Application and method of pectin methylesterase inhibitory protein SlPMEI3

Abstract

The invention discloses an application and a method of pectin methylesterase inhibition protein SlPMEI3. The application refers to the application in improving the tomato fruit texture and improving the storage and transportation performance, the tomato fruit texture is improved and the storage and transportation performance is improved by delaying tomato fruit softening, and particularly, the application is realized by knocking out the pectin methylesterase inhibitory protein coding gene SlPMEI3 in tomatoes. The pectin methylesterase inhibitory protein coding gene SlPMEI3 is knocked out, so that the fruit hardness can be remarkably improved, and the softening process is delayed. The method is beneficial for improving the storage and transportation performance of the tomato fruits and reducing the postharvest loss of the tomato fruits.

Description

Technical Field

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

[0002] tomato( Solanum lycopersicum L.) is an economic crop widely grown and consumed worldwide. Softening is an important indicator of tomato fruit quality, which directly affects the palatability of the fruit. Excessive softening of tomato fruit will greatly shorten the storage period, seriously affecting the commodity value and consumer experience. Therefore, improving fruit hardness and delaying the softening process of tomatoes to extend the fruit storage period and reduce losses have become one of the core goals of breeding and cultivation.

[0003] Fruit softening involves the decomposition of cell walls, including pectin depolymerization and hydrolysis, hemicellulose depolymerization, etc. Among them, pectin plays an important role in maintaining the integrity of the cell wall structure and maintaining intercellular adhesion. During the fruit softening process, pectin methylesterase PME (Pectin Methylesterase) participates in catalyzing the demethylation of pectin. The low-methylated pectin produced can be degraded by degrading enzymes such as polygalacturonase PG (Polygalacturonase) and pectin lyase PL (Pectin lyase); on the other hand, the low-methylated pectin produced by demethylation can also react with Ca 2+ The cations are cross-linked to form an "eggshell" structure, which makes the pectin arranged regularly and produces pectin gel, thereby enhancing the rigidity of the cell wall. It can be seen that PME may have a bidirectional regulatory effect on the strength of the cell wall and the hardness of the fruit. The pectin demethylesterification process is not only catalyzed by PME, but also antagonized by the pectin methylesterase inhibitor protein PMEI (Pectin Methylesterase Inhibitor), that is, PMEI prevents the substrate pectin from entering the PME active center by binding to PME to form a complex to inhibit PME activity and jointly regulate the pectin methylesterification process. It can be seen that PMEI plays an important role in balancing the level of pectin methylesterification and maintaining the normal metabolism of pectin in the cell wall. So far, there has been no report on the involvement of PMEI in regulating fruit softening. SlPMEI3 The gene is highly expressed in the early developmental stage, and its expression in the jelly is significantly higher than that in the peel. slpmei3 No attempts to increase tomato fruit firmness have been reported. Summary of the invention

[0004] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide a pectin methylesterase inhibitory protein SlPMEI3 that is involved in regulating the delayed softening of tomato fruit. SlPMEI3Gene knockout to obtain targeted gene-edited tomato mutants slpmei3 That is, by constructing a target SlPMEI3 The vector of sgRNA of the gene was used to genetically transform tomato cotyledons to screen for knockout mutants slpmei3 The sgRNA sequence 1 is shown in SEQ ID No.3 and SEQ ID No.4, the sgRNA sequence 2 is shown in SEQ ID No.5 and SEQ ID No.6, and the sgRNAs are based on SlPMEI3 Nucleotide sequence (SEQ ID NO.1) design.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: 1. Application of a pectin methylesterase inhibitory protein SlPMEI3: The application is to improve the texture of tomato fruits and enhance the storage and transportation properties.

[0006] The application is to improve the texture of tomato fruit and enhance the storage and transportation properties by regulating and delaying the softening of tomato fruit.

[0007] The improving tomato fruit texture is to improve the texture of tomato fruit from unripe green fruit to red ripe fruit.

[0008] The application is to improve the texture of tomato fruit and enhance the storage and transportation properties by delaying the softening of tomato fruit.

