CN115896164A - Method for improving storage stability of plant fruit - Google Patents

Abstract

The invention discloses a method for improving the storage stability of plant fruits. The invention utilizes CRISPR/Cas9 genome multi-target editing technology to target a key gene for degrading tomato chlorophyll, namely the chlorophyll-stay green gene 1 (SGR 1), and is also a key gene for synthesizing lycopene, thereby creating a gene-edited tomato. Compared with wild tomatoes, the gene-edited tomatoes have better storage resistance and transportation resistance of fruits. The method provided by the invention provides a new strategy and method for improving tomato germplasm and improving economic benefits of tomato transportation.

Description

Method for improving storage stability of plant fruit

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for improving the storage stability of plant fruits.

Background

Tomatoes are important vegetable crops in the world, are rich in nutrition, have sour and sweet mouthfeel, can be eaten fresh or processed deeply, and have high economic benefit. The greenhouse can provide a relatively suitable growth environment for the growth of the tomatoes, reduces the damage of natural disasters and plant diseases and insect pests to plants, and is beneficial to improving the quality and the economic benefit of the tomatoes. Therefore, the facility cultivation of the tomatoes can break through the limitation of seasons and regions, reduce the production difficulty, help to meet the requirement that people can purchase high-quality tomatoes all the year round, and become the main facility for planting tomatoes in winter and early spring.

Tomatoes belong to climacteric fruits, are easy to mature after being picked, and the softening, the decay and the deterioration of the tomatoes are also aggravated by imperfect postharvest storage conditions and techniques, so that the tomatoes are infected by various germs and the physiological quality is reduced, thereby gradually losing the commodity value and causing huge economic loss. Therefore, how to improve the storage stability of tomatoes is an important problem to be solved urgently. Until now, there are many reported methods for improving the storage endurance of tomatoes, mainly selecting a suitable storage environment and applying some chemicals from external sources, but many measures have insignificant effects and also have the problems of high cost, chemical residue, loss of nutrients and the like. The cultivated new tomato germplasm with strong storage stability can improve the characteristics of the tomato germplasm fundamentally, and can more effectively improve the storage stability by combining some safe exogenous physical and chemical measures, maintain the stable quality of the picked fruits and further improve the economic benefit.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the storability of plant fruits and/or how to regulate the weight loss rate of the plant fruits and/or how to regulate the fruit hardness of the plant fruits and/or how to cultivate storage-resistant plants.

In order to solve the above technical problems, the present invention provides a method for improving fruit storability of a plant, comprising reducing or inhibiting the activity of a protein in a target plant or/and the expression level of a gene encoding the protein or/and performing gene editing on the gene encoding the protein or/and mutating the gene encoding the protein, thereby improving fruit storability of the plant.

The protein may be a protein of A1), A2) or A3) as follows:

a1 Protein with the amino acid sequence of sequence 1 in the sequence table;

a2 ) the amino acid sequence shown in the sequence 1 in the sequence table is subjected to substitution and/or deletion of amino acid residues and

or a protein which is derived from A1) and has the same function, or has 80% or more of identity with the protein shown in A1) and has the same function;

a3 A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1) or A2).

The above-described shelf life may be an extension of the shelf life of the fruit after picking. The storability may be a reduction in weight loss rate of the fruit after picking and/or maintenance of fruit firmness.

The method as described above comprises introducing into said plant a substance that reduces or inhibits expression of a gene encoding a protein as described above or a substance that effects gene editing of a gene encoding said protein. The substance may be any of the following c 1) to c 4):

c1 A nucleic acid molecule which inhibits or reduces the expression of a gene encoding a protein according to A1) above;

c2 An expression cassette comprising the nucleic acid molecule according to c 1);

c3 A recombinant vector containing the nucleic acid molecule according to c 1) or a recombinant vector containing the expression cassette according to c 2);

c4 A recombinant microorganism containing the nucleic acid molecule according to c 1), or a recombinant microorganism containing the expression cassette according to c 2), or a recombinant microorganism containing the recombinant vector according to c 3).

