the DNA molecule encoding the sgRNA described herein into an expression vector and then introduce it into the lettuce recipient cells; (2) Directly introduce the Cas9 protein or the mRNA of the Cas9 protein into the lettuce recipient cells, or first construct the DNA molecule encoding the Cas9 protein into an expression vector and then introduce it into the lettuce recipient cells, or fuse the Cas9 protein with a cell-penetrating peptide (a peptide used to promote the uptake and absorption of the Cas9 protein fused thereto and to exert biological functions within the cell, such as the transcriptional trans-activator Tat peptide of the human immunodeficiency virus HIV), and then introduce it into the lettuce recipient cells through the cell-penetrating peptide.

[0038] Those skilled in the art know that the Cas9 protein, the Cas9 protein mRNA, the Cas9 expression vector (a vector containing and expressing the DNA molecule encoding the Cas9 protein), the sgRNA, and the sgRNA expression vector (a vector containing and expressing the DNA molecule encoding the sgRNA) can be transferred into plant cells by various methods known in the art, such as chemical stimulation methods (including methods such as PEG, calcium phosphate, and calcium chloride treatment), electroporation, liposome-mediated method, microinjection method, gene gun method (also known as particle bombardment method), laser microbeam method, pollen tube pathway method, ultrasonic method, pneumatic gun method, and vortex method, etc. The target gene can also be transferred into plant recipient cells using a vector as a medium, such as the Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integrated vector systems and binary vector systems) mediated method.

[0039] When using expression vectors to deliver the sgRNA and the Cas9 protein, the sgRNA and the Cas9 protein can be expressed in different expression vectors or ligated into the same expression vector for expression.

[0040] Further, the method for preparing male sterile lettuce may include the following steps: (1) Targeting LsNP1The coding DNA of the sgRNA of the gene is constructed into a Cas9 expression vector to obtain a CRISPR / Cas9 gene editing vector; (2) Introduce the CRISPR / Cas9 gene editing vector into lettuce; (3) Obtain LsNP1 transgenic lettuce with gene knockout, which is the male sterile lettuce.

[0041] Further, the introduction in step (2) can be carried out by the Agrobacterium-mediated method, which may include the following steps: introducing the CRISPR / Cas9 gene editing vector constructed in step (1) (such as by Ca ion-induced transformation method, polyethylene glycol-mediated transformation method, metal cation-mediated transformation method, electroporation transformation method, phage transduction method, etc.) into Agrobacterium to obtain recombinant Agrobacterium; infecting the callus or explant of the target plant (lettuce) with the recombinant Agrobacterium; inducing and culturing the obtained positive callus or explant through identification to obtain regenerated plants.

[0042] The explants include but are not limited to seeds, roots, leaves, petioles, cotyledons, cotyledon petioles, hypocotyls, stem segments, shoot apical meristems, epidermal parenchyma cells, tubers, stolon segments, embryogenic suspension cells, and protoplasts, etc.

[0043] The methods of screening and identification are known to those skilled in the art. For example, gene-edited plants (including progeny materials of gene-edited plants) can be identified by methods such as PCR detection, Sanger sequencing, high-throughput sequencing, immunoblotting (Western Blot), and Southern blot hybridization (Southern blot).

[0044] Further, the Cas9 expression vector in step (1) contains the Cas9 gene and can express the Cas9 protein. Further, the Cas9 expression vector may also contain one or more of the following elements: replication origin (ori), promoter (such as U6 promoter), enhancer (such as CAG enhancer), tag (such as FLAG tag), terminator (such as bGH poly(A) terminator), resistance gene (such as Kana antibiotic resistance gene, ampicillin resistance gene), promoter of the resistance gene, selection gene (such as bar gene), promoter of the selection gene, promoter of the Cas9 gene (such as Ubi promoter).

[0045] The Cas9 expression vector can be commercially obtained, such as the pBUE411 vector, etc. After designing the sgRNA targeting the target gene, it is very convenient to insert the DNA molecule encoding the sgRNA into the commercial Cas9 expression vector, simultaneously expressing the Cas9 protein and sgRNA, and then editing the target gene. In addition, conventional methods in the art can also be used to construct the Cas9 expression vector. For example, the Cas9 gene can be amplified using the Streptococcus pyogenes genome as a template, and then the Cas9 gene can be cloned into a backbone expression vector (such as pET28a, pET32a, etc.) to obtain the Cas9 expression vector.