[0009] The nucleotide sequence corresponding to the pectin methylesterase inhibitory protein SlPMEI3 is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0010] Specifically, by knocking out the gene encoding pectin methylesterase inhibitory protein in tomato SlPMEI3 to implement the application.

[0011] 2. A method for improving tomato fruit texture and storage and transportation performance, the method steps are: First build SlPMEI3 Gene knockout vector SlPMEI3 The knockout vector was transformed into Agrobacterium, and then SlPMEI3 Agrobacterium carrying a gene knockout vector infects tomato cotyledons, tissue culture is performed, and tomato mutants are screened; the tomato mutants are grown and reproduced until they are ripe, and tomato fruits with improved texture and storage and transportation properties are obtained.

[0012] The transfer SlPMEI3 After Agrobacterium carrying the gene knockout vector infects tomato cotyledons for tissue culture, the hardness of the mature tomato mutant fruits increases, thereby delaying softening, thereby improving the texture of the tomato fruit and improving its storage and transportation properties.

[0013] The nucleotide sequence corresponding to the pectin methylesterase inhibitory protein SlPMEI3 is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0014] The method is specifically constructed using the CRISPR / Cas9 gene editing method SlPMEI3 Gene knockout vector, transferred into SlPMEI3 Agrobacterium carrying the gene knockout vector infected tomato cotyledons and used CRISPR / Cas9 gene editing to SlPMEI3 The gene was knocked out, and then the tomato cotyledons were placed on a culture medium for tissue culture, and then transferred to soil for culture. Finally, the gene editing method was used to knock out the gene in the tomato. SlPMEI3 Gene knockout.

[0015] Said SlPMEI3 The gene is specifically the DNA corresponding to the pectin methylesterase inhibitory protein SlPMEI3.

[0016] Said SlPMEI3 The 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.

[0017] The present invention has the following beneficial effects: The present invention knocks out the gene encoding pectin methylesterase inhibitory protein in tomato SlPMEI3 (Solyc07g042390) obtained mutant slpmei3 The mutant phenotypic analysis showed that the fruit firmness of immature green fruit (IMG), mature green fruit (MG), breaker (BR), and red ripe fruit (Breaker + 7, B7) increased significantly, indicating that SlPMEI3 The gene knockout can improve the firmness of tomato fruit. The invention helps to improve the storage and transportation of tomato fruit and reduce the loss of tomato fruit after harvest. slpmei3 The fruit hardness is significantly improved, the fruit softening process is delayed, and it can be used to improve the texture of tomato fruits and improve the storage and transportation properties. The present invention provides a candidate gene for improving the texture and storage and transportation properties of tomato fruits, providing theoretical support for tomato variety improvement and high-quality breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 yes SlPMEI3 Diagram of gene expression patterns at different developmental stages of tomato fruit.

[0019] Figure 2 It's a mutant slpmei3 Schematic diagram of target editing on nucleotide sequences.

[0020] Figure 3 It's a mutant slpmei3 Figure 1. Results of tomato fruit firmness analysis.

[0021] Figure 4 It's a mutant slpmei3 Schematic diagram of the tomato fruit development process. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0023] The experimental methods used in the following examples, unless otherwise specified, are based on conventional experimental methods.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0025] Embodiments of the present invention are as follows: 1) Plant materials and gene expression patterns: The plant material for this protocol is tomato AC (Ailsa Craig), which was grown in the Zhejiang University Plant Factory under the conditions of 16 h light (26-29°C) and 8 h dark (17-22°C). The transcriptome data of tomato fruit development stages were downloaded from the SGN-TEA database. Figure 1 As shown, SlPMEI3 The (Solyc07g042390) gene was highly expressed in the early stage of fruit development, and its expression in the cavity was significantly higher than that in the peel.

