Application and method of pectin methylesterase inhibitor protein SlPMEI3

Abstract

The present invention discloses the application and method of pectin methylesterase inhibitor protein SlPMEI3. The application is for improving the texture of tomato fruits and enhancing their storage and transportation properties. By delaying the softening of tomato fruits, the texture of tomato fruits is improved and their storage and transportation properties are enhanced. Specifically, the application is achieved by knocking out the pectin methylesterase inhibitor protein-encoding gene SlPMEI3 in tomatoes. Knocking out the pectin methylesterase inhibitor protein-encoding gene SlPMEI3 in the present invention can significantly increase the fruit firmness and thus delay the softening process. The present invention helps to improve the storage and transportation properties of tomato fruits and reduce the post-harvest loss of tomato fruits.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and genetic engineering, and particularly to the application and method of pectin methylesterase inhibitor protein SlPMEI3. Background Art

[0002] Tomato ( Solanum lycopersicum L.) is an economic crop widely planted and consumed globally. Softening is an important indicator of tomato fruit quality, directly affecting the palatability of the fruit. Excessive softening of tomato fruits will greatly shorten the storage period, seriously affecting the commercial value and the experience of consumers. Therefore, improving fruit firmness and delaying the softening process of tomatoes to extend the fruit storage period and reduce losses have become one of the core objectives of breeding and cultivation.

[0003] Fruit softening involves the decomposition of the cell wall, including pectin depolymerization and hydrolysis, and the depolymerization of hemicellulose. Among them, pectin plays an important role in maintaining the structural integrity of the cell wall and keeping cell - to - cell adhesion. During the fruit softening process, pectin methylesterase (PME) participates in catalyzing the demethylation of pectin. The resulting low - methylesterified pectin can, on the one hand, be degraded by degrading enzymes such as polygalacturonase (PG) and pectin lyase (PL); on the other hand, the low - methylesterified pectin produced by demethylation can also cross - link with cations such as Ca 2+ etc. to form an "eggshell" structure, making the pectin arranged regularly to produce pectin gel, thereby enhancing the rigidity of the cell wall. Thus, PME may have a two - way regulatory effect on cell wall strength and fruit firmness. The process of pectin demethylation is not only catalyzed by PME but also antagonized by pectin methylesterase inhibitor (PMEI). That is, PMEI inhibits the activity of PME by binding to PME to form a complex and preventing the substrate pectin from entering the active center of PME, thereby jointly regulating the pectin methylesterification process. It can be seen that PMEI plays an important role in balancing the pectin methylesterification level and maintaining the normal metabolism of pectin in the cell wall. So far, there has been no report on the research of PMEI participating in the regulation of fruit softening. SlPMEI3 The gene is highly expressed in the early stage of development and is significantly higher in the columella than in the pericarp. Regarding the mutant slpmei3 There has been no report on the attempt to improve tomato fruit firmness. Summary of the Invention

[0004] To solve the problems existing in the background art, the purpose of the present invention is to provide a pectin methylesterase inhibitor protein SlPMEI3 that participates in regulating the delayed softening of tomato fruits. The present invention uses the CRISPR / Cas9 technology to SlPMEI3Obtaining Tomato Mutants with Directed Gene Editing by Gene Knockout slpmei3 . That is, by constructing a vector containing sgRNA targeting the SlPMEI3 gene, and through genetic transformation of tomato cotyledons, knockout mutants were screened and obtained slpmei3 . The sgRNA sequence 1 is as shown in SEQ ID No.3 and SEQ ID No.4, and the sgRNA sequence 2 is as shown in SEQ ID No.5 and SEQ ID No.6. The sgRNAs were designed according to the SlPMEI3 nucleotide sequence (SEQ ID NO.1).

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] I. Application of a Pectin Methylesterase Inhibitor Protein SlPMEI3:

[0007] The application is for improving tomato fruit texture and enhancing storage and transportation properties.

[0008] The application improves tomato fruit texture and enhances storage and transportation properties by regulating the delay of tomato fruit softening.

[0009] The improvement of tomato fruit texture is to improve the texture of tomato fruits during the period from immature green fruits to red-ripe fruits.

[0010] The application improves tomato fruit texture and enhances storage and transportation properties by delaying tomato fruit softening.

[0011] The nucleotide sequence corresponding to the pectin methylesterase inhibitor protein SlPMEI3 is as shown in SEQ ID No.1, and the amino acid sequence is as shown in SEQ ID No.2.

[0012] Specifically, the above application is achieved by knocking out the gene encoding the pectin methylesterase inhibitor protein in tomatoes SlPMEI3 .

