CN111778274A - Method for improving tomato storability through gene editing - Google Patents
Method for improving tomato storability through gene editing Download PDFInfo
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- CN111778274A CN111778274A CN202010520046.1A CN202010520046A CN111778274A CN 111778274 A CN111778274 A CN 111778274A CN 202010520046 A CN202010520046 A CN 202010520046A CN 111778274 A CN111778274 A CN 111778274A
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Abstract
The invention provides a method for improving the tomato storage resistance through gene editing, which edits a catalytic synthesis ethylene precursor regulatory gene SlACS2 in a tomato material genome by using a CRISPR/Cas9 system, so that the performance of the catalytic synthesis ethylene precursor regulatory gene SlACS2 is lost, and the tomato storage resistance is improved; the system includes two sgRNA target sites, sgRNA1 and sgRNA 2; the sgRNA1 and sgRNA2 both recognize target sequences that are DNA fragments encoding the SlACS2 protein in the genome of the tomato material. By using the editing sites, the SlACS2 gene of the tomato can be edited under the mediation of endonuclease Cas9, and site-directed mutation of the SlACS2 gene is formed. The invention has very important function for promoting the application of endogenous gene knockout or exogenous gene site-directed integration technology in tomato gene breeding.
Description
Technical Field
The invention belongs to the technical field of plant gene editing, and particularly relates to a method for improving tomato storability through gene editing, in particular to a method for editing double sites (197-215 and 275-257) of a SlACS2 gene of a tomato by using a CRISPR-Cas9 system.
Background
The tomato fruit is easy to be over-ripe and softened, so that the pressure resistance is poor, the storage period is shortened, the pathogenic bacteria resistance is reduced, and the production, storage, transportation and processing quality are seriously influenced.
Crossbreeding is a common approach for improving tomato quality, but requires a long period and is easily limited by poor gene linkage and interspecies reproductive isolation. The exogenous gene is introduced into the tomato to improve the storability, the breeding period is shortened compared with the conventional breeding, the gene source is widened, but the exogenous gene is integrated on the tomato genome, so that the safety of the people is easily worried, and the breeding efficiency and the popularization and application of the genetic engineering are influenced.
In recent years, a genome editing technology established on the basis of various novel high-efficiency DNA targeting endonucleases is used for carrying out site-directed modification on the gene of a plant, so that the breeding efficiency is improved, and the adverse effect possibly generated by conventional transgenes is avoided. Gene editing can be divided into Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered interactive transmitted small palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies. ZFN and TALEN are used for recognizing specific genome target sites by using proteins, two corresponding nucleases are required to be constructed aiming at each mutation site, and the operation is complicated; the CRISPR/Cas utilizes a simpler complementary nucleotide pairing mode to recognize a specific genome target site, has higher editing efficiency than ZFNs and TALENs, and is simpler to construct.
The tomato is a climacteric fruit, and generates a large amount of ethylene along with the climacteric, namely system II ethylene, so as to ripen the fruit. The system II has an autocatalysis mechanism, so that the ripening process of the tomatoes is difficult to control, and the fruits are over-ripe, softened, rotten and deteriorated. Tomato 1-aminocyclopropane-1-carboxylic acid ACC (1-aminocyclopropane-1-carboxylic acid) is a direct precursor for the synthesis of ethylene, and is produced by ACC synthase (ACC synthsase) SlACS catalysis. The tomato SlACS2 catalyzes the generation of a precursor ACC for ethylene synthesis of the system II and is an important rate-limiting enzyme for ethylene synthesis of the system II. The SlACS2 gene is knocked out by using the CRISPR/Cas9 technology, so that the influence of the SlACS2 gene on the ripening, storage performance and other aspects of tomato fruits can be researched, a storage-resistant variety can be further cultivated, and a new way for improving other properties of tomatoes can be established.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for improving the storability of tomatoes by gene editing, which comprises the steps of carrying out SlACS2 gene editing on tomatoes by using a CRISPR-Cas9 system, and providing double editing sites (197-215 and 275-257) capable of efficiently knocking out the SlACS2 gene. The editing site can be used for Cas 9-mediated tomato SlACS2 gene targeting, so that the function of the gene is inactivated, and the storage resistance of tomatoes is improved. The invention designs and screens gene editing sites capable of being cut efficiently aiming at the SlACS2 gene, and provides a new way for constructing tomatoes with exogenous genes knocked out endogenously or knocked into at fixed points of the SlACS2 gene.