[0026] 2) Construction of recombinant vector: based on SlPMEI3 The genome sequence of the gene was used to select a 20 nt single guide RNA (sgRNA) with specificity in the exon region using an online website: sgRNA sequence 1: 5'-GATTGGCAGGATTTAGCTCAAGCCG-3' (SEQ ID NO. 3) 3'-CCGTCCTAAATCGAGTTCGGCCAAA-5' (SEQ ID NO.4) sgRNA sequence 2: 5'-GGTCGGGCTTGAGCTAAATCCTGCG-3' (SEQ ID NO. 5) 3'-CCCGAACTCGATTTAGGACGCCAAA-5' (SEQ ID NO.6) First, the sgRNA sequence was fused with its reverse complementary sequence, and the fusion fragment was connected to the AtU6-18T and AtU3-18T vectors. SlPMEI3 -AtU6-18T and SlPMEI3 -AtU3-18T plasmid was used as template for amplification and SlPMEI3 -AtU6-18T and SlPMEI3 -AtU3-18T product was connected, and the connected product was purified and further connected to CDC45-1300 vector, coated on LB solid medium containing kanamycin and gentamicin, positive clones were screened, and positive clones were selected for sequencing verification. The recombinant plasmid with correct sequencing was transformed into GV3101 Agrobacterium ( Agrobacterium tumefaciens ).

[0027] 3) Genetic transformation by tomato cotyledon infection method: Tissue culture was used to screen tomato mutants through cotyledon infection. slpmei3 , the specific steps are as follows: Tomato seeds were soaked in tap water for 5-6 h, disinfected and washed with 10% sodium hypochlorite solution, inoculated in 1 / 2 MS medium (containing 2.22 g / L MS, 1% Sucrose, 0.8% Agar, 100 mg / L Inositol), and cultured at 25°C in the dark for about 3 days. After germination, they were transferred to a light culture room (25°C, 16 h light / 8 h dark) for about 3 days until the cotyledons were fully expanded. The cotyledons were cut and divided into 2 sections, inverted and pre-cultured in KCMS medium (containing 4.44 g / L MS, 3% Sucrose, 0.8% Agar, 100 mg / L Inositol and 100 μM Acetosyringon) with filter paper for 1 day (overnight, protected from light). Transfer the cotyledons to an empty culture dish, pour in the Agrobacterium solution containing the recombinant plasmid (OD600=1.0), gently shake the culture dish for 2-3 min, pour out and dry the solution. Place the infected cotyledons on KCMS for co-culture for 2 days (protected from light), and then carefully transfer 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 weeks, transfer 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 transfer to new 0.2Z medium every 2-3 weeks. When the regenerated plants grow to 1 cm, they are 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 are transferred to soil for subsequent positive identification.

[0028] 4) Identification of mutant materials and acquisition of homozygous mutants: design SlPMEI3 Transgenic detection primers and Cas9 detection primers.

[0029] SlPMEI3 GMO detection primers: CRISPR- SlPMEI3 -FP: TCAGTGACTACATCATCATCTTCTT (SEQ ID NO.7) CRISPR- SlPMEI3 -RP: GAAAGATTACGAATGTTGCTAGAG (SEQ ID NO.8) Cas9 detection primers: Cas9-FP: CAAGGGCTACAAAGAAGTG (SEQ ID NO.9) Cas9-RP: AGTTCACATATTTGGAGGG (SEQ ID NO.10) The genomic DNA (gDNA) of the transgenic plants was extracted, quantified by Nanodrop and diluted to 20 ng / μL. The above-mentioned Cas9 primers were used for RT-qPCR quantification to analyze and screen Cas9-positive plants. The gDNA of wild-type tomato and Cas9-positive tomato leaves was extracted as templates and the above-mentioned Cas9 primers were used to quantify the Cas9-positive plants. SlPMEI3 PCR amplification was performed with transgenic detection primers, and the PCR products were recovered and sequenced. SlPMEI3 Target sequence alignment, screening of transgenic plants with effectively edited target sequences. Homozygous mutant screening uses T1 generation plant gDNA as a template, and the rest of the steps are the same as above. SlPMEI3 Gene tomato fruit) was used as a negative control, and the remaining steps were the same as above to analyze and screen Cas9-negative homozygous mutant plants.

