A method for increasing tomato fruit yield and lycopene content in tomato fruit under weak light conditions

Abstract

The present invention discloses a method for increasing tomato fruit yield and lycopene content in tomato fruit under weak light conditions. The present invention uses CRISPR / Cas9 genome multi-target editing technology to target the key gene for tomato chlorophyll degradation, stay-green gene 1 (SGR1), which is also a key gene for lycopene synthesis, and creates gene-edited tomatoes. Compared with wild-type tomatoes, the gene-edited tomatoes have high lycopene and are tolerant to weak light. At the same time, the yield of tomato fruits under weak light facility cultivation conditions is increased by editing the SGR1 gene. The method provided by the present invention can be applied to tomato breeding and quality improvement.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for increasing the yield of tomato fruits and the lycopene content of tomato fruits under weak light conditions. Background Art

[0002] Tomatoes are an important vegetable crop in the world. They are rich in nutrients and have a sweet and sour taste. They can be eaten fresh or processed, and have high economic benefits. Greenhouses can provide a relatively suitable environment for the growth of tomatoes, reduce the damage to plants caused by natural disasters and pests and diseases, and help improve the quality and economic benefits of tomatoes. Therefore, the facility cultivation of tomatoes can break the seasonal and regional restrictions, reduce the difficulty of production, and help meet people's demand for high-quality tomatoes all year round. It has become the main facility for planting tomatoes in winter and early spring.

[0003] However, greenhouse production also highlights some problems, the most prominent of which is insufficient light. Due to reasons such as sunshine hours, weather, facility shading, and light transmittance, the light inside the greenhouse is only 70% or less of that outside. Tomatoes are typical thermophilic and light-loving plants. Such light conditions will reduce flowering and fruiting and hinder the full expansion of the fruit, resulting in reduced production. At present, the main measures to solve the problem of insufficient light in facility cultivation include the use of high-transmittance plastic films and light supplementation technology, but this also means a significant increase in costs. Starting from the plant itself, improving its ability to tolerate weak light can fundamentally solve this problem. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to increase the yield of tomatoes under weak light growth conditions and / or how to increase the lycopene content of tomato fruits under weak light growth conditions and / or how to cultivate weak light resistant tomatoes.

[0005] In order to solve the above technical problems, the present invention first provides a method for increasing the tomato fruit yield and / or the tomato fruit size and / or the longitudinal and transverse lengths of the tomato fruit and / or the lycopene content of the tomato fruit under weak light conditions, comprising reducing or inhibiting the activity of a protein in tomatoes 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 increasing the tomato fruit yield and / or the tomato fruit size and / or the longitudinal and transverse lengths of the tomato fruit and / or the lycopene content of the tomato fruit under weak light conditions.

[0006] The photon flux density under the weak light condition may be less than 0.5 times that under normal light conditions. For example, the photon flux density under the weak light condition may be 200 μmol m -2 s -1 .

[0007] The protein may be the following protein A1), A2) or A3):

[0008] A1) The amino acid sequence is the protein of sequence 1 in the sequence list;

[0009] A2) the amino acid sequence shown in sequence 1 in the sequence list is substituted and / or deleted and

[0010] / or a protein derived from A1) obtained by addition and having the same function or having more than 80% identity with the protein shown in A1) and having the same function;

[0011] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

[0012] The method described above comprises introducing into tomatoes a substance that reduces or inhibits the expression of the protein-coding gene described above or a substance that performs gene editing on the protein-coding gene described above; the substance may be any one of the following substances c1) to c4):

[0013] c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein described in A1 above;

[0014] c2) an expression cassette containing the nucleic acid molecule described in c1);

[0015] c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0016] c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

[0017] In the above method, the nucleic acid molecule described in c1) may be a DNA molecule expressing a gRNA targeting the protein-coding gene described in A1) above or a gRNA targeting the protein-coding gene described in A1) above;

[0018] The target sequence of the gRNA targeting the A1) protein encoding gene can be as shown in positions 378-397 of Sequence 2 in the sequence list and as shown in positions 722-741 of Sequence 2 in the sequence list.