[0046] In this article, the male sterile lettuce is understood to include not only the first-generation transgenic plants obtained by knocking out the LsNP1 gene in lettuce, but also their progeny. The transgenic plants include seeds, callus, whole plants, and cells.

[0047] The reduction of the content and / or activity of the protein LsNP1 in lettuce according to the present invention can be achieved by any technical means capable of achieving this purpose. Although in one or more embodiments provided by the present invention, the CRISPR / Cas9 technology is used for LsNP1 gene knockout, the present invention is not limited to this specific method. As is well known to those skilled in the art, other gene knockout, gene editing, gene mutation, gene knockdown, homologous recombination and other technologies known in the art can be used to make the LsNP1 gene deleted or inactivated in the lettuce genome. These methods can also be used in the present invention. These alternative methods do not depart from the scope of the present invention, and the present invention should include these alternative methods.

[0048] In this article, the lettuce may contain the coding gene of the LsNP1 protein.

[0049] The present invention first reveals the LsNP1 application of the gene and its encoded protein LsNP1 in the preparation of male sterile lettuce. By reducing the content and / or activity of the protein LsNP1 in lettuce (such as knocking out or silencing the LsNP1 gene), male sterile lettuce can be formed, thereby obtaining male sterile lettuce. Experiments show that LsNP1 the number of pollen grains of the mutants obtained after gene knockout is significantly reduced, the pollen activity is significantly decreased, the pollen grain morphology is abnormal, and the anthers cannot normally disperse pollen for fertilization during flowering, resulting in reduced pollen fertility of the mutants. The present invention has successfully created male sterile lettuce, providing valuable gene resources for the innovation of lettuce germplasm resources and genetic improvement, and for LsNP1The application of genes has opened up new fields and provided new materials for the application of lettuce male sterile lines. By using the genes and methods of the present invention, lettuce male sterile line germplasms can be rapidly created, accelerating the cultivation process of new lettuce varieties, and having broad application prospects in the field of agricultural production.

[0050] Term Definition In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, for a better understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0051] The term "expression cassette" generally refers to a nucleic acid construct containing nucleic acid elements sufficient to express a gene of interest. A typical expression cassette contains a promoter, an MCS (multiple cloning site), and / or a terminator. The expression cassette may also include a gene of interest, a marker gene (such as the TK gene, DHFR gene, CAT gene, and NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) signal sequence, and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette can be directly connected or indirectly connected through a linker.

[0052] The term "vector" generally refers to a vector that can transport foreign DNA or a gene of interest into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into the host cell by transformation, transduction, or transfection, enabling the genetic material elements it carries to be amplified and / or expressed in the host cell. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the nature of the recipient cell. The vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., cosmid plasmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, polyomaviruses (such as SV40), herpesviruses (such as herpes simplex virus)). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.

[0053] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria may come from the genus Escherichia ( Escherichia sp. ), such as Escherichia coli, the genus Erwinia ( Erwinia sp. ), the genus Agrobacterium ( Agrobacterium sp. ), such as Agrobacterium tumefaciens, the genus Flavobacterium ( Flavobacterium sp. ), the genus Alcaligenes ( Alcaligenes sp. ), the genus Pseudomonas ( Pseudomonas sp. ), and the genus Bacillus ( Bacillus sp. ), such as Bacillus subtilis, etc. The viruses may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papovavirus (such as SV40), and herpesvirus (such as herpes simplex virus), etc. The fungi may come from the genus Saccharomyces ( Saccharomyces sp. ), such as Saccharomyces cerevisiae, Pichia methanolica, Pichia pastoris, the genus Fusarium ( Fusarium sp. ), the genus Rhizoctonia ( Rhizoctonia sp. ), the genus Verticillium ( Verticillium sp. ), the genus Penicillium ( Penicillium sp. ), the genus Aspergillus ( Aspergillus sp. ), and Cephalosporium ( Cephalosporium sp. ), etc. The actinomycetes may come from the genus Streptomyces ( Streptomyces sp. ), such as Streptomyces. The algae may come from Cyanophyta (such as cyanobacteria), the genus Fucus ( Fucus sp. ), the genus Achnanthes ( Achnanthes sp. ), the genus Amphiprora ( Amphiprora sp. ), the genus Amphora ( Amphora sp. ), the genus Ankistrodesmus ( Ankistrodesmus sp. ), the genus Asterocystis ( Asteromonas sp. ), and the genus Chrysosphaera ( Boekelovia sp. ), etc.