[0030] After screening, the present invention obtained two homozygous and Cas9-negative knockout mutants slpmei3 , respectively named slpmei3#1 , slpmei3#2 . Mutants slpmei3 The nucleotide sequence target mutations are as follows Figure 2 As shown, the target editing situation is shown in Figure 2 .

[0031] Mutants slpmei3 The amino acid sequences of the truncated SlPMEI3 with loss of function are shown in SEQ ID No.11 and SEQ ID No.12.

[0032] 5) CRISPR / Cas9 knockout mutants slpmei3 Determination of firmness of tomato fruit: Note: Immature green fruit stage (Immaturegreen, IMG); Mature green stage (Maturegreen, MG); Color change stage (Breaker, BR); Red ripe stage (Breaker + 7, B7).

[0033] When the fruit diameter is 1 cm (about 10 days after flowering), it is marked. The fruit that is 1 cm in diameter is an immature green fruit 13-15 days after fruiting, the fruit that is 1 cm in diameter is a ripe green fruit 25-27 days after fruiting, the fruit that turns red at the top is a color-changing fruit, and the fruit that is red and ripe 7 days after color changing is a ripe red fruit. Figure 4 The wild type was used as the control (i.e., not knocked out SlPMEI3Genetically modified tomato fruit) slpmei3#1 and slpmei3#2 represent SlPMEI3 Two knockout lines of the gene. Fruit firmness was determined using a TA-XT plus texture analyzer (Stable Micro Systems, UK), and each fruit was subjected to a whole-fruit squeeze test (P100 plate with a diameter of 100 mm). Each group had 8 single-fruit replicates, and the unit of hardness was expressed in Newton (N).

[0034] The results of phenotypic analysis showed that Figure 3 and Figure 4 As shown, compared with wild-type fruit (i.e., non-knockout SlPMEI3 gene tomato fruit) compared to knockout SlPMEI3 The gene significantly increased the firmness of tomato fruit at the IMG, MG, BR, and B7 stages, but did not affect the fruit development process. SlPMEI3 The gene can be used as a candidate gene for improving tomato texture and enhancing storage and transportation properties.

[0035] It can be seen from the above embodiments that the present invention provides a pectin methylesterase inhibitory protein SlPMEI3 involved in regulating tomato fruit softening and its application, which is transformed into SlPMEI3 After gene knockout, the hardness of tomato fruit at IMG, MG, BR, and B7 stages was significantly improved.