In the above method, the nucleic acid molecule of c 1) is a DNA molecule that expresses a gRNA targeting the gene encoding the protein of A1) above or a gRNA targeting the gene encoding the protein of A1) above;

the target sequence of the gRNA of the gene coding the protein of the targeting A1) can be shown as 378 th to 397 th of a sequence 2 in a sequence table and 722 th to 741 th of the sequence 2 in the sequence table.

In the above method, the inhibiting or reducing the expression of the gene encoding the protein described above or the gene editing of the gene encoding the protein in the plant may be carried out by subjecting the gene encoding the protein represented by sequence 2 in the plant genome to the following mutations:

inserting a base G between 380 th and 381 th nucleotides of the sequence 2, and inserting a nucleotide C between 738 th and 739 th nucleotides of the sequence 2;

the plant may be a tomato.

In order to solve the above-mentioned problems, the present invention also provides a use of any one of a substance that regulates an activity or a content of a protein and/or a substance that regulates an expression level of a gene encoding the protein and/or a substance that edits a gene encoding the protein and/or a substance that mutates a gene encoding the protein, the use of the substance being:

p1, the application of the substance in regulating and controlling the storage stability of plant fruits,

p2, the use of said substances in plant breeding or quality improvement;

p3, application of the biological material in regulation and control of plant fruit weight loss rate;

p4, application of the biological material in regulating and controlling the hardness of plant fruits;

the protein may be a protein of A1), A2) or A3) as follows:

a1 Protein of which the amino acid sequence is a sequence 1 in a sequence table;

a2 Substitution and/or deletion of amino acid residue of amino acid sequence shown in sequence 1 in sequence table and

or a protein which is derived from A1) and has the same function, or has 80% or more of identity with the protein shown in A1) and has the same function;

a3 A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1) or A2). In the above application, the substance for regulating the activity or content of the protein may be a substance for knocking out a gene encoding the protein and/or a substance for regulating the expression of a gene encoding the protein.

In the above application, the substance for regulating gene expression may be a substance for regulating at least one of the following 6: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In the above application, the regulation of gene expression may be the inhibition or reduction of gene expression, and the inhibition or reduction of gene expression may be achieved by gene knockout or by gene silencing.

The gene knock-out (geneknockout) refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a specific target gene by a change in the DNA sequence.

The gene silencing refers to the phenomenon that a gene is not expressed or is under expression under the condition of not damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not altered, resulting in no or low expression of the gene. Gene silencing can occur at two levels, one is at the transcriptional level due to DNA methylation, differential staining, and positional effects, and the other is post-transcriptional gene silencing, i.e., inactivation of a gene at the post-transcriptional level by specific inhibition of a target RNA, including antisense RNA, co-suppression (co-suppression), gene suppression (quelling), RNA interference (RNAi), and micro-RNA (miRNA) -mediated translational suppression, among others.

In the above application, the substance for regulating gene expression may be an agent for inhibiting or reducing the gene expression. The agent that inhibits or reduces the expression of the gene can be an agent that knocks out the gene, such as an agent that knocks out the gene by homologous recombination, or an agent that knocks out the gene by CRISPR-Cas 9. The agent that inhibits or reduces expression of the gene may comprise a polynucleotide that targets the gene, such as an siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

In the above method or use, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity.

The term "identity" refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences. The having 90% or more than 90% identity can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

Any of the following uses of the protein-related biomaterials described above is also within the scope of the present invention:

p1, application of the biological material in regulation and control of plant fruit storability;

p2, the use of the biological material in plant breeding or quality improvement;

p3, application of the biological material in regulation and control of plant fruit weight loss rate;

p4 and application of the biological material in regulating and controlling the hardness of the plant fruits.

The biomaterial may be any one of:

d1 Nucleic acid molecules encoding the proteins described hereinabove;

d2 An expression cassette comprising a nucleic acid molecule according to D1);

d3 A recombinant vector containing the nucleic acid molecule according to D1) or a recombinant vector containing the expression cassette according to D2);

d4 A recombinant microorganism containing the nucleic acid molecule according to D1), or a recombinant microorganism containing the expression cassette according to D2), or a recombinant microorganism containing the recombinant vector according to D3);

d5 A transgenic plant cell line containing the nucleic acid molecule according to D1) or a transgenic plant cell line containing the expression cassette according to D2);

d6 A transgenic plant tissue containing the nucleic acid molecule according to D1) or a transgenic plant tissue containing the expression cassette according to D2);

d7 A transgenic plant organ containing the nucleic acid molecule according to D1) or a transgenic plant organ containing the expression cassette according to D2);

d8 Nucleic acid molecules which inhibit or reduce the expression of a gene coding for a protein as described above or the activity of a protein as described above or/and nucleic acid molecules which carry out gene editing of a gene coding for a protein as described above;

d9 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing a nucleic acid molecule according to D8).