[0013] II. A method for improving tomato fruit texture and enhancing storage and transportation properties, the method steps are as follows:

[0014] First, construct a SlPMEI3 gene knockout vector, and then transfer the SlPMEI3 gene knockout vector into Agrobacterium tumefaciens. Subsequently, use the Agrobacterium tumefaciens transferred with the SlPMEI3 gene knockout vector to infect tomato cotyledons, carry out tissue culture, and screen to obtain tomato mutants; the tomato mutants are grown and propagated until the mature fruits are picked to obtain tomato fruits with improved texture and enhanced storage and transportation properties.

[0015] The transfer of the SlPMEI3After the cotyledons of tomato are infected with Agrobacterium carrying the gene knockout vector and then subjected to tissue culture, the fruit hardness of the tomato mutant increases after maturity, thereby delaying softening, improving the texture of tomato fruits and enhancing storage and transportation properties.

[0016] The nucleotide sequence corresponding to the pectin methylesterase inhibitor protein SlPMEI3 is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No.2.

[0017] Specifically, the CRISPR / Cas9 gene editing method is used to construct SlPMEI3 the gene knockout vector, which is transferred into SlPMEI3 Agrobacterium carrying the gene knockout vector to infect the cotyledons of tomato. Through the CRISPR / Cas9 gene editing method, SlPMEI3 the gene is knocked out. Then the tomato cotyledons are placed on the culture medium for tissue culture, and then transferred to the soil for cultivation. Finally, through the gene editing method, SlPMEI3 the gene in tomato is knocked out.

[0018] The SlPMEI3 gene is specifically the DNA corresponding to the pectin methylesterase inhibitor protein SlPMEI3.

[0019] The SlPMEI3 gene knockout vector is obtained by connecting to the CDC45-1300 vector through sgRNA sequence 1 and sgRNA sequence 2. The sgRNA sequence 1 is shown as SEQ ID No.3 and SEQ ID No.4, and the sgRNA sequence 2 is shown as SEQ ID No.5 and SEQ ID No.6.

[0020] The beneficial effects of the present invention are as follows:

[0021] In the present invention, the gene encoding the pectin methylesterase inhibitor protein SlPMEI3 (Solyc07g042390) is knocked out in tomato to obtain a mutant slpmei3 . The phenotypic analysis of the mutant shows that the fruit hardness at the immature green stage (IMG), mature green stage (MG), breaker stage (BR), and breaker + 7 stage (B7) is significantly increased, indicating that SlPMEI3 after gene knockout, the fruit hardness of tomato can be improved. The present invention helps to enhance the storage and transportation properties of tomato fruits and reduce the post-harvest loss of tomato fruits, that is, the fruit hardness of the tomato mutant slpmei3 is significantly increased, and the fruit softening process is delayed, which can be applied to improving the texture of tomato fruits and enhancing storage and transportation properties. The present invention provides a candidate gene for improving the texture and storage and transportation properties of tomato fruits, providing a theoretical support for tomato variety improvement and high-quality breeding. Brief Description of the Drawings

[0022] Figure 1 is SlPMEI3 The expression pattern diagram of genes at different development stages of tomato fruits.

[0023] Figure 2 is a mutant slpmei3 Schematic diagram of the target editing situation on the nucleotide sequence.

[0024] Figure 3 is a mutant slpmei3 The result diagram of the hardness analysis of tomato fruits.

[0025] Figure 4 is a mutant slpmei3 Schematic diagram of the situation during the development process of tomato fruits. Specific implementation manners

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0027] The experimental methods used in the following embodiments, unless otherwise specified, are all in accordance with conventional experimental methods.

[0028] The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0029] Embodiments of the present invention are as follows:

[0030] 1) Plant materials and gene expression patterns:

[0031] The plant materials in this scheme are tomato AC (Ailsa Craig), which are planted in the plant factory of Zhejiang University under the conditions of 16 h light (26 - 29 °C) and 8 h darkness (17 - 22 °C). Transcriptome data of tomato fruit development stages are downloaded from the SGN-TEA database. As Figure 1 shown, SlPMEI3 The gene (Solyc07g042390) has a relatively high expression in the early stage of fruit development, and its expression in the locule is significantly higher than that in the pericarp.