[0054] The term "host cell" is also called recipient cell, and generally refers to any type of cell that can be used to introduce a vector, such as plant cells and animal cells. The host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or alterations, the progeny may not have to be exactly the same as the original parental cell, but is still included within the scope of the host cell. Suitable host cells are known in the art, among which: the plant cells can be Arabidopsis thaliana ( Arabidopsis thaliana ), tobacco ( Nicotiana tabacum ), maize ( Zea mays ), rice ( Oryza sativa ), wheat ( Triticum aestivum), and other plant cells, but not limited thereto; the animal cells may be mammalian cells (such as Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell sub-strains (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (such as chicken or duck cells), amphibian cells (such as Xenopus laevis ( Xenopus laevis )cells or Andrias davidianus ( Andrias davidianus )cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5), etc., but not limited thereto.

[0055] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by ligating an exogenous target gene and a vector in vitro, which can be constructed in any suitable manner as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the ability for the exogenous target gene to replicate, integrate, amplify and / or express in the recipient cell.

[0056] The term "recombinant microorganism" generally refers to a microorganism whose genes are manipulated and modified to obtain a recombinant microorganism with changed functions. For example, an exogenous target gene or a recombinant vector is introduced into the target microorganism, or the endogenous genes of the target microorganism are directly gene-edited.

[0057] The term "recombinant host cell" generally refers to a host cell whose genes are manipulated and modified to obtain a recombinant host cell with changed functions. For example, an exogenous target gene or a recombinant vector is introduced into the host cell, or the endogenous genes of the host cell are directly gene-edited.

[0058] The term "ligation" generally refers to the association of two or more molecules. The ligation can be covalent or non-covalent. The ligation described herein can be directly ligated through a peptide bond or ligated through a linker.

[0059] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, and is usually expressed as a percentage. The identity described herein may refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies with exactly the same sequence have 100% identity. Those skilled in the art know that the identity of an amino acid sequence or a nucleotide sequence can be determined using identity search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search, the identity value (%) of the amino acid sequence can be calculated, and then the identity value can be obtained. In addition, it can be determined using sequence analysis software (such as CLC Main Workbench and MegAlign TM )), for example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The identity of more than 80% described herein may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% identity.

[0060] The term "conservative substitution" generally refers to the replacement of one amino acid residue with another amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (such as Met, Ala, Val, Leu and Ile), between neutral hydrophilic side chain residues (such as Cys, Ser, Thr, Asn and Gln), between acidic side chain residues (such as Asp, Glu), between basic side chain amino acids (such as His, Lys and Arg) or between aromatic side chain residues (such as Trp, Tyr and Phe). It is known in the art that conservative substitutions generally do not cause significant changes in the conformational structure of proteins and basically do not change the biological activity of proteins. Conservative substitutions that are expected to have minimal or no effect on the structure or function of a protein sequence can be easily designed by those of ordinary skill in the art.

[0061] The term "introduction" generally refers to the transfer of exogenous genes into recipient cells such as eukaryotic recipient cells or prokaryotic recipient cells. There is no particular limitation on the method of introduction, and any known transformation method can be used as long as it can transfer the target gene (such as the DNA molecule of the present invention) into the recipient cell. The method of introduction may include any one of the following: (1) introducing the target gene or the recombinant vector containing the target gene into the host bacterium by chemical transformation methods (such as Ca ion-induced transformation method, polyethylene glycol-mediated transformation method or metal cation-mediated transformation method, etc.) or physical transformation methods (such as electroporation transformation method). (2) Transducing the target gene into the host bacterium by phage transduction. (3) Transferring the target gene into plant recipient cells by physical or chemical methods, such as gene gun method (also known as particle bombardment method or biolistic method), chemical stimulation method, electroshock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube pathway method, ultrasonic method, pneumatic gun method and eddy current method, etc. (4) Transferring the target gene into plant recipient cells by means of a vector, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integrated vector system and binary vector system) mediated method (Agrobacterium-mediated method), plant virus vector-mediated transformation method, etc.