In the above application, the nucleic acid molecule may be a DNA molecule as shown below:

b1 The coding sequence is a DNA molecule shown as a sequence 2 in a sequence table;

b2 The nucleotide sequence is a DNA molecule shown as a sequence 4 in a sequence table;

b3 A DNA molecule having 90% or more identity to the nucleotide sequence defined in b 1) or b 2) and encoding the protein of claim 1;

b4 A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined under b 1), b 2) or b 3) and which codes for a protein as claimed in claim 1.

D8 The nucleic acid molecule may be a DNA molecule that expresses a gRNA targeting a gene encoding a protein described in A1) above or a gRNA targeting a gene encoding a protein described in A1) above.

The target sequence of the gRNA targeting the protein coding gene of the A1) can be shown as 378 th to 397 th of a sequence 2 in a sequence table and 722 th to 741 th of the sequence 2 in the sequence table.

In the above biological materials, the expression cassette containing a nucleic acid molecule according to D2) is a DNA capable of expressing the protein in the above application in a host cell, and the DNA may include not only a promoter for initiating transcription of a gene encoding the protein but also a terminator for terminating transcription of a gene encoding the protein. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.

The recombinant expression vector containing the protein coding gene expression cassette can be constructed by using the existing plant expression vector.

In the above biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

The plant described above may be any of:

c1 A dicotyledonous plant such as a dicotyledonous plant,

c2 A monocotyledonous plant such as a monocotyledonous plant,

c3 A plant of the order tubuliformes,

c4 A plant of the family Solanaceae,

c5 A plant of the genus Solanum),

c6 ) tomatoes.

The application of the method in the preparation of products for improving the storage stability of plant fruits also belongs to the protection scope of the invention.

The biological materials described above also fall within the scope of the present invention.

The invention utilizes CRISPR/Cas9 genome multi-target editing technology, targets chlorophyll degradation key gene-stay green gene 1 (STAY GREEN, SGR1), is also a lycopene synthesis key gene, improves the postharvest storability of tomatoes, and creates a novel tomato germplasm with storability. The invention provides a new strategy and a new method for improving tomato germplasm and improving economic benefits.

Drawings

FIG. 1 shows the fruiting phenotype of wild type tomato (WT) and gene edited tomato (L-1) stored at ambient temperature for different days (0 d, 5d and 10 d). The upper and lower photographing records are respectively carried out on each group, and the scale is 2.5cm

FIG. 2 is a comparison of weight loss and firmness during storage of wild type and L-1 gene edited tomatoes. A is the comparison of weight loss rate of wild type and L-1 gene during the storage period of the tomato; b is a comparison of the hardness of wild type and L-1 gene edited tomatoes during storage. * Indicating significant difference (P < 0.05); * Indicates that the difference was very significant (P < 0.01).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The agrobacterium-mediated tomato stable genetic transformation method (leaf disc method) in the embodiment of the invention is as follows:

aseptic seeding of tomato seeds:

picking plump AC tomato seeds, soaking in 70% ethanol for 2min in an ultra-clean workbench, then soaking in 5% sodium hypochlorite for 10min, and reversing and mixing uniformly for several times; sucking liquid with a gun, washing with sterile water for 3-5 times, placing seeds on sterile filter paper, and sucking water; uniformly placing the sterilized seeds on a 1/2MS culture medium, wherein 7-9 seeds are placed in each bottle; placing the culture bottle in a dark box at 25 ℃ for about 3 days, taking out the culture bottle when the seeds grow white buds of 2-3cm, placing the culture bottle in the light for culture, and using the culture bottle for infection when the seedlings grow until cotyledons are fully extended and true leaves do not really grow out (the seedlings cannot be too old, otherwise, the conversion rate is reduced).