[0019] In the above method, the inhibition or reduction of the expression of the gene encoding the protein mentioned above in tomatoes or gene editing of the gene encoding the protein mentioned above can be to perform the following mutation on the protein encoding gene shown in sequence 2 in the tomato genome:

[0020] A base G is inserted between nucleotides 380 and 381 of sequence 2, and a nucleotide C is inserted between nucleotides 738 and 739 of sequence 2.

[0021] In order to solve the above technical problems, the present invention also provides any of the following applications of a substance for regulating the activity or content of a protein or / and a substance for regulating the expression amount of a gene encoding the protein and / or a substance for gene editing a gene encoding the protein and / or a substance for mutating a gene encoding the protein:

[0022] P1. Application of the substance in regulating tomato fruit yield.

[0023] P2. Use of the substance in increasing tomato fruit size.

[0024] P3, application of the substance in increasing the longitudinal and transverse diameters of tomato fruits,

[0025] P4. Use of the substance in increasing the lycopene content of tomato fruit;

[0026] P5. Application of the substance in tomato breeding or quality improvement.

[0027] The protein may be the following protein A1), A2) or A3):

[0028] A1) The amino acid sequence is the protein of sequence 1 in the sequence list;

[0029] A2) a protein derived from A1) or having more than 80% identity with the protein shown in A1) and having the same function, obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 in the sequence list;

[0030] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and the C-terminus of A1) or A2). In the above application, the substance that regulates the activity or content of the protein can be a substance that knocks out the gene encoding the protein and / or a substance that regulates the expression of the gene encoding the protein and / or a substance that performs gene editing on the gene encoding the protein and / or a substance that mutates the gene encoding the protein.

[0031] In the above application, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0032] In the above application, the regulating gene expression may be to inhibit or reduce the gene expression, and the inhibiting or reducing the gene expression may be achieved by gene knockout or gene silencing.

[0033] The gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

[0034] The gene silencing refers to the phenomenon of making the gene non-expressed or low-expressed without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, making the gene non-expressed or low-expressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition.

[0035] In the above application, the substance regulating gene expression may be an agent that inhibits or reduces the expression of the gene. The agent that inhibits or reduces the expression of the gene may 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-Cas9. The agent that inhibits or reduces the expression of the gene may include a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0036] In the above methods or applications, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98% or 99% identity.

[0037] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. The identity of 90% or more can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0038] Any of the following applications of the biomaterials related to the above-mentioned proteins also fall within the protection scope of the present invention:

[0039] P1. Application of the biological material in regulating tomato fruit yield.

[0040] P2. Application of the biological material in increasing tomato fruit size.

[0041] P3, application of the biological material in increasing the longitudinal and transverse diameters of tomato fruits,

[0042] P4. Application of the biological material in increasing the lycopene content of tomato fruit;

[0043] P5. Application of the biological material in tomato breeding or quality improvement.

[0044] The biological material may be any of the following:

[0045] D1) a nucleic acid molecule encoding the protein described above;

[0046] D2) an expression cassette containing the nucleic acid molecule described in D1);

[0047] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0048] 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);

[0049] D5) a transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2);

[0050] D6) transgenic plant tissue containing the nucleic acid molecule described in D1), or transgenic plant tissue containing the expression cassette described in D2);

[0051] 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);

[0052] D8) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the protein mentioned above or the activity of the protein mentioned above or / and nucleic acid molecules that edit the gene encoding the protein mentioned above;

[0053] D9) An expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line containing the nucleic acid molecule described in D8).

[0054] In the above application, the nucleic acid molecule may be a DNA molecule as shown below:

[0055] b1) The coding sequence is the DNA molecule shown in Sequence 2 in the sequence listing;

[0056] b2) the nucleotide sequence is the DNA molecule shown in Sequence 4 in the sequence listing;

[0057] b3) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in b1) or b2) and encodes the protein;

[0058] b4) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in b1), b2) or b3) and encodes the protein.

[0059] D8) The nucleic acid molecule may be a DNA molecule expressing a gRNA targeting the protein-coding gene described in A1) above, or a gRNA targeting the protein-coding gene described in A1) above.