[0062] The term "gene knock-down" also known as gene knock-low, generally refers to a technique that inactivates gene expression or silences genes at the post-transcriptional level or translational level, while the DNA sequence of the gene remains unchanged. Gene knock-down includes RNA interference technology, Morpholino interference technology, antisense nucleic acid technology and ribozyme technology, etc.

[0063] The term "gene editing" generally refers to a technique that can complete the alteration of a specific gene sequence in any cell including somatic cells, and can cause base deletion, duplication, insertion, frameshift mutation and replacement and knockout of the target gene, achieving replacement, deletion, cleavage and single-base alteration of the genomic sequence, that is, the technique of arbitrarily "editing" the genomic sequence or the sequence of a specific gene. Gene editing includes zinc finger nuclease gene knockout technology, TALEN gene editing technology and CRISPR gene editing technology.

[0064] The term "gene knock-out" generally refers to a technique that uses exogenous mutated genes to replace the endogenous normal homologous genes by homologous recombination, thereby inactivating the endogenous genes, including complete gene knock-out (such as complete mutation of the target gene based on replacement-type targeting vector or insertion-type targeting vector) and conditional gene knock-out (such as tissue-specific knock-out based on Cre-LoxP recombinase system or FLP-FRT recombinase system).

[0065] The term "RNA interference (RNAi)" generally refers to a technology that uses double-stranded RNA (dsRNA) to induce the degradation of the mRNA of a target gene that is homologous and complementary to it, silencing the expression of the gene and thus triggering post-transcriptional gene silencing (PTGS) to achieve the purpose of preventing gene expression.

[0066] The term "Morpholino interference technology" generally refers to replacing the pentose ring on traditional nucleotides with morpholine, and the original phosphate group also changes, making the whole molecule carry no charge and unable to be recognized and degraded by RNase and DNase, with extremely strong stability. Its principle is the same as that of antisense nucleic acid technology. It binds to the mRNA molecule by complementary pairing with the homologous sequence of the target gene mRNA, thus hindering the binding of other molecules and proteins to the specific mRNA nucleic acid sequence, and ultimately preventing the translation of the target gene mRNA into protein.

[0067] The term "antisense nucleic acid technology" generally refers to a technology that uses the principle that antisense RNA can complementarily bind to a specific mRNA molecule with a homologous sequence, thereby inhibiting the processing and translation of the mRNA. By artificially synthesizing antisense RNA or introducing its gene into cells, the expression of a specific gene is inhibited. Antisense nucleic acid technology mainly includes antisense RNA (asRNA) and antisense oligonucleotide (AON).

[0068] The term "ribozyme technology" generally refers to a technology that uses ribozymes to cleave and degrade target RNA molecules. Ribozymes are a class of RNA molecules with biological catalytic activity that can specifically bind to and cleave target RNA molecules, enabling the degradation of specific RNA molecules and thus inhibiting the expression of target genes. Ribozymes include hammerhead ribozymes, hairpin ribozymes, hepatitis delta virus ribozymes, VS (Varkud satellite) ribozymes, and group I intron ribozymes, etc.

[0069] The term "Cas9 protein" generally refers to the Cas endonuclease of the type II CRISPR system that forms a complex with crRNA and tracrRNA or with guide RNA, and is used to specifically recognize and cleave all or part of a DNA target sequence. The Cas9 protein has two distinct domains: the HNH domain and the RuvC domain. The HNH domain is responsible for cleaving the DNA strand (target strand) complementary to the crRNA (or gRNA), while the RuvC domain is responsible for cleaving the non-complementary strand (non-target strand). The Cas9 protein is not limited to a specific protein as long as it can be used in conjunction with sgRNA (gRNA). The Cas9 protein can be derived from bacterial species.