Infection:

the secondarily activated recombinant agrobacterium EHA105/Cas9-SGR1 bacterial liquid (OD) _600nm = 0.6-0.8) centrifuging and collecting bacteria, removing supernatant, adding a small amount of MS liquid, blowing, beating and resuspending, continuously adding MS liquid to dilute the bacteria liquid to OD _600nm 0.2-0.4, adding acetosyringone with final concentration of 100-200 μ M, and shaking or standing at 28 deg.C while preparing for infection of material.

The tomato cotyledon is transversely cut into two sections (two ends are removed), the two sections are placed in a conical flask added with a proper amount of MS liquid, the MS liquid is slowly poured out after the material preparation is completed, and the prepared bacterial liquid is added to ensure that all leaf tissues are immersed in the bacterial liquid without wall hanging. Shaking slowly at 28 deg.C for 10min, standing for 10min, or standing in a workbench by hand for several times. Pouring out the bacterial liquid, spreading the leaves on filter paper, quickly spreading on a co-culture medium after the water film on the surface of the leaves disappears, sealing with a sealing film, and culturing in the dark of a tissue culture room for 2-3d.

Screening and culturing:

after the completion of co-cultivation, the leaf pieces were gently transferred to a selection medium (hygromycin 5 mg/L), cultured in the dark for 1 week, and then left to incubate in the light. After callus and adventitious shoots are produced, if contamination does not occur, transfer to subculture medium is not urgent. The adventitious bud generated by induction grows gradually along with the gradual decrease of the content of the zeatin in the culture medium. If the seedling generated by the adventitious bud is higher than 1cm, the seedling can be directly transferred to a rooting medium. Nutrient depletion or water depletion in the culture medium is not necessary to worry about in the culture process.

Subculturing:

taking larger adventitious buds from the screening culture medium, removing callus, inserting into subculture medium, and culturing under illumination. In the step, the callus needs to be cleaned, otherwise, endogenous hormones generated by the callus interfere with the hormone level of the whole plant, so that adventitious buds are difficult to grow up, and large callus is generated at the base of the plant. If necessary, the callus can be removed of browned part and moved back to the screening medium to continue inducing adventitious buds. If the adventitious bud is small (< 2 mm), it is difficult to survive and can be discarded. About 2-3 weeks, the adventitious bud will grow into a plant.

Rooting culture:

larger plants (stems are clearly visible, higher than 1 cm) are selected from the subculture medium, callus and tillering clumps at the base are removed, and the plants are inserted into a rooting medium. If the conditions exist, the lower quarter of the plant can be cut off to adjust the hormone level of the plant, so that the plant is easier to root. For larger shoots in the screening medium, they can also be transferred directly to rooting medium, but the callus needs to be cleaned. Care should be taken to wipe off flower buds at any time during the cultivation process.

For positive plants, the side branches generated at the top of the plant and after the mother plant can be cut and inserted into a rooting medium for rapid propagation.

Potting and planting:

taking out the plant in the culture bottle, removing the culture medium at the root (the step is critical, if the removal is not clean enough, the seedling is difficult to survive) and the tiller cluster, planting in sterilized culture soil, watering thoroughly, covering with a plastic film, sealing with rubber band, and moving to normal light after 3 days in dark. The plants are not cut in the planting process and are exposed in the air for too long, which is easy to cause wilting. After 1 week, the plastic film can be peeled off at one corner, and after 2 weeks, complete peeling can be tried, i.e. T is obtained ₀ And (3) simulating transgenic tomato plant plantlets.

The planting conditions of the tomatoes in the embodiment of the invention are as follows: the tomato seeds are sown in small square pots of 10 x 10cm after being mixed with nutrient soil, turf and vermiculite (2, 1, v/v/v), two tomatoes are planted in each pot, the pots are cultivated in a plant growth chamber after being covered with a film and kept moist, the film is opened with a small opening after germination, the film is removed after one week, and the seedlings are transplanted in a sunlight greenhouse when growing to about 15 cm. The cultivation environment with weak light has a temperature of 25 deg.C/18 deg.C (day/night), a photoperiod of 12h/12h (day/night), and an optical density of 100-200 μmol m ^-2 s ^-1 。

All experiments in the examples of the present invention included more than three replicates, and data processing and differential significance analysis were performed using Excel and SPSS 24.