The invention provides a method for improving the tomato storage resistance through gene editing, which edits a catalytic synthesis ethylene precursor regulatory gene SlACS2 in a tomato material genome by using a CRISPR/Cas9 system, so that the performance of the catalytic synthesis ethylene precursor regulatory gene SlACS2 is lost, and the tomato storage resistance is improved;
the CRISPR/Cas9 system includes two sgRNA target sites, designated sgRNA1 and sgRNA2, respectively;
the sgRNA1 and the sgRNA2 recognize target sequences which are DNA fragments encoding SlACS2 proteins in the tomato material genome.
Preferably, the sgRNA1 target site sequence is: 5 '-GGATTAAGAGAAACCCAAAAGG-3';
the sequence of the sgRNA2 target site is as follows: 5 '-TAATCTTGAAAGTTGGCAATGG-3'.
Preferably, the editing method is to introduce a tomato genome editing vector into the tomato material;
the tomato genome editing vector contains the sgRNA1 target site sequence, the sgRNA2 target site sequence and a Cas9 protein encoding gene.
Preferably, the method further comprises the step of screening for homozygous mutants of slocs 2.
The invention provides a method for obtaining a storage-resistant tomato material, which comprises the following steps: selfing the tomato material obtained by the method to obtain selfed progeny, and selecting the selfed progeny which is homozygously mutated by SlACS2 and does not carry exogenous DNA fragments, namely the storage-resistant tomato material.
The invention provides the use of the above method in the cultivation of storage-resistant tomato material.
The present invention provides a biomaterial as described in any one of (1) to (3) below:
(1) the target site sequence described above;
(2) the tomato genome editing vector;
(3) a microbial transformant comprising the tomato genome editing vector described above.
The invention provides the application of the vector or the microbial transformant or the target site sequence in improving the storage-resistant performance of tomatoes;
or, the application of the carrier or the microbial transformant or the target site sequence in cultivating the storage-resistant tomato;
or, the vector or the microbial transformant or the target site sequence is applied to tomato breeding.
The invention provides a method for identifying whether a tomato to be detected is a storage-resistant tomato obtained by the method or a progeny thereof, which comprises the following steps:
respectively extracting genomic DNAs of a tomato wild plant and a tomato transformant to be detected, carrying out PCR amplification on the genomic DNA of the tomato to be detected by using an upstream primer and a downstream primer to respectively obtain PCR amplification products, judging whether the tomato to be detected is the storage-resistant tomato obtained by the method or the progeny thereof according to the sequencing result of the PCR amplification products, wherein the judgment method comprises the steps of comparing the sequencing result of the transformant plant with the sequencing result of the wild plant, and judging whether the tomato to be detected is the storage-resistant tomato obtained by the method or the progeny thereof if the deletion or insertion of a base occurs near the sgRNA1 or between the sgRNA 2;
the upstream primer is as follows: CTCTTACACCATAACACAAC, respectively;
the downstream primer is as follows: CCAGCCATAACAACTCTTTC are provided.
The invention provides a product for identifying or identifying whether a tomato to be detected is a storage-resistant tomato obtained by the method or a progeny product thereof, which is any one of the following (1) to (3):
(1) the above-mentioned upstream primer and downstream primer;
(2) PCR reagents comprising the forward primer and the reverse primer of (1);
(3) a kit comprising the forward primer and the reverse primer described in (1) or the PCR reagent described in (2).
In the case of determining the upstream primer and the downstream primer, methods for constituting the PCR reagent and the PCR kit are well known in the art and will not be described in detail herein.
Compared with the prior art, the invention has the beneficial effects that:
by using the editing sites (197-215 and 275-257) provided by the invention, the SLACS2 gene of the tomato can be edited with high efficiency under the mediation of endonuclease Cas9, and site-directed mutation of the SlACS2 gene is formed. The invention has very important effect on promoting the application of endogenous gene knockout or exogenous gene site-specific integration technology in the research and production of tomato gene breeding.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic structural diagram of a plant expression vector pSlACS 2-DsgRNA.