[0060] The target sequence of the gRNA targeting the A1) protein encoding gene can be as shown in positions 378-397 of Sequence 2 in the sequence list and as shown in positions 722-741 of Sequence 2 in the sequence list.

[0061] In the above-mentioned biological material, the expression cassette containing nucleic acid molecules described in D2) refers to a DNA capable of expressing the protein described in the above-mentioned application in a host cell, and the DNA may include not only a promoter for initiating transcription of a protein-coding gene, but also a terminator for terminating transcription of a protein-coding gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters.

[0062] Existing plant expression vectors can be used to construct a recombinant expression vector containing the protein encoding gene expression cassette.

[0063] In the above biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

[0064] The use of the method described above in preparing a product for increasing tomato fruit yield and / or tomato fruit size and / or tomato fruit longitudinal and transverse diameters and / or plant tomato lycopene content also falls within the scope of protection of the present invention.

[0065] The proteins described above also fall within the protection scope of the present invention.

[0066] The present invention uses CRISPR / Cas9 genome multi-target editing technology to target the key gene for chlorophyll degradation - STAY GREEN 1 (SGR1), which is also a key gene for lycopene synthesis, to create a new tomato germplasm with high lycopene and tolerance to weak light, and improve the yield of tomato fruits cultivated in weak light facilities. In addition, the germplasm also shows stronger post-harvest storage resistance. The present invention provides a new strategy and method for improving tomato germplasm and improving economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The growth and development of wild type (WT) and gene-edited tomato (L-1) fruits. A is the fruit phenotype of wild type and L-1 cultured in semi-shading, with a scale of 3.5 cm; B is the fruit phenotype of tomato cultured in semi-shading, with a scale of 2.5 cm; C is the average single fruit weight and single plant yield of wild type and L-1 under semi-shading cultivation conditions; D is the longitudinal and transverse diameter length of wild type and L-1 under semi-shading cultivation conditions. ** indicates extremely significant difference (P<0.01).

[0068] Figure 2 Comparison of lycopene content in mature fruits of wild-type (WT) and gene-edited tomatoes (L-1). DETAILED DESCRIPTION

[0069] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0070] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0071] The wild-type tomato AC (Ailsa Craig) in the embodiment of the present invention was provided by Zhu Hongliang's research group of the College of Food Science and Nutritional Engineering, China Agricultural University (related literature: Ma L, Yang Y, Wang Y, et al. SlRBP1 promotes translational efficiency via SleIF4A2 to maintain chloroplast function into mato [J]. The Plant Cell, 2022); pYLCRISPR / Cas9 and pYLsgRNA plasmids were provided by Liu Yaoguang's research group of the State Key Laboratory of Conservation and Utilization of Subtropical Agricultural Biological Resources, College of Life Sciences, South China Agricultural University (related literature: Zeng Dongchang, Ma Xingliang, Xie Xianrong, et al. Operation method for construction of plant CRISPR / Cas9 multi-gene editing vector and mutation analysis. Chinese Science: Life Sciences, 2018, 48: 783–794; Ma X, Zhang Q, Zhu Q, et al. A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants [J]. Molecular plant,2015,8(8):1274-1284).

[0072] The planting conditions of tomatoes in the embodiment of the present invention are as follows: tomato seeds are sown in a 10×10 cm small square pot mixed with nutrient soil, peat, and vermiculite (2:1:1, v / v / v), two plants are planted in each pot, and the seeds are covered with a film for moisture retention and cultured in a plant growth room. After germination, a small opening is opened in the film, and the film is removed after another week. When the seedlings grow to about 15 cm, they are transplanted into a solar greenhouse. The temperature of the weak light cultivation environment is 25°C / 18°C (day / night), the photoperiod is 12h / 12h (day / night), and the optical density is 100-200μmol m -2 s -1 .

[0073] All experiments in the examples of the present invention were repeated three times or more, and Excel and SPSS24 were used for data processing and difference significance analysis.

