Dmd1 gene for regulating plant mitochondrial inheritance and application thereof

Abstract

This invention provides a DMD1 gene for regulating plant mitochondrial inheritance and its applications. The provided DMD1 gene has the sequence shown in SEQ ID NO:1 or a sequence complementary to SEQ ID NO:1; or a sequence with more than 80% homology to that sequence. By regulating the provided gene, maternally derived mitochondrial DNA can be degraded, thereby regulating the mitochondrial inheritance pattern of plants and obtaining new plant varieties with different characteristics.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a DMD1 gene that regulates plant mitochondrial genetics and its application in the regulation of plant mitochondrial genetics. Background Technology

[0002] Mitochondria are organelles that inherit their genetic information in a non-Mendelian manner, typically exhibiting strict uniparental inheritance in plants and animals. The genes encoded by the mitochondrial genome are primarily involved in energy and metabolic processes such as ATP synthesis and electron transport, directly influencing plant signal transduction and performance under stress. However, due to the low rate of genetic variation in mitochondrial genes, traits regulated by mitochondrial genes are rarely identified. The most well-known example is cytoplasmic male sterility, whose genetic basis lies in variations in the mitochondrial genome. Therefore, mitochondrial genetic regulation is crucial for plant and animal offspring and has always been a hot topic in genetics research. Cloning related genes has significant scientific and applied value.

[0003] Cucumbers belong to the genus *Cucumis* in the family Cucurbitaceae. Like other members of the same genus, *Cucumis*, cucumbers exhibit paternal inheritance of mitochondrial genes. Furthermore, the large and easily observable mitochondrial genomes of both cucumbers and melons make them ideal materials for studying mitochondrial genetic regulation. However, to date, no gene clones controlling or influencing mitochondrial inheritance patterns have been reported. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. This invention provides a DMD1 (Defective in Mitochondrial DNA Degradation 1) gene capable of regulating the genetic traits of plant mitochondria and its application in the genetic regulation of plant mitochondria. The DMD1 gene is derived from cucumber and can regulate the degradation of mitochondrial DNA in cucumber. By regulating this gene and its encoded protein, the genetic traits of cucumber mitochondria can be regulated. Furthermore, it can be applied to other plants, such as melons and bananas, to regulate the genetic traits of mitochondria in these plants.

[0005] Therefore, the present invention provides the following technical solution:

[0006] According to a first aspect of the present invention, an separable DMD1 gene is provided, the gene having: (a) the sequence shown in SEQ ID NO:1 or a sequence complementary to SEQ ID NO:1; or (b) a sequence having 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the sequence shown in SEQ ID NO:1 or a sequence complementary to SEQ ID NO:1. The inventors of the present invention have discovered a DMD1 gene derived from cucumber, which can be used to regulate the degradation of mitochondrial DNA in cucumber, thereby enabling the regulation of mitochondrial genetic material in plants through regulation of this gene.

[0007] According to a second aspect of the present invention, a polypeptide or protein sequence is provided, said polypeptide or protein sequence having: (c) the sequence shown in SEQ ID NO:3; or (d) a sequence having 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the sequence shown in SEQ ID NO:3. By regulating the protein encoded by the DMD1 gene, maternally derived mitochondrial DNA can be degraded. After loss of function, maternal mitochondrial DNA can be passed on to offspring, creating cucumber varieties with mitochondrial DNA from both parents. This not only facilitates the transmission of cucumber cytoplasmic sterility genes but also facilitates the creation of cucumber mitochondrial DNA mutants and improves the stress resistance of cucumbers. Similarly, it can be applied to a variety of plant varieties.

[0008] According to an embodiment of the present invention, the polypeptide or protein sequence is encoded by the separable DMD1 gene described in the first aspect of the present invention.

[0009] According to embodiments of the present invention, the polypeptide or protein sequence has endonuclease activity. This polypeptide or protein, possessing endonuclease activity, can degrade maternally derived mitochondrial DNA.

[0010] According to a third aspect of the present invention, a gene mutant is provided that, compared with the sequence shown in SEQ ID NO:2, contains a single nucleotide deletion mutation (SNP) at 908 bp. This single nucleotide deletion mutation at 908 bp causes a frameshift mutation, resulting in premature termination of the protein sequence encoded by SEQ ID NO:2. The sequence shown in SEQ ID NO:2 is derived from wild-type cucumber material.

[0011] According to a fourth aspect of the present invention, the present invention provides an expression vector capable of regulating the expression of the separable DMD1 gene described in the first aspect of the present invention.

[0012] According to embodiments of the present invention, the expression vector is at least one of a plasmid, a virus, or a bacteriophage. According to embodiments of the present invention, the plasmid is selected from at least one of pBSE402 or pKSE402.

[0013] According to an embodiment of the present invention, the expression vector comprises: (1) an expression cassette for expressing Cas9 protein, including a promoter, a signal peptide sequence targeting the DMD1 gene, the Cas9 gene, and a terminator; (2) an expression cassette for expressing sgRNA, including a promoter, a target sequence, and a gRNA sequence; wherein the target sequence is a sequence homologous to the DMD1 gene with a length of 16-24 bp. According to an embodiment of the present invention, the Cas9 gene sequence is shown in SEQ ID NO:7; according to an embodiment of the present invention, the signal peptide sequence targeting the DMD1 gene is shown in SEQ ID NO:8; the provided expression vector only needs to be able to achieve non-expression or loss of function of the DMD1 gene.

[0014] According to a fifth aspect of the present invention, the present invention provides a recombinant cell comprising the expression vector described in the fourth aspect of the present invention.

[0015] According to embodiments of the present invention, the recombinant cells are bacterial cells, fungal cells, or algal cells. For example, they can be Escherichia coli cells, Agrobacterium cells, etc.

[0016] According to a sixth aspect of the present invention, the present invention provides a kit comprising the expression vector described in the fourth aspect of the present invention.

[0017] According to a seventh aspect of the present invention, the present invention provides a method for cultivating plants with mitochondrial biparental inherited traits, comprising:

[0018] Obtain a transgenic plant in which the DMD1 gene is not expressed or is lost to function;

[0019] Using the transgenic plant as the maternal parent and the wild-type plant as the paternal or maternal parent, hybrid plants with mitochondrial parental genetic traits are obtained by crossbreeding.