[0036] The genetic sequences involved in the present invention are as follows: SEQ ID NO.1: name: SlPMEI3 Nucleotide sequence DNA type: genomic DNA source: Solanum lycopersicum ATGAAAACTCTACTTGTTTCTCTGTTTCTAATTTCATCAGTGACTACATCATCATCTTCTTCTGCTAGTGACATTGTCCGCTCTTCCTGCGTGCACGCCAGCTATCCCACAATTTGCGTCAGAACACTCTCTTCATACTCGGACACACCCATTAACACCCCGCAGGATTTAGCTCAAGCCGCGGTGAAAATTACCCTTTCTCGAGCAGGTAAAGCGTCTGGGTTCCTGTCTCGGGTGAAAGTGGAAAGCAAAAGAGAGAAAGGGGCATTGAGTGATTGTATCGAACAGATGGGTGACTCGATGGAGGAGCTGAGGAAGAGTTTGTCAGAGCTTAAACATGTACGCAGGGGAAATGCATTTAAGTGGCAAATGAGCAATTTGGAGACGTGGGTAAGTGCTGCTTTGACAAATGAAGATACATGTCTTGATGGATTCAAGGAAATTGATGGCAAAATCAGGTCTGATGTGAAACGAAAGATTACGAATGTTGCTAGAGTTACTAGTAATGCACTTTACCTTATCAATCGACTGGATGATTCTGCAAACAAAATTACTCATCCTTGA SEQ ID NO.2: Name: SlPMEI3 Amino acid sequence Amino acid type: AA Source: Solanum lycopersicum MKTLLVSLFLISSVTTSSSSSASDIVRSSCVHASYPTICVRTLSSYSDTPINTPQDLAQAAVKITLSRAGKASGFLSRVKVESKREKGALSDCIEQMGDSMEELRKSLSELKHVRRGNAFKWQMSNLETWVSAALTNEDTCLDGFKEIDGKIRSDVKRKITNVARVTSNALYLINRLDDSANKITHP* SEQ ID NO.3: Name: sgRNA sequence 1 (5'-3') RNA type: other RNA Source: Synthetic Construct GATTGGCAGGATTTAGCTCAAGCCG SEQ ID NO.4: Name: sgRNA sequence 1 (3'-5') RNA type: other RNA Source: Synthetic Construct CCGTCCTAAATCGAGTTCGGCCAAA SEQ ID NO.5: Name: sgRNA sequence 2 (5'-3') RNA type: other RNA Source: Synthetic Construct GGTCGGGCTTGAGCTAAATCCTGCG SEQ ID NO.6: Name: sgRNA sequence 2 (3'-5') RNA type: other RNA Source: Synthetic Construct CCCGAACTCGATTTAGGACGCCAAA SEQ ID NO.7: Name: CRISPR-SlPMEI3-FP DNA type: other DNA Source: Synthetic Construct TCAGTGACTACATCATCATCTTCTT SEQ ID NO.8: Name: CRISPR-SlPMEI3-RP DNA type: other DNA Source: Synthetic Construct GAAAGATTACGAATGTTGCTAGAG SEQ ID NO.9: Name: Cas9-FP DNA type: other DNA Source: Synthetic Construct CAAGGGCTACAAAGAAGTG SEQ ID NO.10: Name: Cas9-RP DNA type: other DNA Source: Synthetic Construct AGTTCACATATTTGGAGGG SEQ ID NO.11: name: slpmei3#1 Truncated amino acid sequence Amino acid type: AA source: Solanum lycopersicum MKTLLVSLFLISSVTTSSSSSASDIVRSSCVHASYPTICVRTLSSYSDTPINTPQPR* SEQ ID NO.12: name: slpmei3#2 Truncated amino acid sequence Amino Acid Type: AA source: Solanum lycopersicum MKTLLVSLFLISSVTTSSSSSASDIVRSSCVHASYPTICVRTLSSYSDTPINTRGENYPFSSR*. * indicates a stop codon.

Claims

1. Application of pectin methylesterase inhibitory protein SlPMEI3, characterized in that: The application is to improve the texture of tomato fruits and enhance the storage and transportation properties.

2. The use of the pectin methylesterase inhibitory protein S1PMEI3 according to claim 1, characterized in that: The application is to improve the texture of tomato fruit and enhance the storage and transportation properties by regulating the softening of tomato fruit.

3. The use of the pectin methylesterase inhibitory protein S1PMEI3 according to claim 2, characterized in that: The application is to improve the texture of tomato fruit and enhance the storage and transportation properties by delaying the softening of tomato fruit.

4. The use of the pectin methylesterase inhibitory protein S1PMEI3 according to claim 2, characterized in that: The amino acid sequence of the pectin methylesterase inhibitory protein SlPMEI3 is shown in SEQ ID No.

2.

5. The use of the pectin methylesterase inhibitory protein S1PMEI3 according to claim 2, characterized in that: The application is achieved by knocking out the pectin methylesterase inhibitory protein encoding gene SlPMEI3 in tomatoes.

6. 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 was constructed, and then the SlPMEI3 gene knockout vector was transferred into Agrobacterium. Subsequently, tomato cotyledons were infected with Agrobacterium transferred with the SlPMEI3 gene knockout vector, and tissue culture was performed to screen to obtain tomato mutants. The tomato mutants were grown and reproduced until maturity, and tomato fruits with improved tomato fruit texture and improved storage and transportation properties were obtained.

7. A method for improving tomato fruit texture and storage and transportation properties according to claim 6, characterized in that: The amino acid sequence of the pectin methylesterase inhibitory protein SlPMEI3 is shown in SEQ ID No.

2.

8. The method for improving the texture of tomato fruit and enhancing the storage and transportation properties according to claim 6, 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.

9. A method for improving tomato fruit texture and storage and transportation properties according to claim 8, 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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