[0074] Example 1: Application of tomato SGR1 protein in regulating tomato fruit yield and lycopene under weak light conditions

[0075] 1. Construction of CRISPR-Cas9 recombinant plasmid

[0076] The amino acid sequence of the tomato SGR1 protein is shown in Sequence 1 in the sequence listing, and the CDS coding sequence of its cDNA is the nucleotide sequence shown in Sequence 2 in the sequence listing.

[0077] The CRISPR expression vector was constructed according to the method of Zeng Dongchang et al. (2018):

[0078] 1.1 Obtaining gRNA fragments

[0079] Design the target site of SGR1 gene on the website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR#, select the target sequence with high score and no off-target, and design and synthesize the primers for constructing the expression cassette.

[0080] The nucleotide sequence of target 1 of SGR1 is 5'-GGCCTCCACTAATGTGGCAA-3', and the nucleotide sequence of target 2 is 5'-CCCCAGTGAGTGTTATGCCT-3', which correspond to nucleotides 378-397 and 722-741 of sequence 2 in the sequence table of the SGR1 gene, respectively.

[0081] 1.2 Construction of CRISPR-Cas9 recombinant plasmid

[0082] After the PCR reaction, the PCR product was recovered and the two targets were constructed into the corresponding sgRNA vector (pYLsgRNA plasmid) to obtain two complete sgRNA expression cassettes. Reaction system: Q5 buffer 5μL, dNTP mix 2.5μL, pYLsgRNA vector template 1μL, upstream and downstream primers 1μL each, Q5 enzyme 0.25μL, ddH2O fill 25μL; PCR reaction conditions: 98℃3min; 98℃10s, 55℃30s, 72℃30s / kb, 34cycles; 72℃5min. The upstream and downstream primer sequences corresponding to target 1 are:

[0083] S1-F: 5'-GGCCTCCACTAATGTGGCAAgttttagagctagaaat-3';

[0084] S1-R: 5'-TTGCCACATTAGTGGAGGCCTgaccaatggtgctttg-3'.

[0085] The upstream and downstream primer sequences corresponding to target 2 are:

[0086] S2-F: 5'-CCCCAGTGAGTGTTATGCCTgttttagagctagaaat-3';

[0087] S2-R: 5'-AGGCATAACACTCACTGGGGTgaccaatggtgctttg-3'.

[0088] The universal primers required are:

[0089] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3';

[0090] gR-R: 5'-CGGAGGAAAATTCCATCCAC-3'.

[0091] The sgRNA expression cassette was connected to the pYLCRISPR / Cas9 expression vector using the Golden Gate method to obtain the ligation product. Reaction system: 10×Cutsmart buffer 1.5μL, 10×T4 DNA ligase buffer 1.5μL, pYLCRISPR / Cas9 plasmid 60ng, each sgRNA expression cassette 10ng, Bsa I 1μL, T4 DNA ligase 1μL, ddH2O 15μL; reaction conditions: 37℃5min, 10℃5min, 20℃5min, 20cycles; 37℃5min.

[0092] 1.3 Obtaining recombinant Agrobacterium

[0093] The ligation product was transformed into DH5α Escherichia coli, spread on LB plates containing kanamycin resistance, and cultured overnight at 37°C. After obtaining positive clones, they were sent to the company for sequencing. The correctly sequenced bacterial solution was shaken to extract the plasmid, and the CRISPR-Cas9 recombinant plasmid Cas9-SGR1 was obtained.

[0094] The recombinant plasmid Cas9-SGR1 was transformed into the competent Agrobacterium EHA105 to obtain the recombinant Agrobacterium, named EHA105 / Cas9-SGR1, which was used for subsequent stable genetic transformation experiments.

[0095] 2. Obtaining transgenic tomato plants

[0096] 2.1 Agrobacterium-mediated stable genetic transformation of tomato (leaf disc method)

[0097] Sterile sowing of tomato seeds:

[0098] Pick up plump AC tomato seeds, soak them in 70% ethanol for 2 minutes in a clean bench, then soak them in 5% sodium hypochlorite for 10 minutes, inverting several times to mix; use a gun to suck out the liquid and rinse with sterile water for 3-5 times, place the seeds on sterile filter paper, and absorb the moisture; place the sterilized seeds evenly on 1 / 2MS culture medium, 7-9 seeds per bottle; place the culture bottle in a dark box at 25°C for about 3 days, and when the seeds grow 2-3cm white buds, take out the culture bottle and culture it under light. When the seedlings grow to the point where the cotyledons are fully extended but the true leaves have not yet grown, they can be used for infection (the seedlings cannot be too old, otherwise the conversion rate will decrease).