[0020] According to an embodiment of the present invention, the plant is selected from at least one of cucumber, melon, and banana.

[0021] According to embodiments of the present invention, the DMD1 gene in the transgenic plant is rendered non-expressed or loses function through the following methods: CRISPR-Cas9 technology, ZFNs technology, or TALENs technology. These technologies, as commonly used genome editing techniques, can introduce mutations at specific DNA sites, enabling stable inheritance of phenotypes and serving as powerful tools for functional genomics research and precision breeding. The principle of genome editing technology is to use modifiable endonucleases to introduce DNA double-strand breaks at specific sites in the genome and utilize cellular repair mechanisms to achieve DNA sequence alterations in the target genomic region. ZFNs and TALENs technologies both rely on nuclease molecules composed of designed, sequence-specific DNA-binding elements and non-specific DNA-cutting domains to cut specific sequences. These two gene editing technologies are relatively complex and costly due to the need for complex sequence-specific binding elements and the acquisition of specific fusion proteins. The CRISPR-Cas system (represented by the CRISPR-Cas9 system) relies on gRNA forming a complex with the Cas enzyme to edit the target gene. It is simpler to design, lower in cost, and more efficient, and is also a commonly used gene editing system.

[0022] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0023] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0024] Figure 1 This invention relates to parental materials and a genetic population construction process for genetic mapping according to embodiments of the present invention. The maternal parent material is P3A, with normal cotyledons and true leaves (non-wrinkled, nonmosaic); the paternal parent material is MSC16, with wrinkled cotyledons and true leaves (mosaic), a mutant phenotype caused by the deletion of the mitochondrial gene rps7. The F1 generation exhibits normal cotyledons and true leaves (nonmosaic); the F2 generation also exhibits normal cotyledons and true leaves (nonmosaic). Individual plants from the F2 generation are used as maternal parent materials and crossed with MSC16. The segregation ratio of the hybrid offspring is observed and statistically analyzed to infer the genotype of the F2 individual plants.

[0025] Figure 2 This is a localization map of the DMD1 gene provided according to an embodiment of the present invention. In the map, a and b are preliminary localization results obtained using the BSA cluster separation method and Indel markers, with the target gene DMD1 located within approximately 2483 kb between molecular markers Indel-16 and Indel-38; c is the fine localization result of the DMD1 gene, with the target gene DMD1 located within approximately 326 kb between molecular markers KASP-13 and KASP-16; d is the structural diagram of the candidate gene CsaV3_3G040940 and the mutation type annotation of the parental P3A sequence; e is the CDS and protein multiple sequence alignment results of the candidate gene CsaV3_3G040940 across multiple materials.

[0026] Figure 3 The results of nuclease activity analysis of the DMD1 gene-encoded protein provided according to embodiments of the present invention are shown. In the figures, a is a schematic diagram of vector sequence construction; b, c, and d are the expression, isolation, and purification results of the DMD1-His recombinant protein after IPTG induction, respectively; e is the recombinant protein activity detection result; and f is the metal ion-dependent nuclease activity detection result.

[0027] Figure 4 Analysis of the results of hybridization progeny of DMD1 gene knockout plants and MSC16 provided according to embodiments of the present invention. Wherein, a represents the CRISPR / Cas9 target site sequence and location information; b represents the sequencing information of the gene knockout lineage and background material; c represents the phenotypic segregation results of homozygous and heterozygous individuals of the gene knockout, as well as the hybrid progeny of the background material and MSC16. Detailed Implementation

[0028] To facilitate understanding by those skilled in the art, some terms appearing in this document are explained and described. It should be noted that these explanations and descriptions are for convenience of understanding only and should not be regarded as a limitation on the scope of protection of this invention.

[0029] The term "homology" as used in this article refers to the degree of similarity between the shown nucleic acid or protein sequences.

[0030] The term "wild type" as used in this article refers to those that have not undergone artificial genetic modification. When wild type is mentioned, it is generally in contrast to genetically modified organisms.

[0031] Normally, mitochondrial inheritance in melon plants follows a strictly paternal pattern, with maternal mitochondrial DNA not being passed on to hybrid offspring. However, the inventors of this invention discovered during their research that a protein composed of the amino acid sequence shown in SEQ ID NO:3 can degrade maternally derived mitochondrial DNA, resulting in paternal inheritance. When the gene sequence encoding the sequence shown in SEQ ID NO:3 mutates, causing a loss of function, maternal mitochondrial DNA can be inherited by hybrid offspring, achieving biparental inheritance of mitochondrial DNA. This is beneficial for creating mitochondrial DNA mutant materials and improving the stress resistance of hybrid offspring.

[0032] Therefore, according to one aspect of the present invention, an separable DMD1 gene is provided, the gene having: (a) the sequence shown in SEQ ID NO:1 or a sequence complementary to SEQ ID NO:1; or (b) a sequence having 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the sequence shown in SEQ ID NO:1 or a sequence complementary to SEQ ID NO:1. The inventors of the present invention have discovered a DMD1 gene derived from cucumber, which can be used to regulate the degradation of mitochondrial DNA in cucumber, thereby enabling the regulation of mitochondrial genetic material in plants through regulation of this gene.

[0033] According to another aspect of the present invention, a polypeptide or protein sequence is provided, said polypeptide or protein sequence having: (c) the sequence shown in SEQ ID NO:3; or (d) a sequence having 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the sequence shown in SEQ ID NO:3. By regulating the protein encoded by the DMD1 gene, maternally derived mitochondrial DNA can be degraded. After loss of function, maternal mitochondrial DNA can be passed on to offspring, creating cucumber varieties with mitochondrial DNA from both parents. This not only facilitates the transmission of cucumber cytoplasmic sterility genes but also facilitates the creation of cucumber mitochondrial DNA mutants and improves the stress resistance of cucumbers. Similarly, it can be applied to a variety of plant varieties. The sequences mentioned that are homologous to the sequence shown in SEQ ID NO:1 may be due to differences in species or to the degeneracy of the codons themselves. In short, any sequence that has a similar function to the sequence shown in SEQ ID NO:1 is acceptable.