[0070] The term "sgRNA (single-guide RNA)" generally refers to a structure of a single RNA formed by artificially modifying the crRNA / tracrRNA complex (gRNA) with a dual RNA structure, by directly (or through a linker) connecting the crRNA and tracrRNA. The sgRNA is a component of the CRISPR-Cas9 system and is responsible for guiding the Cas9 protein to recognize and cleave the target nucleic acid molecule. In actual gene editing applications, the sgRNA can be synthesized directly, or obtained by plasmid expression or in vitro transcription.

[0071] The term "explant" generally refers to a part of a plant body used as an in vitro culture material in plant tissue culture. After appropriate treatment, under suitable conditions, it can regenerate into a whole plant. In actual operations, those skilled in the art select appropriate explants for transformation according to different plants. The explants include seeds, roots, leaves, petioles, cotyledons, cotyledon petioles, hypocotyls, stem segments, shoot apical meristems, epidermal parenchyma cells, tubers, stolon segments, embryogenic suspension cells, and protoplasts, etc.

[0072] The term "callus" generally refers to newly formed tissue on the wound surface after local trauma stimulation of the original plant body. It is composed of living parenchyma cells and can originate from living cells of various tissues in any organ of the plant body. In plant tissue culture, it can refer to a mass of disorganized and vigorously dividing parenchyma cells formed from explants. Culturing callus on an appropriate medium can induce it to form a whole plant.

[0073] The term "comprising" is not intended to be restrictive, is intended to be inclusive and means that there may be other elements in addition to the listed elements, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". In this article, the terms "comprising" and "including" can be used interchangeably. Brief Description of the Drawings

[0074] Figure 1 Amino acid sequence alignment result of lettuce LsNP1 protein in Example 1

[0075] Figure 2 For lettuce in Example 1 LsNP1 Expression analysis results of genes in different tissues

[0076] Figure 3 For the construction in Example 2 LsNP1 Positions of editing targets designed when constructing the gene knockout vector

[0077] Figure 4 Process diagram of Agrobacterium-mediated genetic transformation in Example 2 Figure 4 In A, aseptic sowing of lettuce seeds Figure 4 In B, Agrobacterium infection of cotyledons Figure 4 In C, co-culture Figure 4 In D, screening culture Figure 4 In E, rooting culture Figure 4 In F, soil culture transplantation

[0078] Figure 5 Sequencing results of gene editing in Example 2

[0079] Figure 6 For Example 3 LsNP1 Phenotypes of gene knockout mutant plants

[0080] Figure 7 For Example 3 LsNP1 CT scan results of gene knockout mutant plants

[0081] Figure 8 For Example 3 LsNP1 Results of I2-KI staining experiment of gene knockout mutant plants

[0082] Figure 9 For Example 3 LsNP1 Cryo-scanning electron microscope photos of gene knockout mutant plants Detailed implementation manners

[0083] The present invention will be further described in detail below in conjunction with the specific implementation manners. The given examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0084] 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 this field or according to the product specifications. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0085] The vector pZKD673 in the following examples is described in the following literature: Pan W, Liu X, Li D, ZhangH. Establishment of an Efficient Genome Editing System in Lettuce WithoutSacrificing Specificity. Front Plant Sci . 2022 Jun 22;13:930592. It can be obtained from the applicant, and this biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0086] Example 1, Lettuce LsNP1 Gene Sequence and Its Expression Characteristic Analysis 1. Lettuce LsNP1 Gene Sequence Through extensive and in-depth research, the inventors of the present application screened and identified the lettuce male sterility gene, and the sequence of this gene is as follows: LsNP1 The nucleotide sequence of the coding region (CDS) of the gene is shown in SEQ ID NO:2, and it encodes a protein with the amino acid sequence shown in SEQ ID NO:1. The name of this protein is LsNP1.

[0087] Download the amino acid sequences of monocotyledonous plants (maize ZmIPE1, rice OsNP1, wheat TaNP-A1 / B1 / D1) and dicotyledonous plants (Arabidopsis thaliana AtNP1, alfalfa MtNP1) from the NCBI website, and use DNAMAN to perform amino acid sequence alignment to determine the position of the GMC conserved domain of the LsNP1 protein. The amino acid sequence alignment result is as Figure 1 shown. It is found through amino acid sequence alignment that LsNP1 is a glucosylmethionine (GMC) oxidoreductase, and the GMC conserved domain is marked by the red horizontal line in the figure.