[0032] 2) Construction of recombinant vectors:

[0033] Based on SlPMEI3 the genomic sequence, 20 nt specific single guide RNA (sgRNA) is selected in the exon region using an online website:

[0034] sgRNA sequence 1: 5’-GATTGGCAGGATTTAGCTCAAGCCG-3’ (SEQ ID NO.3)

[0035] 3’-CCGTCCTAAATCGAGTTCGGCCAAA-5’ (SEQ ID NO.4)

[0036] sgRNA sequence 2: 5’-GGTCGGGCTTGAGCTAAATCCTGCG-3’ (SEQ ID NO.5)

[0037] 3’-CCCGAACTCGATTTAGGACGCCAAA-5’ (SEQ ID NO.6)

[0038] First, the sgRNA sequence was fused with its reverse complementary sequence, and the fusion fragment was ligated to the AtU6-18T and AtU3-18T vectors. The plasmids with correct sequencing results were extracted SlPMEI3 -AtU6-18T and SlPMEI3 -AtU3-18T plasmids were used as templates for amplification, and the SlPMEI3 -AtU6-18T and SlPMEI3 -AtU3-18T products were ligated. After purification of the ligation product, it was further ligated to the CDC45-1300 vector, spread on LB solid medium containing kanamycin and gentamicin, positive clones were screened, and positive clones were picked for sequencing verification. The recombinant plasmid with correct sequencing was transformed into Agrobacterium tumefaciens GV3101 ( Agrobacterium tumefaciens ).

[0039] 3) Genetic transformation by cotyledon infection method in tomato:

[0040] Through the cotyledon infection method, tissue culture was carried out to screen and obtain tomato mutants slpmei3 , and the specific steps are as follows:

[0041] Tomato seeds were soaked in tap water for 5 - 6 h, then disinfected with 10% sodium hypochlorite solution and washed. They were inoculated into 1 / 2 MS medium (containing 2.22 g / L MS, 1% Sucrose, 0.8% Agar, 100 mg / L Inositol) and cultured under dark conditions at 25℃ for about 3 d. After germination, they were transferred to a light culture room (25℃, 16 h light / 8 h dark) for about 3 d until the cotyledons were fully expanded. The cotyledons were excised and cut into 2 segments, which were inverted and pre-cultured on KCMS medium (containing 4.44 g / L MS, 3% Sucrose, 0.8% Agar, 100 mg / L Inositol and 100 μM Acetosyringon) with filter paper attached for 1 d (overnight, in the dark). The cotyledons were transferred to an empty petri dish, and the Agrobacterium suspension containing the recombinant plasmid (OD600 = 1.0) was poured in. The petri dish was gently shaken for 2 - 3 min, and then the suspension was poured out and blotted dry. After co-culturing the infected cotyledons on KCMS for 2 d (in the dark), they were carefully transferred to 2Z medium (containing 4.44 g / L MS, 3% Sucrose, 0.8% Agar, 2 mg / L Zeatin, 320 mg / L Timentin and 3 mg / L Hygromycin). After culturing for 2 - 3 w, they were transferred to 0.2Z medium (containing 4.44 g / L MS, 3% Sucrose, 0.8% Agar, 0.2 mg / L Zeatin, 320 mg / L Timentin and 3 mg / L Hygromycin), and then transferred to fresh 0.2Z medium every 2 - 3 w. When the regenerated plants grew to 1 cm, they were transferred to rooting medium (containing 4.44 g / L MS, 3% Sucrose, 0.8% Agar, 320 mg / L Timentin and 3 mg / L Hygromycin). The rooted seedlings were transplanted into soil for subsequent positive identification.

[0042] 4) Identification of mutant materials and obtaining of homozygous mutants:

[0043] Design SlPMEI3 Transgenic detection primers and Cas9 detection primers.

[0044] SlPMEI3 Transgenic detection primers:

[0045] CRISPR- SlPMEI3 -FP: TCAGTGACTACATCATCATCTTCTT (SEQ ID NO.7)

[0046] CRISPR- SlPMEI3-RP: GAAAGATTACGAATGTTGCTAGAG (SEQ ID NO.8)

[0047] Cas9 detection primers:

[0048] Cas9-FP: CAAGGGCTACAAAGAAGTG (SEQ ID NO.9)

[0049] Cas9-RP: AGTTCACATATTTGGAGGG (SEQ ID NO.10)

[0050] Extract the genomic DNA (gDNA) of transgenic plants, quantify it by Nanodrop and dilute it to 20 ng / μL. Use the above Cas9 primers for RT-qPCR quantification to analyze and screen Cas9 positive plants. Extract the gDNA of wild-type tomato and Cas9 positive tomato leaves as templates, and use the above SlPMEI3 transgenic detection primers for PCR amplification. Recover and sequence the PCR products. Compare the sequencing results with SlPMEI3 the target sequence to screen transgenic plants with effective editing of the target sequence. For screening homozygous mutants, use the gDNA of T1 generation plants as a template, and the remaining steps are the same as above. At the same time, use wild-type fruits (i.e., fruits of tomatoes without the knocked-out SlPMEI3 gene) as a negative control, and the remaining steps are the same as above to analyze and screen homozygous mutant plants that are Cas9 negative.