[0099] Infection:

[0100] The recombinant Agrobacterium EHA105 / Cas9-SGR1 culture (OD 600nm =0.6-0.8) to collect the bacteria by centrifugation, discard the supernatant, add a small amount of MS solution to resuspend by pipetting, and continue to add MS liquid to dilute the bacterial solution to OD 600nm The concentration of acetosyringone was 0.2-0.4, and the final concentration was 100-200 μM acetosyringone. The mixture was shaken slowly at 28°C or allowed to stand, during which time the infection materials were prepared.

[0101] Cut the tomato cotyledons into two sections horizontally (remove the two ends) and place them in a conical flask with an appropriate amount of MS liquid. After the materials are prepared, slowly pour out the MS liquid and add the previously prepared bacterial solution to ensure that all leaf tissues are immersed in the bacterial solution without hanging on the wall. Shake slowly at 28℃ for 10 minutes, then let it stand for 10 minutes, or let it stand on the workbench, shaking it manually several times during the period. Pour out the bacterial solution, spread the leaves on the filter paper, and after the water film on the surface of the leaves disappears, quickly spread them on the co-culture medium, seal them with sealing film, and culture them in the dark in the tissue culture room for 2-3 days.

[0102] Screening culture:

[0103] After the co-cultivation, gently transfer the leaves to the screening medium (hygromycin 5mg / L), culture in the dark for 1 week, and then culture under the light. After the callus and adventitious buds are produced, if there is no contamination, there is no need to rush to transfer to the subculture medium. As the zeatin content in the culture medium gradually decreases, the induced adventitious buds gradually grow. If the seedlings produced by the adventitious buds are higher than 1cm, they can be directly transferred to the rooting medium. There is no need to worry about the exhaustion of nutrients or water in the culture medium during the culture process.

[0104] Subculture:

[0105] Take the larger adventitious buds from the screening medium, remove the callus, insert them into the subculture medium, and culture them under light. The callus needs to be removed in this step, otherwise the endogenous hormones produced by the callus will interfere with the overall hormone level of the plant, making it difficult for the adventitious buds to grow and producing large pieces of callus at the base of the plant. If necessary, remove the browned part of the callus and move it back to the screening medium to continue inducing adventitious buds. If the adventitious buds are small (<2mm), they are difficult to survive and can be discarded. In about 2-3 weeks, the adventitious buds will grow into plants.

[0106] Rooting culture:

[0107] Select larger plants (stems are clearly visible and taller than 1 cm) from the subculture medium, remove the callus and tiller clusters at the base, and insert them into the rooting medium. If conditions permit, cut off a quarter of the lower part of the plant to adjust the plant's hormone level and make it easier to root. For larger buds in the screening medium, they can also be directly transferred to the rooting medium, but the callus needs to be removed. During the culture process, be careful to remove the flower buds at any time.

[0108] For plants that test positive, the top of the plant and the side branches produced behind the mother plant can be cut and inserted into rooting medium for rapid propagation.

[0109] Planting in pots:

[0110] Take out the plant from the culture bottle, remove the culture medium at the root (this step is very critical, if it is not removed cleanly, the seedling will not survive) and the tiller cluster, plant it in sterilized culture soil, water it thoroughly, cover it with plastic film, seal it with a rubber band, and move it to normal light after 3 days away from light. During the planting process, do not expose the plant to the air for too long, which can easily cause wilting. After 1 week, you can peel off a corner of the plastic film, and after 2 weeks, you can try to peel it off completely, and you will get T0 generation transgenic tomato plant seedlings.