[0088] 2. Tissue Expression Analysis (1) Sample Preparation Lettuce plant samples (bracts, petals, pappus, anthers, stigmas, ovaries) are placed in a 2 mL grinding tube, an appropriate amount of steel beads are added, and they are quickly frozen in liquid nitrogen, and then placed in a grinder for sample treatment until all samples become powder state.

[0089] (2) Total RNA Extraction Use the Novizan FastPure® Plant Total RNA Isolation Kit for polysaccharide- and polyphenol-rich plant RNA extraction, with the product number RC401-01. Refer to the instruction manual for the specific operation steps.

[0090] (3)Reverse transcription to synthesize cDNA Use the Novizan HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (product number: R223-01) to reverse transcribe total RNA into cDNA.

[0091] (4)Real-time quantitative PCR Use the Novizan AceQ® Universal SYBR qPCR Master Mix, a universal high-specificity dye-based quantitative PCR detection kit with the product number Q511-02. Refer to the instruction manual for the specific operation steps. After thoroughly mixing all components and sealing the membrane, centrifuge the quantitative plate at high speed for 1 min, and perform the quantitative reaction on a Bio-Rad CFX96 instrument. Using the lettuce LsActin gene as an internal reference gene, use the 2 -ΔΔCT method to calculate the relative expression change of the gene.

[0092] The results are as Figure 2 shown. The quantitative results show that LsNP1 the gene is expressed in reproductive organs such as anthers, stigmas, and ovaries, and it is speculated that LsNP1 the gene is involved in the regulation process of lettuce reproductive development.

[0093] Example 2, LsNP1 Obtaining and identification of gene knockout mutant plants 1. Construction of CRISPR vector and location of editing target (1)Gene cloning Use the Novizan 2×Phanta Flash Master Mix (Dye Plus) high-fidelity enzyme to amplify the LsNP1 gene using lettuce cDNA as a template. The sequences of the amplification primers are as follows: LsNP1-F: 5’-ATGCTCTCAACACATAATAAACTCAC-3’, LsNP1-R: 5’-TCAATTGGGTCTAACGGTTGC-3’.

[0094] Sequence the obtained PCR product, and the gene sequence that is consistent with the comparison result with the reference sequence (SEQ ID NO:2) is LsNP1 the gene sequence.

[0095] (2) Construction of gene knockout vector The CRISPR / Cas9 technology was used to create knockout mutant materials. According to LsNP1 the gene sequence (SEQ ID NO:2), the CRISPR / Cas9 target sites were designed. The designed sgRNA target sequences are as follows (the edited target site positions are shown in Figure 3 ): sgRNA target sequence: 5’-atcgttggcccaatatccac-3’ (SEQ ID NO:3).

[0096] The synthesized DNA fragment (SEQ ID NO:3) was cloned into the pZKD673 gene editing vector by restriction enzyme digestion and ligation to obtain a recombinant vector for knocking out LsNP1 the gene, named pZKD673- LsNP1 . The recombinant vector pZKD673- LsNP1 was transformed into Escherichia coli, and monoclonal sequencing was performed to obtain the correct recombinant vector.

[0097] The recombinant vector pZKD673- LsNP1 contains the coding gene of the editing target site (SEQ ID NO:3) and the Cas9 protein on the vector. After being introduced into the receptor, the transcribed guide RNA (sgRNA) can target the target sequence near the PAM of the receptor genome through base complementary pairing, that is, targeting LsNP1 the gene, and the Cas9 protein causes LsNP1 double-strand break of the DNA at the gene target site. Through the DNA damage repair response mechanism of the organism itself, gene mutations occur in the sheared region during the repair process, resulting in frameshift mutations or premature termination of translation of the coding gene, thereby achieving the knockout of LsNP1 the gene.

[0098] 2. Agrobacterium-mediated genetic transformation The recombinant vector pZKD673- LsNP1 was transformed into competent EHA105 Agrobacterium cells to obtain recombinant Agrobacterium EHA105 / pZKD673- LsNP1 . Single colonies were picked for electrophoresis detection, and monoclonal colonies with correct band sizes could be used for Agrobacterium infection.