[0051] After screening, two homozygous and Cas9 negative knockout mutants slpmei3 were obtained in the present invention slpmei3#1 , slpmei3#2 . The nucleotide sequence target mutation situation of mutant slpmei3 is as shown in Figure 2 , that is, the target editing situation is shown in Figure 2 .

[0052] The truncated SlPMEI3 amino acid sequence with loss of function in mutant slpmei3 is as shown in SEQ ID No.11 and SEQID No.12.

[0053] 5) CRISPR / Cas9 knockout mutant slpmei3 Tomato fruit firmness measurement:

[0054] Note: Immature green fruit stage (IMG); Mature green stage (MG); Breaker stage (BR); Red ripe stage (Breaker + 7, B7).

[0055] When the fruit diameter reached 1 cm (about 10 days after flowering), the fruits were tagged. The fruits were immature green fruits 13 - 15 days after the 1 cm fruits, green - ripe fruits 25 - 27 days after the 1 cm fruits, turning - color fruits when the top of the fruit turned red, and red - ripe fruits 7 days after turning - color. Figure 4 Among them, the wild - type was used as the control (i.e., the tomato fruits without SlPMEI3 gene knockout), slpmei3#1 and slpmei3#2 represented SlPMEI3 two knockout lines of the

[0056] gene. The fruit firmness was measured using a TA - XT plus texture analyzer (Stable Micro Systems, UK). A whole - fruit compression test (P100 plate with a diameter of 100 mm) was performed on each fruit. Each group had 8 single - fruit replicates, and the firmness unit was expressed in Newtons (N). Figure 3 and Figure 4 As shown in SlPMEI3 gene - knockout), compared with the wild - type fruits (i.e., the tomato fruits without SlPMEI3 gene knockout), knocking out the SlPMEI3 gene significantly increased the firmness of tomato fruits at the IMG, MG, BR, and B7 stages, but did not affect the fruit development process. Therefore, knocking out the

[0057] gene can be used as a candidate gene for improving the texture of tomatoes and enhancing their storability and transportability. SlPMEI3 As can be seen from the above examples, the present invention provides a pectin methylesterase inhibitor protein SlPMEI3 involved in regulating tomato fruit softening and its application. After knocking out the

[0058] gene by the CRISPR / Cas9 technology, the firmness of tomato fruits at the IMG, MG, BR, and B7 stages was significantly increased.

[0059] SEQ ID NO.1:

[0060] Name: SlPMEI3 Nucleotide sequence

[0061] DNA type: genomic DNA

[0062] Source: Solanum lycopersicum

[0063] ATGAAAACTCTACTTGTTTCTCTGTTTCTAATTTCATCAGTGACTACATCATCATCTTCTTCTGCTAGTGACATTGTCCGCTCTTCCTGCGTGCACGCCAGCTATCCCACAATTTGCGTCAGAACACTCTCTTCATACTCGGACACACCCATTAACACCCCGCAGGATTTAGCTCAAGCCGCGGTGAAAATTACCCTTTCTCGAGCAGGTAAAGCGTCTGGGTTCCTGTCTCGGGTGAAAGTGGAAAGCAAAAGAGAGAAAGGGGCATTGAGTGATTGTATCGAACAGATGGGTGACTCGATGGAGGAGCTGAGGAAGAGTTTGTCAGAGCTTAAACATGTACGCAGGGGAAATGCATTTAAGTGGCAAATGAGCAATTTGGAGACGTGGGTAAGTGCTGCTTTGACAAATGAAGATACATGTCTTGATGGATTCAAGGAAATTGATGGCAAAATCAGGTCTGATGTGAAACGAAAGATTACGAATGTTGCTAGAGTTACTAGTAATGCACTTTACCTTATCAATCGACTGGATGATTCTGCAAACAAAATTACTCATCCTTGA

[0064] SEQ ID NO.2:

[0065] Name: SlPMEI3 Amino acid sequence

[0066] Amino acid type: AA

[0067] Source: Solanum lycopersicum

[0068] MKTLLVSLFLISSVTTSSSSSASDIVRSSCVHASYPTICVRTLSSYSDTPINTPQDLAQAAVKITLSRAGKASGFLSRVKVESKREKGALSDCIEQMGDSMEELRKSLSELKHVRRGNAFKWQMSNLETWVSAALTNEDTCLDGFKEIDGKIRSDVKRKITNVARVTSNALYLINRLDDSANKITHP*