[0111] 2.2 Identification of gene-edited plants

[0112] The T0 generation tomato plants that have taken root in the rooting medium are numbered, and a leaf of appropriate size and close to the growth point is gently cut with scissors in the clean bench. The leaf is placed in a 1.5mL centrifuge tube with the corresponding number using tweezers to extract leaf DNA; upstream and downstream primers for amplifying the target fragment are designed around 200-300bp before and after the target point, and the target fragment is amplified by PCR and sequenced. The target gene editing method is confirmed by using the online website DSDecodeM (http: / / skl.scau.edu.cn / home / ) combined with the sequencing peak diagram. The detection primers for the two targets are as follows:

[0113] Upstream primer: 5′-gctcatgacgcatgtcgaaatc-3′;

[0114] Downstream primer: 5′-ggcacaacccaacttacaataattg-3′.

[0115] After testing, the present invention obtained a total of 2 gene-edited T0 generation plants, one of which had a heterozygous mutation in the SGR1 gene sequence corresponding to target 1, and a homozygous mutation in the SGR1 gene sequence corresponding to target 2; that is, a base G was inserted between the 380-381 nucleotides of sequence table sequence 2 corresponding to the SGR1 gene of one chromosome in the tomato genome, and the other chromosome did not mutate; and at the same time, the 738th nucleotide G of sequence table sequence 2 corresponding to the SGR1 genes in two chromosomes in the tomato genome was lost.

[0116] The other tree had a heterozygous mutation in the SGR1 gene sequence corresponding to target 1, and a biallelic mutation in the SGR1 gene sequence corresponding to target 2; that is, a base G was inserted between the 380-381 nucleotides of sequence list sequence 2 corresponding to the SGR1 gene of one chromosome in the tomato genome, and the other chromosome did not mutate; and at the same time, a nucleotide C was inserted between the 738-739 nucleotides of sequence list sequence 2 corresponding to the SGR1 gene of one chromosome in the tomato genome, and the nucleotide G at position 738 of sequence list sequence 2 corresponding to the SGR1 gene of the other chromosome was deleted.

[0117] One of the T0 generation gene-edited plants was self-pollinated for two generations to obtain a T2 generation gene-edited plant, and a homozygous mutant was obtained, which was named L-1. Compared with the wild-type tomato, L-1 had a base G inserted between the 380-381 nucleotides of sequence 2 in the sequence list corresponding to the SGR1 genes of the two chromosomes in the tomato genome, and at the same time, a nucleotide C was inserted between the 738-739 nucleotides of sequence 2 in the sequence list corresponding to the SGR1 genes of the two chromosomes in the tomato genome, resulting in premature termination, thereby knocking out the SGR1 gene.

[0118] In the L-1 plant, the nucleotide sequence of the mutant SGR1 gene is shown as Sequence 4 in the sequence listing, and the amino acid sequence of the mutant SGR1 protein is shown as Sequence 3 in the sequence listing.

[0119] 3. Phenotypic Observation

[0120] 3.1 Observation of fruit weight and size

[0121] The 4-week-old tomato seedlings were transplanted to the greenhouse for semi-shading (weak light) cultivation until maturity. The photoperiod was 12h / 12h (day / night).

[0122] The light quantum flux density under semi-shading condition, i.e. weak light condition, is 200 μmol m -2 s-1 The light quantum flux density under normal lighting conditions is 400 μmol m -2 s -1 .

[0123] By observing the semi-shading condition, i.e. weak light condition (photoperiod is 12h / 12h (day / night), the light quantum flux density under weak light condition is 200μmol m -2 s -1 ; Normal facility cultivation optical density 400μmol m -2 s -1 ) under wild-type tomato ( Figure 1 WT) and gene-edited tomato fruit ( Figure 1 The development of L-1) in the middle of the study found that the gene-edited tomatoes produced significantly more fruit and were larger ( Figure 1 A and B). Compared with the wild type, the single fruit weight, single plant yield, and fruit longitudinal and transverse diameters of gene-edited tomatoes were significantly increased (P<0.01) ( Figure 1 C and D).

[0124] Therefore, by gene editing the tomato SGR1 gene, the SGR1 homozygous mutant gene-edited plants obtained are more tolerant to low-light cultivation than wild-type tomatoes, and can be used to increase the size and yield of tomato fruit under facility cultivation conditions.