[0034] According to embodiments of the present invention, the polypeptide or protein sequence is obtained by encoding the aforementioned separable DMD1 gene. For example, it is obtained by encoding the sequence shown in SEQ ID NO:1 or a sequence homologous to the sequence shown in SEQ ID NO:1.

[0035] According to embodiments of the present invention, the polypeptide or protein sequence has endonuclease activity. This polypeptide or protein, possessing endonuclease activity, can degrade maternally derived mitochondrial DNA.

[0036] According to another aspect of the present invention, an expression vector is provided that can regulate the expression of the aforementioned separable DMD1 gene. The expression vector is a recombinant gene knockout vector, which knocks out the DMD1 gene, causing the DMD1 gene to be unexpressed or lose its function. The expression vector can be a plasmid, virus, or bacteriophage, etc., and the plasmids mentioned include, but are not limited to, at least one of pBSE402 or pKSE402.

[0037] In one embodiment of the present invention, the recombinant gene knockout vector is specifically a segment of sgRNA encoding the DMD1 inserted between two cloning sites BsaI of the plant gene knockout vector pBSE402, to obtain a gene knockout recombinant vector (pBSE402-DMD1).

[0038] According to a specific embodiment of the present invention, the expression vector comprises: (1) an expression cassette for expressing Cas9 protein, including a first promoter, a signal peptide sequence targeting the DMD1 gene, the Cas9 gene, and a terminator; (2) an expression cassette for expressing sgRNA, including a second promoter, a target sequence, and a gRNA sequence; according to a specific embodiment, the target sequence is a sequence approximately 20 bp in length homologous to the DMD1 gene. The Cas9 gene sequence is shown in SEQ ID NO:7; the signal peptide sequence targeting the DMD1 gene is shown in SEQ ID NO:8. The first promoter may be a 35S promoter, and the second promoter may be an Arabidopsis U6 promoter. The terminator may be a NOS terminator. The signal peptide sequence targeting the DMD1 gene enables the Cas enzyme to enter the mitochondria, thereby achieving the editing of plant mitochondrial genes. According to a specific embodiment, the gRNA coding sequence is shown in SEQ ID NO:9.

[0039] According to another aspect of the present invention, the present invention provides a kit comprising the expression vector described above.

[0040] According to another aspect of the present invention, the present invention provides a method for cultivating plants with mitochondrial biparental inherited traits, comprising:

[0041] Obtain a transgenic plant in which the DMD1 gene is not expressed or is lost to function;

[0042] Using the aforementioned transgenic plant as the female parent and a wild-type plant as the male parent, hybrid plants with mitochondrial genetic traits from both parents are obtained through hybridization.

[0043] For example, CRISPR / Cas9 or similar methods can be used to mutate the DMD1 gene sequence, causing the encoded protein product to lose its function, thus obtaining transgenic material with DMD1 function deficiency. This transgenic material, used as the female parent, can be crossed with wild-type material to obtain plants that inherit the mitochondrial DNA of both parents. The aforementioned lack of expression or loss of function of the DMD1 gene refers to the lack of expression or loss of function of the protein encoded by the DMD1 gene compared to wild-type plants. According to a specific embodiment of the present invention, the lack of expression or loss of function of the DMD1 gene refers to the DMD1 gene encoding a protein sequence of SEQ ID NO:3 or homologous to SEQ ID NO:3.

[0044] According to embodiments of the present invention, the plant is selected from at least one of cucumber, melon, and banana. For example, the homologous gene sequence of the DMD1 gene in melon is shown in SEQ ID NO:12. After knocking out this gene, when hybridized with other melons, the maternally derived mitochondrial genome can also be detected in the offspring, indicating that the gene has consistent function within the genus Cucurbita.

[0045] According to embodiments of the present invention, the DMD1 gene in the transgenic plant is not expressed or is lost in function by the following methods: CRISPR-Cas technology, ZFNs technology, or TALENs technology.

[0046] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. The MSC16 and P3A materials mentioned are from the Havey Laboratory at the University of Wisconsin, USA; the Calypso material is a commercially available material.

[0047] Example 1: Obtaining DMD1 gene knockout cucumber

[0048] Example 1 provides a method for obtaining DMD1 gene knockout cucumbers. It includes the following:

[0049] I. Construction of the recombinant knockout vector pBSE402-DMD1

[0050] 1. Discovery of the DMD1 gene

[0051] Previous studies have found that MSC16 material (mitochondrial rps7) - The mutant material, exhibiting a wrinkled MSC phenotype (Mosaic) in both cotyledons and true leaves (a mutant phenotype caused by the deletion of the mitochondrial gene rsp7), was used as the male parent and crossed with other materials (Calypso). The offspring showed a large number of MSC plants, with only a small percentage (4%) exhibiting the wild-type phenotype (WT). Experiments confirmed that these MSC phenotype plants only contain the paternally derived mitochondrial genome (rsp7). - This result is consistent with the paternal inheritance pattern of mitochondria in *Melastoma* plants, indicating that maternal mitochondria have degraded, retaining only paternally derived mitochondria. Further experiments confirmed that normal phenotype plants contain maternally derived mitochondrial genomes (rps7). + That is, the maternally leaked mitochondria are complementary to the paternal mitochondria rps7. -The cause of phenotypic defects caused by mutations. Therefore, the inventors creatively conceived of using MSC16 material as the male parent to cross with other materials. By observing the phenotype of the cotyledons or true leaves of the offspring, they could screen for mutants with lost maternal mitochondrial DNA degradation function and obtain key genes that regulate mitochondrial inheritance.