The wild type tomato AC (Ailsa Craig) in The embodiment of The invention is provided by The subject group of The Chinese college of agriculture university food science and nutrition engineering Zhu Hongliang (relevant documents: ma L, yang Y, wang Y, et al. SlRBP1 proteins transfer relational efficacy via SleIF4A2 to main chloride function in tomato [ J ]. The Plant Cell, 2022); both the pYLCRISPR/Cas9 and pYLsgRNA plasmids are provided by the subject group of subtropical agriculture biological resource protection and utilization of national emphasis laboratory Liu Yaoguang (related documents: zeng Dongchang, ma Xingliang, xie Xianrong, etc.. Methods for the construction and mutation analysis of plant CRISPR/Cas9 multigene editing vectors, chinese science: life sciences, 2018,48 783-794 Ma X, zhang Q, zhu Q, arob CRISPR/Cas9 system for concomittant, high-efficiency multiplex gene editing in monocation and dicot plants [ J ]. Molecular plant,2015,8 (1274-1284).

All experiments in the examples of the present invention included more than three replicates, and data processing and differential significance analysis were performed using Excel and SPSS 24.

Example 1 Gene editing of SGR1 Gene to improve tomato storability

Construction of CRISPR-Cas9 recombinant plasmid

The amino acid sequence of the tomato SGR1 protein is shown as a sequence 1 in a sequence table, and the CDS coding sequence of cDNA is nucleotide shown as a sequence 2 in the sequence table.

The construction of CRISPR expression vectors is carried out with reference to the method of Zeng Dongchang et al (2018).

1.1 acquisition of fragments of gRNA

Designing SGR1 gene target on http:// criprp. Hzau.edu.cn/cgi-bin/CRISPR2/CRISPR # website, selecting target sequence with high score and without off-target, and designing and synthesizing primer for constructing expression cassette.

The nucleotide sequence of the target 1 of the SGR1 is 5'-GGCCTCCACTAATGTGGCAA-3', the nucleotide sequence of the target 2 is 5'-CCCCAGTGAGTGTTATGCCT-3', and the target 1 and the target 2 correspond to nucleotides 378-397 and 722-741 of a sequence 2 in a sequence table of the SGR1 gene respectively.

1.2 construction of CRISPR-Cas9 recombinant plasmids

And recovering PCR products after PCR reaction, and respectively constructing two target spots on corresponding sgRNA vectors (pYLsgRNA plasmids) to obtain two complete sgRNA expression cassettes. Reaction system: q5 buffer 5. Mu.L, dNTP mix 2.5. Mu.L, pYLsgRNA vector template 1. Mu.L, upstream and downstream primers 1. Mu.L each, Q5 enzyme 0.25. Mu.L, ddH ₂ Supplementing 25 mu L of O; the PCR reaction conditions were: 3min at 98 ℃;98 ℃ 10s,55 ℃ 30s,72 ℃ 30s/kb,34cycles; 5min at 72 ℃. Wherein the upstream and downstream primer sequences corresponding to the target 1 are respectively as follows:

S1-F：5’-GGCCTCCACTAATGTGGCAAgttttagagctagaaat-3’；

S1-R：5’-TTGCCACATTAGTGGAGGCCTgaccaatggtgctttg-3’。

the upstream and downstream primer sequences corresponding to the target 2 are respectively:

S2-F：5’-CCCCAGTGAGTGTTATGCCTgttttagagctagaaat-3’；

S2-R：5’-AGGCATAACACTCACTGGGGTgaccaatggtgctttg-3’。

the required universal primers are:

U-F：5’-CTCCGTTTTACCTGTGGAATCG-3’；

gR-R：5’-CGGAGGAAAATTCCATCCAC-3’。

and connecting the sgRNA expression cassette to a pYLCISPR/Cas 9 expression vector by using a Golden Gate method to obtain a connection product. Reaction system: 10 XCutsmart buffer 1.5. Mu.L, 10 XT 4 DNA ligase buffer 1.5. Mu.L, pYRCISPR/Cas 9 plasmid 60ng, each sgRNA expression cassette 10ng, bsa I1. Mu.L, T4 DNA ligase 1. Mu.L, ddH ₂ Supplementing O to 15 μ L; reaction conditions are as follows: 5min at 37 ℃, 5min at 10 ℃, 5min at 20 ℃,20cycle; 5min at 37 ℃.