[0125] The determination methods of single fruit weight, single plant yield and vertical and horizontal diameters of tomato fruits are as follows:

[0126] The fruit weight was measured using a 1 / 10,000 electronic balance, and the vertical and horizontal diameters of the fruit were measured using a digital vernier caliper (0-150 mm, 0.01 mm). Five plants were selected from each WT and CR line, and 10 fruits were randomly selected from each plant to take the average value. The whole fruit was harvested to calculate the yield of a single plant, and the average value of the five plants was taken.

[0127] 3.2 Determination of lycopene in fruits

[0128] The method for determining lycopene in tomato fruit is as follows:

[0129] 1) Take a tissue block (0.1g-0.5g), rinse it in ice-cold PBS, wipe it dry with filter paper, weigh it accurately, and put it into a 5ml homogenate tube;

[0130] 2) Add an appropriate amount of homogenization medium (0.05 mol / L Tris-HCl, pH 7.4 phosphate buffer (PBS)) into the homogenization tube at a ratio of weight (g): volume (mL) = 1:9, and cut the tissue blocks into pieces as quickly as possible with ophthalmic scissors under ice water bath conditions;

[0131] 3) Hold the homogenizing tube with your left hand and insert the lower end into the container containing the ice-water mixture. With your right hand, insert the pestle vertically into the casing, rotate it up and down for dozens of times (6 to 8 minutes), grind it thoroughly to make a 10% homogenate, and then centrifuge it at 3000 rpm for 10 to 15 minutes;

[0132] 4) Take the supernatant and use the plant lycopene ELISA detection kit of Shanghai Qiyi Biotechnology Co., Ltd. to determine the lycopene content.

[0133] The results showed that the lycopene content of SGR1 gene-edited tomatoes was significantly increased compared with wild-type AC tomatoes ( Figure 2 , P<0.01).

[0134] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for increasing the size of tomato fruits under weak light conditions, comprising reducing or inhibiting the activity of SGR1 protein in tomatoes or / and reducing the expression of a gene encoding the SGR1 protein, thereby increasing the size of tomato fruits under weak light conditions; The SGR1 protein is a protein whose amino acid sequence is sequence 1 in the sequence list; The low light condition is 25°C for 12 h of light and 18°C ​​for 12 h of darkness, with an optical density of 100-200 µmol m -2 s -1 .

2. The method according to claim 1, characterized in that: The method comprises introducing any one of the following substances c1) to c4) into tomatoes: c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the SGR1 protein according to claim 1; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

3. The method according to claim 2, characterized in that: c1) the nucleic acid molecule is a DNA molecule expressing a gRNA targeting the gene encoding the SGR1 protein described in claim 1 or a gRNA targeting the gene encoding the SGR1 protein described in claim 1; The target sequence of the gRNA is shown in positions 378-397 of sequence 2 in the sequence list and in positions 722-741 of sequence 2 in the sequence list.

4. The method according to claim 1, characterized in that: The method of reducing the expression level of the gene encoding the SGR1 protein is to perform the following mutation on the gene encoding the SGR1 protein shown in sequence 2 in the tomato genome: A base G is inserted between nucleotides 380 and 381 of sequence 2, and a nucleotide C is inserted between nucleotides 738 and 739 of sequence 2.

5. Use of the biological material related to the SGR1 protein of claim 1 in increasing the size of tomato fruit under weak light conditions, characterized in that: The biological material is any of the following: D1) a nucleic acid molecule that reduces the expression level of the gene encoding the SGR1 protein of claim 1 or inhibits or reduces the activity of the SGR1 protein of claim 1; D2) an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule described in D1); The low light condition is 25°C for 12 h of light and 18°C ​​for 12 h of darkness, with an optical density of 100-200 µmol m -2 s -1 .

6. The use according to claim 5, characterized in that: The nucleic acid molecule is a DNA molecule expressing a gRNA targeting the gene encoding the SGR1 protein of claim 1 or a gRNA targeting the gene encoding the SGR1 protein of claim 1.

Images