[0052] like Figure 1 As shown, through extensive testcross experiments with MSC16, we found that the offspring (F1 generation) of hybrids between germplasm material P3A (with normal cotyledons and true leaves, nonnmosaic, non-wrinkled, used as maternal material) and MSC16 (used as paternal material) exhibited a large number (95-100%) of wild-type phenotype plants (non-wrinkled, nonnmosaic), and these wild-type plants all contained mitochondrial genomes derived from P3A. Therefore, these results confirm that P3A contains gene mutations that prevent maternal mitochondrial DNA degradation 1 (dmd1) in the hybrid offspring, thus transmitting it to the hybrid offspring and restoring the phenotypic variations caused by paternal mitochondrial mutations. Normal-type plants were randomly selected from the F1 generation for self-pollination to create the F2 population. Individual plants in the segregating population exhibited normal cotyledons and true leaves (nonnmosaic). Individual plants from the F2 population were used as maternal material and crossed with MSC16. The segregation ratio of the hybrid offspring was observed and statistically analyzed to infer the genotype of the F2 individual plants. Analysis of genetic segregation data showed that this trait conformed to a Mendelian segregation ratio of 1:2:1 (χ²). 2 =0.87, P=0.65), confirming that this trait is controlled by a single gene. Further analysis using the F2 population located the gene within the 325kb region of chromosome 3. Comparative analysis of resequencing data from germplasm materials identified the candidate gene as CsaV3_3G040940, whose functional annotation indicates it is a DNA lyase, consistent with mitochondrial DNA degradation function. Therefore, this gene was selected as a candidate gene for DMD1.

[0053] like Figure 2 As shown. Figure 2This is a localization map of the DMD1 candidate gene provided according to an embodiment of the present invention. In the map, a and b are preliminary localization results obtained using BSA cluster separation and Indel markers, with the target gene DMD1 located within approximately 2483 kb between molecular markers Indel-16 and Indel-38; c is the fine localization result of the DMD1 gene, with the target gene DMD1 located within approximately 326 kb between molecular markers KASP-13 and KASP-16; d is the structural diagram of the candidate gene CsaV3_3G040940 and annotation of the mutation type of the parental P3A sequence; e is the CDS and protein multiple sequence alignment results of the candidate gene CsaV3_3G040940 across multiple materials.

[0054] 2. Obtaining the DMD1 gene

[0055] Fresh leaves from parental materials P3A and MSC16 were ground into powder using liquid nitrogen. Total RNA was obtained from both parents using an RNA extraction kit from Tiangen Biotech, and cDNA was obtained by reverse transcription using a reverse transcription kit from Takara. PCR amplification was then performed using the primers listed below to obtain the alleles of the annotation gene CsaV3_3G040940 in the parental materials. Sequencing of these alleles revealed that their sequences differed from the CsaV3_3G040940 annotation sequence. The parental sequence differences were manifested in the presence of a SNP deletion in P3A material, with the sequences shown in Sequence 1 (SEQ ID NO:1) of the sequence listing, and this gene was named DMD1. The sequence in the parental MSC16 material is shown in Sequence 2 (SEQ ID NO:2) of the sequence listing. The proteins encoded by the DMD1 gene are shown in Sequence 3 (SEQ ID NO:3) and Sequence 4 (SEQ ID NO:4) of the sequence listing. The SNP deletion in Sequence 1 caused a frameshift mutation, resulting in premature termination of the protein sequence; therefore, Sequence 3 and Sequence 4 showed significant differences. Sequence 3 shows the corresponding encoded amino acid. A SNP occurs at 908 bp (the bold and underlined base T) of SEQ ID NO:2, causing a frameshift mutation, which prematurely terminates the encoded amino acid sequence.

[0056] SEQ ID NO:1 is shown below:

[0057] ATGCGGAGCGCAACGGTCTGGGTCTGGGTCTGGTCTGTTTTGCAATTCTGGTCTAGGACTTTCCTTC

[0058] ATTCCTTCTTCAGCCTTGCAGTCAATCAGAGTAGGCTGAGAGTTGCAACATTGATACGACCTTTGCA

[0059] AAAGAATGCAATGTCGTCGAGGTTGAATCAATCCTCTGTACCTTTATTCACAAAAGAATTCAGAGCCT

[0060] AGCAGGGCGGGAGAGGTATAAGTGTGCGAAGGAATTTCCAATGAGACTGTTGTCCAGGAACATGA

[0061] TTGTAAAGTGGACATTCAAAGTTTTAAGGATGACCCGTCGAAAATTGAGGCTATGACGGTCCAAAA

[0062] ACTGAGAATGACATTAAGAAGCTTGGGTCTTCTAGCCAAAGGGCTTAAGCGTGATCTTGTAACTGC

[0063] ACTGCAAAGCTTTGTGGAGAATGAAACAGTTGTAGAAAATCATAGAAACACAACAGACAGAGAGAA

[0064] ATTCTAATGTATCAGCTTCTGATGGTGACACTGTGAAAGCTGAAACAAAAATCCTAACACCAAAGG

[0065] AGAGGCAGTCAGCTGAATCTAACAAGGTTTCAAGTGGTGCAATAGGCAGTAACCCATCTAGCAGA

[0066] AAGAGAAAAGATTCCTCAGACGTTGTTTCCAGCATTGTGAAGCAAGAGGATGGAGTAGAGGGAT

[0067] GCAAAATGAACCATGGGTTGTTCTTGCTCATAAGAAGCCTCAAAAAGGTTGGATCCCGTATAACCC

[0068] AAGAATCATGAGGCCTAAACCTCTTTCCAAGGATACAAAATCTGTGAAAATTTTATCTTGGAATGTC

[0069] AATGGGTTAAGAGCCTTACTTAAGGGGTCTTCAGCAGTGGAACTTGCTGAAAGGGAAGATTTTGAC

[0070] GTATTGTGCTTACAAGAGACCAAATTGCAGGAAAAGGACATATTAAATATACAAAATCTCTTGTGGA

[0071] TGGATATCATTATACCTACTGGACGTGCAGCGTTTCTAAACTTGGCTATTCTGGAACTGCAATCATAT

[0072] CCCGGATAAAGCCAATTTCAGTTAGATATGGTTTAGGCATATCAGAACATGATGGTGAAGGTCGGGT

[0073] TGTGATGGTGGAGTTCGATTCTTTTTTTCTATTGAATGTTTATGTTCCTAATTCTGGAGATGGATTAAA

[0074] GAGACTGTCATACAGGATTACCCAGTGGGATCCATCTCTCAGTAACTATATTAAGGAGTTGGAAAAG

[0075] TCGAAGCCCGTGATTTTGACTGGTGATCTAAATTGTGCACATCAGGAGATAGACCTCTATAATCCTG

[0076] CGGGAAACCGAAAAAGTGCTGGTTTCACCAACGAAGAAAGGCAATCATTTGAGACCAACTTTCTG

[0077] CAAAAGGGGTTTGTTGATACTTTTAGACAAAAGCATCCTGATGTTGTTGGCTATACATATTGGGGTTA

[0078] TCGTCATGGTGGACGCAAAACAAACAAAGGATGGCGGTTGGACTATTTTCTTGTATCAGAGAGGGT

[0079] GGCAGAGAAGGTACATGACTCTTACATACTCCCCGATGTGGGAGGTAGTGATCACTGTCCCATTGG

[0080] CCTTGTTCTCAAGCTTTAG

[0081] Wherein SEQ ID NO: 2 is shown below:

[0082] ATGCGGAGCGCAACGGTCTGGGTCTGGGTCTGGTCTGTTTTGCAATTCTGGTCTAGGACTTTCCTTC

[0083] ATTCCTTCTTCAGCCTTGCAGTCAATCAGAGTAGGCTGAGAGTTGCAACATTGATACGACCTTTGCA

[0084] AAAGAATGCAATGTCGTCGAGGTTGAATCAATCCTCTGTACCTTTATTCACAAAGAATTCAGAGCCT

[0085] AGCAGGGCGGGGAGAGGTATAAGTGTGCGAAGGAATTTCAATGAGACTGTTGTCCAGGAACATGA

[0086] TTGTAAAGTGGACATTCAAAGTTTTAAGGATGACCCGTCGAAAATTGAGGCTATGACGGTCCAAAA

[0087] ACTGAGAATGACATTAAGAAGCTTGGGTCTTCTAGCCAAAGGGCTTAAGCGTGATCTTGTAACTGC

[0088] ACTGCAAAGCTTTGTGGAGAATGAAACAGTTGTAGAAAATCATAGAACACAACAGACAGAGAGAA

[0089] ATTCTAATGTATCAGCTTCTGATGGTGACACTGTGAAAGCTGAAACAAAAATCCTAACACCAAAGG

[0090] AGAGGCAGTCAGCTGAATCTAACAAGGTTTCAAGTGGTGCAATAGGCAGTAACCCATCTAGCAGA

[0091] AAGAGAAAAGATTCCTCAGACGTTGTTTCCAGCATTGTGAAGCAAGAGGATGGAGTAGAGGGAT

[0092] GCAAAATGAACCATGGGTTGTTCTTGCTCATAAGAAGCCTCAAAAAGGTTGGATCCCGTATAACCC

[0093] AAGAATCATGAGGCCTAAACCTCTTTCCAAGGATACAAAATCTGTGAAAATTTTATCTTGGAATGTC

[0094] AATGGGTTAAGAGCCTTACTTAAGGGGTCTTCAGCAGTGGAACTTGCTGAAAGGGAAGATTTTGAC

[0095] GTATTGTGCTTACAAGAGACCAAATTGCAGGAAAAGGACATATTAAATA T TACAAAATCCTTGTGG

[0096] ATGGATATCATTATACCTACTGGACGTGCAGCGTTTCTAAACTTGGCTATTCTGGAACTGCAATCATA

[0097] TCCCGGATAAAGCCAATTTCAGTTAGATATGGTTTAGGCATATCAGAACATGATGGTGAAGGTCGGG

[0098] TTGTGATGGTGGAGTTCGATTCTTTTTTTCTATTGAATGTTTATGTTCCTAAATTCTGGAGATGGATTAA

[0099] AGAGACTGTCATACAGGATTACCCAGTGGATCCATTCTCTCAGTAACTATATTAAGGAGTTGGAAAA

[0100] GTCGAAGCCCGTGATTTTGACTGGTGATCTAAATTGTGCACATCAGGGAGATAGACCTCTATAATCCT

[0101] GCGGGAAACCGAAAAAGTGCTGGTTTCACCAACGAAGAAAGGCAATCATTTGAGACCAACTTTCT

[0102] GCAAAAGGGGTTTGTTGATACTTTTAGACAAAAGCATCCTGATGTTGTTGGCTATACATATTGGGGT

[0103] TATCGTCATGGTGGACGCAAAACAAACAAAGGATGGCGGTTGGACTATTTTCTTGTATCAGAGAGG

[0104] GTGGCAGAGAAGGTACATGACTCTTACATACTCCCCGATGTGGGAGGTAGTGATCACTGTCCCATTG

[0105] GCCTTGTTCTCAAGCTTTAG

[0106] Wherein SEQ ID NO: 3 is shown as follows:

[0107] MRSATVWVWVWSVLQFWSRTFLHSFFSLAVNQSRLRVATLIRPLQKNAMSSRLNQSSVPLFTKNSEPS

[0108] RAGRGISVRRNFNETVVQEHDCKVDIQSFKDDPSKIEAMTVQKLRMTLRSLGLLAKGLKRDLVTALQS

[0109] FVENETVVENHRTQQTERNSNVSASDGDTVKAETKILTPKERQSAESNKVSSGAIGSNPSSRKRKDSSD

[0110] VVSSIVKQEDGVEGMQNEPWVVLAHKKPQKGWIPYNPRIMRPKPLSKDTKSVKILSWNVNGLRALLKGSSAVELAEREDFDVLCLQETKLQEKDILNIQNLLWMDIIIPTGRAAFLNLAILELQSYPG*

[0111] Wherein SEQ ID NO: 4 is shown as follows:

[0112] MRSATVWVWVWSVLQFWSRTFLHSFFSLAVNQSRLRVATLIRPLQKNAMSSRLNQSSVPLFTKNSEPS

[0113] RAGRGISVRRNFNETVVQEHDCKVDIQSFKDDPSKIEAMTVQKLRMTLRSLGLLAKGLKRDLVTALQS

[0114] FVENETVVENHRTQQTERNSNVSASDGDTVKAETKILTPKERQSAESNKVSSGAIGSNPSSRKRKDSSD

[0115] VVSSIVKQEDGVEGMQNEPWVVLAHKKPQKGWIPYNPRIMRPKPLSKDTKSVKILSWNVNGLRALLK

[0116] GSSAVELAEREDFDVLCLQETKLQEKDILNITKSLVDGYHYTYWTCSVSKLGYSGTAIISRIKPISVRYG

[0117] LGISEHDGEGRVVMVEFDSFFLLNVYVPNSGDGLKRLSYRITQWDPSLSNYIKELEKSKPVILTGDLNC

[0118] AHQEIDLYNPAGNRKSAGFTNEERQSFETNFLQKGFVDTFRQKHPDVVGYTYWGYRHGGRKTNKGWRLDYFLVSERVAEKVHDSYILPDVGGSDHCPIGLVLKL*

[0119] 3. Function of the DMD1 gene

[0120] The DMD1 gene sequences 1 and 2 obtained in the above steps were double-digested with restriction enzymes 1 and 2, and the digested fragments were recovered. These fragments were then ligated to the backbone fragment of the prokaryotic expression vector pET28a, which had undergone the same double-digestion process, to obtain the prokaryotic expression vectors. Enzyme digestion and sequencing confirmed the presence of the target gene fragment, and these vectors were named pET8a-DMD1 and pET28a-DMD1(T303Q), respectively. Figure 3 As shown in Figure a, Figure 3 In the first part, DMD1-His represents the DMD1 protein carrying the His signal peptide; DMD1-His(T303Q) represents the DMD1 protein carrying the His signal peptide and having the T303Q mutation.

[0121] The expression strain was obtained by transforming *E. coli* DH5α and verifying it through double enzyme digestion and sequencing. The expression of the target protein was promoted using IPTG inducer. The supernatant was obtained after lysis and purified by SDS-PAGE to obtain the target protein solutions, which were named DMD1-His and DMD1-His(T303Q) protein solutions, respectively. Figure 3 As shown in Figures b, c, and d, b, c, and d represent the expression, isolation, and purification results of the DMD1-His recombinant protein after IPTG induction, respectively.

[0122] The above protein solution was added to a solution containing circular plasmid DNA, and the mixture was incubated at 37°C for 10, 20, 30, and 60 minutes, respectively. The integrity of the plasmid was then assessed using gel electrophoresis. Figure 3 As shown in d, this is the result of the detection of nuclease activity by metal ions.

[0123] Experiments confirmed that the recombinant DMD1-His protein possesses endonuclease activity and can rapidly degrade plasmid DNA, while DMD1-His(T303Q) can only linearize circular plasmids and cannot degrade DNA. This further confirmed CsaV3_3G040940 as a candidate gene for DMD1.

[0124] 4. Construction of the CRISPR-Cas9 gene knockout vector pBSE402-CRDMD1

[0125] Candidate target sites were designed based on the genomic sequence corresponding to the DMD1 gene. A target sequence (5'-AGGCCTAAACCTCTTTCCA-3', i.e., the target sequence) with a high score in the upstream region of the gene coding region and a low off-target probability was selected. Adapter sequences were added to both ends of the sequence, and upstream and downstream primers were synthesized.

[0126] CR940F: ATTG AGGCCTAAACCTCTTTCCA(SEQ ID NO:5)

[0127] CR940R: AAAC TGGAAAGAGGTTTAGGCCT(SEQ ID NO:6)

[0128] Using BsaI restriction enzyme digestion and ligation, the target sequence was inserted into the backbone of the plant gene editing vector pBSE402 to obtain a recombinant plasmid. Sequencing verification confirmed the presence of a 19 bp target fragment, which was named pBSE402-CRDMD1. Figure 4 As shown in Figure a.

[0129] The sequence of SEQ ID NO:7 (Cas9 gene sequence) is as follows:

[0130] GACAAGAAGTACTCGATCGGCCTCGATATTGGGACTAACTCTGTTGGCTGGGCCGTGATCACCGACGAG

[0131] TACAAGGTGCCCTCAAAGAAGTTCAAGGTCCTGGGCAACACCGATCGGCATTCCATCAAGAAGAATCTC

[0132] ATTGGCGCTCTCCTGTTCGACAGCGGCGAGACGGCTGAGGCTACGCGGCTCAAGCGCACCGCCCGCAG

[0133] GCGGTACACGCGCAGGAAGAATCGCATCTGCTACCTGCAGGAGATTTTCTCCAACGAGATGGCGAAGGT

[0134] TGACGATTCTTTCTTCCACAGGCTGGAGGAGTCATTCCTCGTGGAGGAGGATAAGAAGCACGAGCGGCA

[0135] TCCAATCTTCGGCAACATTGTCGACGAGGTTGCCTACCACGAGAAGTACCCTACGATCTACCATCTGCGG

[0136] AAGAAGCTCGTGGACTCCACAGATAAGGCGGACCTCCGCCTGATCTACCTCGCTCTGGCCCACATGATTA

[0137] AGTTCAGGGGCCATTTCCTGATCGAGGGGGATCTCAACCCGGACAATAGCGATGTTGACAAGCTGTTCAT

[0138] CCAGCTCGTGCAGACGTACAACCAGCTCTTCGAGGAGAACCCCATTAATGCGTCAGGCGTCGACGCGAA

[0139] GGCTATCCTGTCCGCTAGGCTCTCGAAGTCTCGGCGCCTCGAGAACCTGATCGCCCAGCTGCCGGGCGA

[0140] GAAGAAGAACGGCCTGTTCGGGAATCTCATTGCGCTCAGCCTGGGGCTCACGCCCCAACTTCAAGTCGAA

[0141] TTTCGATCTCGCTGAGGACGCCAAGCTGCAGCTCTCCAAGGACACATACGACGATGACCTGGATAACCT

[0142] CCTGGCCCAGATCGGCGATCAGTACGCGGACCTGTTCCTCGCTGCCAAGAATCTGTCGGACGCCATCCTC

[0143] CTGTCTGATATTCTCAGGGTGAACACCGAGATTACGAAGGCTCCGTCTCAGCCTCCATGATCAAGCGCT

[0144] ACGACGAGCACCATCAGGATCTGACCCTCCTGAAGGCGCTGGTCAGGCAGCAGCTCCCCGAGAAGTAC

[0145] AAGGAGATCTTCTTCGATCAGTCGAAGAACGGCTACGCTGGTACATTGACGGCGGGCCTCTCCAGGAG

[0146] GAGTTCTACAAGTTCATCAAGCCGATTCTGGAGAAGATGGACGGCACGGAGGAGCTGCTGGTGAAGCT

[0147] CAATCGCGAGGACCTCCTGAGGAAGCAGCGGACATTCGATAACGGCAGCATCCCACACCAGATTCATCT

[0148] CGGGGAGCTGCACGCTATCCTGAGGAGGCAGGAGGACTTCTACCCTTTCCTCCAAGGATAACCGCGAGAA

[0149] GATCGAGAAGATTCTGACTTTCAGGATCCCGACTACGTCGGCCCACTCGCTAGGGGCAACTCCCGCTTC

[0150] GCTTGGATGACCCGCAAGTCAGAGGAGACGATCACGCCGTGGAACTTCGAGGAGGTGGTCGACAAGGG

[0151] CGCTAGCGCTCAGTCGTTCATCGAGAGGATGACGAATTTCGACAAGAACCTGCCAAATGAGAAGGTGCT

[0152] CCCTAAGCACTCGCTCCTGTACGAGTACTTCACAGTCTACAACGAGCTGACTAAGGTGAAGTATGTGAC

[0153] CGAGGGCATGAGGAAGCCGGCTTTCCTGTCTGGGGAGCAGAAGAAGGCCATCGTGGACCTCCTGTTCA

[0154] AGACCAACCGGAAGGTCACGGTTAAGCAGCTCAAGGAGGACTACTTCAAGAAGATTGAGTGCTTCGAT

[0155] TCGGTCGAGATCTCTGGCGTTGAGGACCGCTTCAACGCCTCCCTGGGGACCTACCACGATCTCCTGAAG

[0156] ATCATTAAGGATAAGGACTTCCTGGACAACGAGGAGAATGAGGATATCCTCGAGGACATTGTGCTGACA

[0157] CTCACTCTGTTCGAGGACCGGGAGATGATCGAGGAGCGCCTGAAGACTTACGCCCATCTCTTCGATGAC

[0158] AAGGTCATGAAGCAGCTCAAGAGGAGGAGGTACACCGGCTGGGGGAGGCTGAGCAGGAAGCTCATCA

[0159] ACGGCATTCGGGACAAGCAGTCCGGGAAGACGATCCTCGACTTCCTGAAGAGCGATGGCTTCGCGAAC

[0160] CGCAATTTCCATGCAGCTGATTCACGATGACAGCTCACATTCAAGGGAGGATATCCAGAAGGCTCAGGTG

[0161] AGCGGCCAGGGGGACTCGCTGCACGAGCATATCGCGAACCTCGCTGGCTCGCCAGCTATCAAGAAGGG

[0162] GATTCTGCAGACCGTGAAGGTTGTGGACGAGCTGGTGAAGGTCATGGGCAGGCCACAAGCCTGAGAACA

[0163] TCGTCATTGAGATGGCCCGGGAGAATCAGACCACGCAGAAGGGCCAGAAGAACTCACGCGAGAGGATG

[0164] AAGAGGATCGAGGAGGGCATTAAGGAGCTGGGGTCCCAGATCCTCAAGGAGCACCCCGGTGGAGAACAC

[0165] GCAGCTGCAGAATGAGAAGCTCTACCTGTACTACCTCAGAATGGCCGCGATATGTATGTGGACCAGGA

[0166] GCTGGATATTAACAGGCTCAGCGATTACGACGTCGATCATATCGTTCCACAGTCATTCCTGAAGGATGACT

[0167] CCATTGACAACAAGGTCCTCACCAGGTCGGACAAGAACCGGGCAAGTCTGATAATGTTCCTTCAGAGG

[0168] AGGTCGTTAAGAAGATGAAACTACTGGCGCCAGCTCCTGAATGCCAAGCTGATCACGCAGCGGGAAG

[0169] TTCGATAACCTCACAAAGGCTGAGAGGGGCGGGCTCTCTGAGCTGGACAAGGCGGGCTTCATCAAGAG

[0170]

[0171] The sequence of SEQ ID NO:8 (signal peptide sequence targeting the DMD1 gene) is as follows:

[0172] GAGGCCTAAACCTCTTTCCA

[0173] The sequence of SEQ ID NO:9 (gRNA coding sequence) is as follows:

[0174] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAGTGGCACCGAGTCGGTGC

[0175] II. Obtaining DMD1 gene knockout cucumbers

[0176] The gene knockout vector pBSE402-CRDMD1 obtained in step one above was transformed into the cotyledonary nodes of cucumber variety CU1 using Agrobacterium GV3101. Transgenic plants were obtained through differentiation and regeneration. The transgenic plants were grown on a medium containing herbicides, and resistant seedlings containing GFP signals were screened and then identified and confirmed by molecular biology experiments.

[0177] Identification of CU1-positive transgenic cucumber plants: DNA was extracted from the leaves of two T0 generation transgenic cucumber plants screened using the above steps. The target fragment was amplified by PCR, and the positive transgenic plants were detected by sequencing. Primers used for amplifying the target fragment:

[0178] 940cxF:TCCAGCATTGTGAAGCAAGAG(SEQ ID NO:10)

[0179] 940cxR:GCAGTTTCCAGAATAGCCAAGTT(SEQ ID NO:11)

[0180] The amplification product is 1014bp.

[0181] The T0 generation (heterozygous) positive cucumber CU1, confirmed by the above identification to have gene editing sites, was self-crossed and then hybridized with CU1 to obtain homozygous knockout lines dmd1-line1 and dmd1-line2, and heterozygous lines dmd1-line1×DMD1-WT and dmd1-line2×DMD1-WT, respectively, which isolated the Cas9 sequence. Figure 4 As shown in b, b represents the sequencing information of the gene knockout lineage and background material.

[0182] The gene-edited plants and the control CU1 plants were crossed with MSC16, and the phenotypic segregation ratio and mitochondrial genome origin of the hybrid offspring were analyzed. Phenotypic identification showed that the offspring of lines dmd1-line1 and dmd1-line2 crossed with MSC16 were all wild-type; while the offspring of lines dmd1-line1×DMD1-WT and dmd1-line2×DMD1-WT crossed with MSC16 showed a 1:1 segregation ratio, and mitochondrial genomes derived from CU1 were detectable in all wild-type plants. As a control, the offspring of CU1×MSC16 were all MSC-type, with only mitochondrial genomes from the paternal line (MSC16) detectable. Figure 4 As shown in Figure c, c represents the phenotypic segregation results of homozygous individuals and hybrids with gene knockout, as well as the offspring of the background material and MSC16.