[0069] SEQ ID NO.3:

[0070] Name: sgRNA Sequence 1 (5'-3')

[0071] RNA Type: other RNA

[0072] Source: Synthetic Construct

[0073] GATTGGCAGGATTTAGCTCAAGCCG

[0074] SEQ ID NO.4:

[0075] Name: sgRNA Sequence 1 (3'-5')

[0076] RNA Type: other RNA

[0077] Source: Synthetic Construct

[0078] CCGTCCTAAATCGAGTTCGGCCAAA

[0079] SEQ ID NO.5:

[0080] Name: sgRNA Sequence 2 (5'-3')

[0081] RNA Type: other RNA

[0082] Source: Synthetic Construct

[0083] GGTCGGGCTTGAGCTAAATCCTGCG

[0084] SEQ ID NO.6:

[0085] Name: sgRNA Sequence 2 (3'-5')

[0086] RNA Type: other RNA

[0087] Source: Synthetic Construct

[0088] CCCGAACTCGATTTAGGACGCCAAA

[0089] SEQ ID NO.7:

[0090] Name: CRISPR-SlPMEI3-FP

[0091] DNA Type: other DNA

[0092] Source: Synthetic Construct

[0093] TCAGTGACTACATCATCATCTTCTT

[0094] SEQ ID NO.8:

[0095] Name: CRISPR-SlPMEI3-RP

[0096] DNA type: other DNA

[0097] Source: Synthetic Construct

[0098] GAAAGATTACGAATGTTGCTAGAG

[0099] SEQ ID NO.9:

[0100] Name: Cas9-FP

[0101] DNA type: other DNA

[0102] Source: Synthetic Construct

[0103] CAAGGGCTACAAAGAAGTG

[0104] SEQ ID NO.10:

[0105] Name: Cas9-RP

[0106] DNA type: other DNA

[0107] Source: Synthetic Construct

[0108] AGTTCACATATTTGGAGGG

[0109] SEQ ID NO.11:

[0110] Name: slpmei3#1 Truncated Amino Acid Sequence

[0111] Amino Acid type: AA

[0112] Source: Solanum lycopersicum

[0113] MKTLLVSLFLISSVTTSSSSSASDIVRSSCVHASYPTICVRTLSSYSDTPINTPQPR*

[0114] SEQ ID NO.12:

[0115] Name: slpmei3#2 Truncated Amino Acid Sequence

[0116] Amino Acid Type: AA

[0117] Source: Solanum lycopersicum

[0118] MKTLLVSLFLISSVTTSSSSSASDIVRSSCVHASYPTICVRTLSSYSDTPINTRGENYPFSSR*. * represents the stop codon.

Claims

1. A method for improving the texture of tomato fruit and enhancing storage and transportation properties, characterized in that: The method comprises the following steps: First, a SlPMEI3 gene knockout vector is constructed, and then the SlPMEI3 gene knockout vector is transferred into Agrobacterium, and then tomato cotyledons are infected with the Agrobacterium transferred with the SlPMEI3 gene knockout vector, tissue culture is performed, and tomato mutants are screened and obtained; the tomato mutants are grown and propagated until they mature to obtain tomato fruits with improved tomato fruit texture and improved storage and transportation properties; The improvement of tomato fruit texture and storage and transportation performance is achieved by delaying the softening of tomato fruit; The amino acid sequence of the SlPMEI3 gene is shown in SEQ ID No.

2.

2. The method for improving the texture of tomato fruit and enhancing the storage and transportation properties according to claim 1, characterized in that: The method specifically adopts a gene editing method to construct a SlPMEI3 gene knockout vector, infects tomato cotyledons with Agrobacterium transferred with the SlPMEI3 gene knockout vector, knocks out the SlPMEI3 gene through gene editing, and then places the tomato cotyledons on a culture medium for tissue culture, and then transfers them to soil for culture.

3. The method for improving the texture of tomato fruit and enhancing the storage and transportation properties according to claim 2, characterized in that: The SlPMEI3 gene knockout vector is obtained by connecting sgRNA sequence 1 and sgRNA sequence 2 to the CDC45-1300 vector, wherein the sgRNA sequence 1 is shown in SEQ ID No.3 and SEQ ID No.4, and the sgRNA sequence 2 is shown in SEQ ID No.5 and SEQ ID No.6.

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