1.3 obtaining recombinant Agrobacterium

And transforming the ligation product into DH5 alpha escherichia coli, coating the Escherichia coli on an LB (lysogeny broth) plate containing kanamycin resistance, carrying out overnight culture at 37 ℃, obtaining positive clones, sending to a company for sequencing, and shaking the bacterial liquid with correct sequencing to extract plasmids to obtain the CRISPR-Cas9 recombinant plasmid Cas9-SGR1.

The recombinant plasmid Cas9-SGR1 is transformed into agrobacterium-infected EHA105 to obtain recombinant agrobacterium, which is named as EHA105/Cas9-SGR1 and used for a subsequent stable genetic transformation test.

2. Obtaining transgenic tomato plants

Transforming the recombinant Agrobacterium obtained in step 1 into wild type tomato AC using leaf disc method to obtain T ₀ And (5) editing plants by generation genes. The identification method of the gene editing plant comprises the following steps:

rooting the rooted T in the rooting culture medium ₀ Numbering plants of the tomatoes, slightly shearing a blade with proper size and growth position close to a growth point by using scissors in a superclean bench, and using tweezersPutting the leaves into a 1.5mL centrifuge tube with a corresponding number, and extracting DNA of the leaves; respectively designing upstream and downstream primers for amplifying a target fragment at about 200-300bp before and after a target spot, amplifying the target fragment by PCR, sequencing, and confirming a target gene editing mode by using an online website DSDecodeM (http:// skl.scau.edu.cn/home /) in combination with a sequencing peak map. The detection primers for both targets were as follows:

an upstream primer: 5'-gctcatgacgcatgtcgaaatc-3';

a downstream primer: 5'-ggcacaacccaacttacaataattg-3'.

Through detection, the invention obtains 2 gene editing T ₀ Generating plants, wherein one plant is heterozygous mutant of the SGR1 gene sequence corresponding to the target point 1, and homozygous mutant of the SGR1 gene sequence corresponding to the target point 2; inserting a base G between 380 th to 381 th nucleotides in a sequence 2 in a sequence table corresponding to SGR1 gene of one chromosome in a tomato genome, wherein the other chromosome is not mutated; and simultaneously loses the 738 th nucleotide G of the sequence 2 in the sequence table corresponding to SGR1 genes in two chromosomes in a tomato genome. The other SGR1 gene sequence corresponding to the target point 1 is subjected to heterozygous mutation, and the SGR1 gene sequence corresponding to the target point 2 is subjected to biallelic mutation; inserting a base G between 380 th to 381 th nucleotides in a sequence 2 in a sequence table corresponding to SGR1 gene of one chromosome in a tomato genome, wherein the other chromosome is not mutated; and simultaneously, a nucleotide C is inserted between 738 th-739 th nucleotides of a sequence table sequence 2 corresponding to the SGR1 gene of one chromosome in the tomato genome, and the 738 th nucleotide G of the sequence table sequence 2 corresponding to the SGR1 gene of the other chromosome is deleted.

One strain T ₀ Plants edited by generation genes are respectively selfed for 2 generations to obtain T ₂ Generating gene edited plants to obtain homozygous mutants, and naming the homozygous mutants as L-1; compared with wild tomato, L-1 is formed by inserting a base G between 380 th to 381 th nucleotides of a sequence table sequence 2 corresponding to SGR1 genes of two chromosomes in a tomato genome, and simultaneously inserting a base G between 738 th to 739 th nucleotides of the sequence table sequence 2 corresponding to SGR1 genes of two chromosomes in the tomato genomeNucleotide C, leading to premature termination, thereby knocking out the SGR1 gene.

In the L-1 plant, the nucleotide sequence of the mutant SGR1 gene is shown as a sequence 4 in the sequence table, and the amino acid sequence of the mutant SGR1 protein is shown as a sequence 3 in the sequence table.