FIG. 2 is a gene editing form of tomato mutants.
FIG. 3 shows the result of PCR detection electrophoresis of ACS2 gene of T0 plant. Wherein, -is a negative blank control, WT is an untransformed wild-type plant, M is maker (DL2000), and 1-8 are T0 generation tomato plants.
FIG. 4 shows the T0 generation of gene-edited plant.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
Example 1 design and synthesis of sgRNA for specifically targeting SlACS2 Gene in CRISPR/Cas 9-specific knockout of tomato SlACS2 Gene
1. Design of sgRNA targeting tomato SlACS2 gene
An online tool is used for designing the SlACS2-sgRNA, and the specific steps are as follows:
firstly, logging in a GenBank website, searching and downloading a SlACS2 gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO. 4.
Secondly, opening a target prediction website (https:// criprpr.dbcls.jp /), inputting a gene sequence in a text box by using a target site online design tool CRISPRdirect, selecting a tomato species to obtain all target sites of the gene sequence, and finally selecting a group of target sites for targeting by comprehensively considering a scoring result, GC content, specificity of the target sites and the distance between the two target sites.
③ the oligonucleotide sgRNA core sequence with the length of 19nt is designed according to the GG (N)19NGG sequence.
The sequence 1 of the obtained sgRNA target site is as follows: 5' -GGATTAAGAGAAACCCAAAAGG-3
sgRNA target site sequence 2 is: 5 '-TAATCTTGAAAGTTGGCAATGG-3'.
The sgRNA target site sequences 1 and 2 are located on exon 2 of the coding region of SlACS2 on chromosome 1 of the tomato genome at the chromosome coordinates of 78221289-78221271 and 78221211-78221229 and at the coding region coordinates of (197-215 and 275-257).
2. Construction of oligonucleotides for sgRNA
And designing a sense strand primer and an antisense strand primer according to the selected sgRNA core sequence, wherein a sequence containing an endonuclease AarI recognition site is added to the 5' ends of the two primers, and the sequence added to the sense strand is ATATCACCTGCACACTTTGG so as to be complementary with the cohesive end of the vector plasmid. The sense strand primer has a sequence GGATTAAGAGAAACCCAAA with an editing site 197-215 at the 3 'end of the added sequence, and GTTTCAGAGCTATGCTGGAA is added at the 3' end of the editing site; the 5 ' end of the antisense strand is added with the sequence ATATCACCTGCACACAAAC, the 3 ' end of the added sequence is added with the reverse complement sequence TTGCCAACTTTCAAGATTA of the editing site 275-257, and the 3 ' end of the editing site is added with CCAAACTACACTGTTAGATTC.
The finally obtained sense strand primer and antisense strand primer are respectively:
sense strand primer: 5 '-ATATCACCTGCACACTTTGGGGATTAAGAGAAACCCAAAGTTTCAGAGCTATGCTGGAA-3' (SEQ ID NO: 5);
antisense strand primer: 5 '-ATATCACCTGCACACAAACTTGCCAACTTTCAAGATTACAAACTACACTGTTAGATTC-3' (SEQ ID NO: 6).
EXAMPLE 2 construction of plant expression vectors
The CP185 vector and the CP178 vector are provided by vegetable and flower research institute of Chinese academy of agricultural sciences, and the vectors are public vectors. PCR amplification is carried out by using a CP185 vector as a template and a sense strand primer (SEQ ID NO:5) and an antisense strand primer (SEQ ID NO:6) to obtain a DNA fragment containing the double sgRNA, the DNA fragment is connected to pEasy-blunt, after the sequencing is correct, the DNA fragment is connected to a CP178 vector by AarI enzyme digestion to form a plant expression vector pSlACS2-DsgRNA (the structure is shown in figure 1, and the nucleotide sequence is SEQ ID NO: 3). The Cas9 gene on the plant expression vector was driven by 35S, and both sgRNA genes were driven by the tomato U6 promoter. The constructed plant expression vector is transformed into an escherichia coli DH5 alpha strain by a heat shock method for amplification, the vector sequence is verified to be correct by sequencing, and then the agrobacterium LBA4404 strain is transformed by a freeze-thaw method for amplification for infection utilization.