[0183] Example 2: DMD1 gene and its functional verification in melon

[0184] Based on sequence homology, a homologous gene of the DMD1 gene was cloned from *Melastoma candida*, a plant in the genus *Melastoma*. The sequence of this homologous gene is SEQ ID NO:12. After knocking out this gene, hybridization with other *Melastoma* species revealed maternally derived mitochondrial genomes in the offspring, confirming the consistent function of this gene within the *Melastoma* genus.

[0185] The sequence of SEQ ID NO:12 is as follows:

[0186] ATGTCGTCGAGGTTGAATCGTTCCCTGTACCTTTATTCACACAGAATTCAGAGCCTAGAGGGGCG

[0187] GGAAGAGGTGTAAGTGTGCGAAGGAATTCCAATGAGGCTGTTGTCGAGGAACATGATTGTAAAG

[0188] TGGACATTCAAAGTTTTAGGGATGACCCGTCGAAAATTGAAGCTATGACAGTCCAAAAATTGAGA

[0189] ATGACATTAAGAAGCGTGGGTCTTCTAGCCAAAGGGCTTAAGCGTGACCTTGTAGCTGCACTGCA

[0190] AAGCTTTGTGGAGAATGGAACAGTTGGTGGTTTTATTGATGCAGTAGAAAATCAGAGCACACAAC

[0191] AGACAGAGAGAAATTCTAGTGTATCAGCTTCTTATGGTGACACTGTGAAAGCTGAAACAAAAATC

[0192] TTAACACCAAAGAAGAGGCAGTCAGCTGAATCTGACAGGGTTTCAAGTGGTGCAATAGGCAGTA

[0193] ACCCATCTAGCAGAAAGATAAATGGATCCTCAGAAGTTGTTTCCAGCATTGTAAAGCAAGAGGAT

[0194] GGAGTAGAGGGGATGCAAAATGAACCATGGGCTGTTCTTGCTCATAAGAAGCCTAAAAAAGGTT

[0195] GGATCCCGTATAACCCAAGAATCATGAGGCCTAAACCTCTTTCCAAGGATACAAAATCTGTGAAA

[0196] ATTTTATCTTGGAATGTCAATGGGTTAAGAGCCTTACTTAAGGGGTCTTCAGCAGTGGAACTTGCT

[0197] GAAAGGGAAGATTTTGACATATTGTGCTTACAGGAGACCAAATTGCAGGAAGGAGACATACTAA

[0198] CTATTACAAAATCTCTCATGGATGGATATCATTATACCTACTGGACATGCAGCGTTTCTAAACTTG

[0199] GTTATTCTGGAACTGCAATCATATCCCGGATAAAACCAATTTCAGTTAGATATGGTTTAGGCATAT

[0200] CAGAACATGATGGTGAAGGGCGGGTTGTGATGGTGGAGTTTGATTCTTTCTTTTTATTGAATGTTT

[0201] ATGTTCCTAATTCTGGAGACGGATTGAAGAGACTGTCATACAGGATTACCGAGTGGGATCCATCT

[0202] CTCAGTAACTATATGAAGGAGTTGGAAAAGTTGAAGCCCGTGATTTTGACTGGTGATCTAAATTG

[0203] TGCTCATCAAGAGATAGACATCTATAATCCTGCGGGAAACCGAAAAAGTGCTGGTTTCACCATCG

[0204] AAGAAAGGCAATCATTTGAGACCAACTTTCTGGAAAAGGGGTTTGTTGATACTTTTAGACAAAAG

[0205] CATCCTGATGTTGTTGGCTATACATATTGGGGTTTATCGTCATGGTGGACGCAAAACAAACAAAGG

[0206] ATGGCGGCTGGACTATTTTCTTGTATCAGATAGGATGGCAGAGAAGGTACACGACTCTTACATAC

[0207] TCCCTGATGTGGGAGGTAGTGATCACTGTCCCATTGGCCTTGTTCTCAAGCTTTAG

[0208] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "implementation," or "specific implementation," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0209] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A mutant of DMD1 gene, characterized in that, The DMD1 gene mutant is the sequence shown in SEQ ID NO:1 or the sequence complementary to SEQ ID NO:

1.

2. A polypeptide or protein sequence, characterized in that, The polypeptide or protein sequence is shown in SEQ ID NO:3; Optionally, the polypeptide or protein sequence is encoded by a gene in the DMD1 gene mutant of claim 1; Optionally, the polypeptide or protein sequence has endonuclease activity.

3. An expression carrier, characterized in that, The expression vector includes: (1) The expression cassette for Cas9 protein, including the first promoter, the signal peptide sequence targeting the DMD1 gene, the Cas9 gene, and the terminator; (2) The expression cassette of the sgRNA, including the second promoter, target sequence and gRNA sequence; The target sequence is a 16-24 bp sequence targeting the DMD1 gene, and the DMD1 gene is the sequence shown in SEQ ID NO:2 or SEQ ID NO:

12. Optionally, the expression vector is at least one of plasmid, virus or bacteriophage; Optionally, the plasmid is selected from at least one of pBSE402 or pKSE402.

4. A recombinant cell, characterized in that, The recombinant cells include the expression vector according to claim 3; Optionally, the recombinant cells are bacterial cells, fungal cells, or algal cells.

5. A reagent kit, characterized in that, The kit includes the expression vector of claim 3 or the recombinant cells of claim 4.

6. A method for cultivating plants with mitochondrial biparental inherited traits, characterized in that, include: A transgenic plant is obtained in which the DMD1 gene is not expressed or is lost in function, wherein the DMD1 gene is the sequence shown in SEQ ID NO:2 or the sequence shown in SEQ ID NO:12; Using the aforementioned transgenic plant as the female parent material and the wild-type plant as the male parent material, hybrid plants with mitochondrial genetic traits from both parents are obtained through hybridization. The plant is selected from at least one of cucumber and melon.

7. The method according to claim 6, characterized in that, The following method was used to prevent the expression or loss of function of the DMD1 gene in the transgenic plant: CRISPR-Cas technology, ZFNs technology, or TALENs technology.

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