3. Determination of relevant indexes of fruit storage-tolerant phenotype

The wild tomato AC (WT) and homozygous gene editing line (L-1) are respectively selected from 5 plants, 10 fruits with consistent maturity, no injury and fruit base are randomly selected from each plant, the surfaces of the fruits are cleaned and then placed in a plastic box paved with a plurality of layers of soft paper, and the fruits are stored at the temperature of 20 ℃. Photographing every 5 days to record the phenotype, simultaneously weighing by using an electronic balance, and calculating the weight loss rate, wherein the calculation formula is as follows: weight loss ratio/% = (m) ₀ -m ₁ )/m ₀ X 100, wherein: m is ₀ Mass before storage, g; m is a unit of ₁ Mass after storage, g.

Fruit hardness was measured using a fruit quality analyzer TAXT Express Texture, and the front and back sides of the center of the skinned fruit were pierced with a TA needle (product number P/2, stable Micro Systems, UK.) to read the data, and the data was introduced into the exposure System software (download site: https:// static Systems. Com/software update Exponent. Html) to analyze.

As can be seen from fig. 1 and 2A, after 5 days of storage, the wild type tomato fruits (WT in fig. 1 and 2) showed slight shrinkage, especially at the bottom, with a weight loss of 17.2%, while the gene-edited tomato fruits (L-1 in fig. 1 and 2) showed no phenotypic change, with a weight loss of 3.5%, with a significant difference (P < 0.01); after 10 days of storage, the wrinkle indentation of the wild tomato fruits is aggravated by one step, the weight loss rate is 31.7%, the gene editing tomato fruits are slightly shrunken, the weight loss rate is 7.3%, and the difference is obvious (P is less than 0.01). Fruit firmness of both types of tomatoes decreased with increasing days of storage, but the decrease in fruit firmness of gene-edited tomatoes was significantly lower than the wild type (B in fig. 2, P < 0.01).

In addition, in order to determine whether fruit size significantly affects wild type and gene-edited tomato storability, fruits of substantially uniform size were selected for further testing, and it can be seen from group 3 tomatoes (represented by representative fruit 3 in fig. 1) that under the same fruit size, the storability of the gene-edited tomato fruit is still significantly better than that of the wild type. Therefore, the storability of the gene-edited tomato fruit is significantly better than that of the wild type.

Therefore, the homozygous gene editing material obtained by carrying out gene editing on the tomato SGR1 gene shows stronger postharvest storage property, and provides a new strategy and method for improving tomato germplasm and improving economic benefits.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims (10)

1. A method for improving the fruit storability of a plant, comprising reducing or inhibiting the activity of a protein in a plant of interest or/and the expression level of a gene encoding said protein or/and performing gene editing on a gene encoding said protein or/and mutating a gene encoding said protein, thereby improving the fruit storability of a plant;

the protein is the protein of A1), A2) or A3) as follows:

a1 Protein of which the amino acid sequence is a sequence 1 in a sequence table;

a2 A protein which is derived from A1) and has the same function, or has more than 80% of identity with the protein shown in A1) and has the same function, and is obtained by substituting and/or deleting and/or adding amino acid residues in an amino acid sequence shown in a sequence 1 in a sequence table;

a3 A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1) or A2).

2. The method of claim 1, wherein: the method comprises introducing into the plant a substance that reduces or inhibits expression of a gene encoding the protein of claim 1 or performing gene editing on a gene encoding the protein; the substance is any one of the following substances c 1) to c 4):

c1 A nucleic acid molecule which inhibits or reduces the expression of a gene encoding a protein according to claim 1) a;

c2 An expression cassette comprising the nucleic acid molecule according to c 1);

c3 A recombinant vector containing the nucleic acid molecule according to c 1) or a recombinant vector containing the expression cassette according to c 2);

c4 A recombinant microorganism containing the nucleic acid molecule according to c 1), or a recombinant microorganism containing the expression cassette according to c 2), or a recombinant microorganism containing the recombinant vector according to c 3).

3. The method according to claim 1 or 2, characterized in that:

c1 The nucleic acid molecule is a DNA molecule for expressing a gRNA targeting the gene encoding the protein A1) of claim 1 or a gRNA targeting the gene encoding the protein A1) of claim 1;

the target sequence of the gRNA targeting the A1) protein coding gene is shown as 378-397 th site of a sequence 2 in a sequence table and 722-741 th site of the sequence 2 in the sequence table.