[0099] Lettuce cotyledons were infected with recombinant Agrobacterium EHA105 / pZKD673- LsNP1 . The genetic transformation process is shown in Figure 4 , and the steps are as follows: (1) Sterile sowing. After lettuce seeds were disinfected with sodium hypochlorite, they were sown on MS medium and grown for 5 days. After the cotyledons unfolded, they were cut off for Agrobacterium infection.

[0100] (2) Infect the cotyledons with Agrobacterium. The OD of Agrobacterium 600 is 0.1, the infection time is 10 min, and after infection, the bacterial solution on the surface of the cotyledons is washed off with sterile water.

[0101] (3) Co-culture. After infecting the cotyledons with Agrobacterium, they are cultured in the dark on the co-culture medium for two days and then transferred to the selection medium.

[0102] (4) Selection culture. After culturing on the selection medium containing kanamycin for about 4 weeks until new buds grow out.

[0103] (5) Rooting culture. The newly grown buds are cut from the callus and transferred to the rooting medium for culturing until roots grow out.

[0104] (6) Transplanting in soil. The tissue-cultured seedlings with roots are washed with running water, and after the medium on the roots is washed clean, they are transplanted into a pot containing nutrient soil and maintained in a normal growth environment.

[0105] 3. Identification of positive plants and gene editing types (1) DNA extraction Take 100 mg of fresh plant tissue, grind it into powder in liquid nitrogen, and transfer it to a 1.5 mL centrifuge tube. Use the MolPure® Plant Plus DNA Kit for polysaccharide and polyphenol plant DNA extraction produced by Yeasen Biotech Co., Ltd. (Product number: 18801ES50) to extract DNA, and the specific operation steps refer to the instruction manual.

[0106] (2) PCR identification Use the Novozyme 2×Rapid Taq Plus Master Mix (Dye Plus) enzyme to amplify the Kan gene and the sequences near the target site using lettuce DNA as a template. The amplification primer sequences are as follows: Kan-F: 5’-GAAGTGAGCTCAGAGCTTTC-3’, Kan-R: 5’-GAATTAACGCCGAATTAATTCG-3’, CR-LsNP1-F: 5’-GCAGGTGGAGCTCGAACTC-3’, CR-LsNP1-R: 5’-GGTTGCAAGTCTCTCACGCAATAAC-3’.

[0107] (3) Sequence alignment The PCR products are sent to a sequencing company for sequencing. Use the SnapGene software to align the sequences near the target site with the LsNP1 DNA sequence (SEQ ID NO:2) to detect the changes in the sequences at the target site. The gene editing sequencing results are shown inFigure 5 。

[0108] After sequencing, the homozygous mutant line (LsNP1-4) was finally selected for subsequent experiments. A base T was inserted at the target site in this mutant LsNP1-4, that is, a T base was inserted after the 1595th position in the CDS region (SEQ ID NO: 2). Correspondingly, a stop codon appeared at the position of the 536th amino acid in the encoded amino acid sequence, resulting in premature termination of translation. This LsNP1 gene knockout mutant was named Lsnp1 -CR.

[0109] Example 3, LsNP1 Phenotypic identification of gene knockout mutant plants 1. Phenotype pictures Five open flower buds of the wild type and LsNP1 the gene knockout mutant ( Lsnp1 -CR) were randomly taken, and the anther tissues were dissected from the inflorescences and observed and photographed under a DVM6 intelligent 3D digital microscope. The results are as Figure 6 shown. Compared with the wild type, the anthers of the mutant could not normally disperse pollen for fertilization during flowering.

[0110] 2. CT scan pictures Anthers were taken from the wild type and LsNP1 the gene knockout mutant ( Lsnp1 -CR) open flowers, quickly put into FAA fixative, and vacuum-treated to make the anthers sink to the bottom. Fixed at 4 °C for more than 21 h, and dehydrated with gradient ethanol (70%, 80%, 90%, 95%, 100%, 15 min for each treatment). After critical point drying with carbon dioxide, the samples were glued to the metal stage, sputter-coated with gold on the metal stage, and then observed under a scanning electron microscope. The results are shown in Figure 7 , compared with the wild type, from the cross-section, both the wild type and the mutant could produce pollen grains; but from the longitudinal section, the number of wild type pollen was significantly higher than that of the mutant, that is LsNP1 after gene knockout, the number of pollen grains was significantly reduced.