4. A method according to any one of claims 1-3, characterized in that: the method for inhibiting or reducing the expression of the gene encoding the protein in the plant, wherein the gene encoding the protein is subjected to gene editing, and the gene encoding the protein is subjected to the following mutations in the plant genome, wherein the mutation is shown as a sequence 2:

inserting a base G between 380 th and 381 th nucleotides of the sequence 2, and inserting a nucleotide C between 738 th and 739 th nucleotides of the sequence 2;

the plant is tomato.

5. The substance for regulating the activity or content of the protein or/and the substance for regulating the expression level of the coding gene of the protein or/and the substance for carrying out gene editing on the coding gene of the protein and/or mutating the coding gene of the protein can be applied to any one of the following applications:

p1, the application of the substance in regulating and controlling the storage stability of plant fruits,

p2, the use of said substances in plant breeding or quality improvement;

p3, application of the biological material in regulation and control of plant fruit weight loss rate;

p4, application of the biological material in regulating and controlling the hardness of plant fruits;

the protein is the protein of A1), A2) or A3) as follows:

a1 Protein of which the amino acid sequence is a sequence 1 in a sequence table;

a2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table, is derived from A1) and has the same function, or has more than 80 percent of identity with the protein shown in A1) and has the same function;

a3 A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1) or A2).

6. Use of a biological material related to a protein according to claim 5 in any of the following applications:

p1, application of the biological material in regulation and control of plant fruit storability;

p2, the use of the biological material in plant breeding or quality improvement;

p3, application of the biological material in regulation and control of plant fruit weight loss rate;

p4, application of the biological material in regulating and controlling the hardness of plant fruits;

the biological material is any one of the following materials:

d1 A nucleic acid molecule encoding the protein of claim 5;

d2 An expression cassette comprising a nucleic acid molecule according to D1);

d3 A recombinant vector containing the nucleic acid molecule according to D1) or a recombinant vector containing the expression cassette according to D2);

d4 A recombinant microorganism containing the nucleic acid molecule according to D1), or a recombinant microorganism containing the expression cassette according to D2), or a recombinant microorganism containing the recombinant vector according to D3);

d5 A transgenic plant cell line containing the nucleic acid molecule according to D1) or a transgenic plant cell line containing the expression cassette according to D2);

d6 A transgenic plant tissue containing the nucleic acid molecule according to D1) or a transgenic plant tissue containing the expression cassette according to D2);

d7 A transgenic plant organ containing the nucleic acid molecule according to D1) or a transgenic plant organ containing the expression cassette according to D2);

d8 A nucleic acid molecule which inhibits or reduces the expression of a gene encoding a protein according to claim 5 or the activity of a protein according to claim 5 or/and a nucleic acid molecule which edits a gene encoding a protein according to claim 5;

d9 An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule according to D8).

7. Use according to claim 3, characterized in that: the nucleic acid molecule is a DNA molecule shown as follows:

b1 The coding sequence is a DNA molecule shown as a sequence 2 in a sequence table;

b2 The nucleotide sequence is a DNA molecule shown as a sequence 4 in a sequence table;

b3 A DNA molecule having 90% or more identity to the nucleotide sequence defined in b 1) or b 2) and encoding the protein of claim 1;

b4 A DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in b 1), b 2) or b 3) and which codes for a protein according to claim 1;

d8 The nucleic acid molecule) is a DNA molecule that expresses a gRNA targeting the gene encoding the protein of A1) above or a gRNA targeting the gene encoding the protein of A1) in claim 5.

8. Use according to any one of claims 5-7, characterized in that: the plant is any one of the following plants:

c1 A dicotyledonous plant such as a dicotyledonous plant,

c2 A monocotyledonous plant such as a monocotyledonous plant,

c3 A plant of the order tubuliformes,

c4 A plant of the family Solanaceae,

c5 A plant of the genus Solanum,

c6 ) tomatoes.

9. Use of a method according to any one of claims 1 to 3 for the preparation of a product for improving the storage stability of a fruit of a plant.

10. The biomaterial of claim 6 or 7.

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