[0111] 3. I2-KI staining experiment Anthers were taken from the wild type and LsNP1 the gene knockout mutant ( Lsnp1 -CR) inflorescences to be opened, placed on a glass slide, the anthers were mashed with forceps to release the pollen grains, 1-2 drops of pollen viability staining solution (I2-KI) were added, covered with a coverslip and observed under a microscope. The results are shown in Figure 8 , compared with the wild type, the pollen activity of the mutant was significantly reduced.

[0112] 4. Cryo-scanning electron microscopy Randomly selected wild-type and LsNP1 gene knockout mutants ( Lsnp1 -CR), the fresh flower buds about to open were gently dissected with forceps to take out the anthers. Under the dissecting microscope, the anthers were opened with forceps to expose the pollen grains and placed on the sample stage. After cryogenic treatment, the morphology of the pollen grains was observed and photographed using a S-4800 FESEM cold field emission scanning electron microscope. The results ( Figure 9 ) showed that the mature pollen grains of the mutants were abnormally shaped and the pollen exine collapsed. The pollen exine plays a role in protecting the male gametophyte during pollen transmission, can resist external biotic and abiotic stresses, and is crucial for protecting the normal development of pollen and pollen-stigma recognition to complete fertilization. It can be seen that LsNP1 gene knockout can cause abnormal pollen grain morphology and defective pollen exine development, thereby leading to reduced pollen fertility of the mutants.

[0113] In summary, the above results indicate that by means of gene editing, knocking out the LsNP1 gene in lettuce can quickly create male sterile materials. This enriches the sterile material resources of lettuce and is conducive to promoting the commercial breeding process of lettuce.

[0114] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. Application of a protein, characterized in that, The application is any one of the following: A1) Application in making lettuce form a male sterile phenotype; A2) Application in preparing male sterile lettuce; The protein is any one of the following: B1) A protein with an amino acid sequence of SEQ ID NO:1; B2) A protein obtained by substitution, deletion, and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO:1, having more than 80% identity with the protein shown in B1) and having the same function; B3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of B1) or B2).

2. Application of biological materials, characterized in that, The application is any one of the following: C1) Application in making lettuce form a male sterile phenotype; C2) Application in preparing male sterile lettuce; The biological material is any one of the following: D1) A nucleic acid molecule encoding the protein described in claim 1; D2) An expression cassette containing the nucleic acid molecule described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) A recombinant host cell containing the nucleic acid molecule described in D1), or a recombinant host cell containing the expression cassette described in D2), or a recombinant host cell containing the recombinant vector described in D3); D6) A transgenic plant tissue containing the nucleic acid molecule described in D1), or a transgenic plant tissue containing the expression cassette described in D2); D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2).

3. The application according to claim 2, wherein The nucleic acid molecule in D1) is a DNA molecule with a coding sequence or nucleotide sequence of SEQ ID NO:

2.

4. Application of a substance for reducing the activity and / or content of the protein described in claim 1 in any one of the following: E1) Application in making lettuce form a male sterile phenotype; E2) Application in preparing male sterile lettuce.

5. The application according to claim 4, wherein The substance includes a substance that inhibits the replication, transcription, translation, post-transcriptional modification, and / or post-translational modification of the coding gene of the protein described in claim 1.

6. The application according to claim 4 or 5, characterized in that, The substance is an sgRNA or a CRISPR / Cas9 system containing the sgRNA, and the sgRNA targets the coding gene of the protein described in claim 1.

7. The application according to claim 6, characterized in that The target sequence of the sgRNA is as shown in SEQ ID NO:

3.

8. A method for preparing male sterile lettuce, characterized in that, The method includes reducing the content and / or activity of the protein described in claim 1 in lettuce to obtain male sterile lettuce.

9. The method according to claim 8, characterized in that, The reduction of the content and / or activity of the protein described in claim 1 in lettuce is achieved by reducing the expression level of the coding gene of the protein described in claim 1 in lettuce.

10. The method according to claim 9, characterized in that, The reduction of the expression level of the coding gene of the protein described in claim 1 in lettuce is carried out using the CRISPR / Cas9 system, and the CRISPR / Cas9 system includes the sgRNA described in claim 6 or 7.

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