Example 3 genetic transformation of tomato and obtaining of plants edited by the T0 generation SlACS2 Gene
1. Obtaining of sterile seedlings
Treating the prepared tomato seeds with 75% alcohol for 30s, washing with sterile water for 2 times, adding 10% bleaching water, placing on a shaking table, shaking for 1 hour, taking out the seeds, washing with double distilled water for 5 times, placing in a refrigerator at 4 ℃ for 12 hours, inoculating to 1/2MS culture medium (without sucrose), and culturing for 6 days to obtain sterile seedlings.
2. Preparation of bacterial liquid
Streaking pSlACS2-DsgRNA glycerobacteria on a kanamycin YEB solid culture medium (5 g/L yeast extract, 5g/L peptone, 5g/L beef extract, 0.5g/L magnesium sulfate heptahydrate and 1g/L sucrose) containing 50mg/L rifampicin and 100mg/L, culturing at 28 ℃ for two days, picking out a single clone, inoculating the single clone into 5mL liquid YEB culture medium containing 50mg/L rifampicin and 100mg/L kanamycin, culturing at 28 ℃ and 220rpm for overnight with shaking at constant temperature, and taking 120 microliter of shaken bacteria liquid to 50mL liquid YEB culture medium (containing 50mg/L rifampicin and 100mg/L kanamycin) the next day to shake bacteria liquid to OD concentration600The collected cells were centrifuged at 5000rpm for 10min at 0.7, and resuspended in MSO liquid medium.
3. Obtaining regenerated plants of T0 generation
Cutting sterile cultured tomato cotyledon, pre-culturing in pre-culture medium for 1d (dark condition), and placing in OD prepared in step 2600pSlACS2-DsgRNA Agrobacterium suspension (0.7) for 15min, after which the excess was blotted off with filter paper and co-cultured in preculture medium for 2d at 28 ℃ (dark condition).
Wherein, the formula of the pre-culture medium is as follows: MS + zeatin (1mg/L) + indoleacetic acid (1 mg/L).
The co-cultured cotyledon is placed on a differentiation medium for differentiation and screening of adventitious buds, when the screened resistant regeneration buds grow to 2cm high, the resistant regeneration buds are transferred to a rooting medium, and the culture conditions are as follows: culturing at 25 + -1 deg.C, light cycle of 16h/d and illumination intensity of 12000lx until rooting to obtain T0 generation regenerated plant.
The formula of the differentiation medium is as follows: MS + zeatin (2mg/L) + indoleacetic acid (1mg/L) + kanamycin (100mg/L) + timentin (300 mg/L).
The formula of the rooting culture medium is as follows: MS + indoleacetic acid (1mg/L) + kanamycin (100mg/L) + timentin (3001 mg/L).
3. Obtaining of T0 generation SlACS2 gene editing plant
The regenerated plants of the T0 generation were numbered, 0.2g of young leaves were taken from each plant to extract genomic DNA, and PCR amplification was performed using a target site upstream primer (SEQ ID NO:7) and a target site downstream primer (SEQ ID NO: 8). After the amplification, the samples were electrophoresed on 1% agarose gels, and the corresponding PCR products were recovered and transformed into E.coli DH 5. alpha. and sequenced (FIG. 2). In the sequencing result, 2 nucleotides (mutation type A) are deleted at the editing sites 197-215 of the SlACS2 gene, 7 nucleotides (mutation type B) are deleted at the editing sites 276-254 of the SlACS2 gene, and the gene is successfully edited to obtain two types of SlACS2 gene editing plants of the T0 generation.
Wherein the content of the first and second substances,
target site upstream primer: CTCTTACACCATAACACAAC, respectively;
target site downstream primer: CCAGCCATAACAACTCTTTC are provided.
The PCR amplification system is as follows: genomic DNA1uL, Premix Taq DNA polymerase Mix 10uL, front and back primers 0.8uL respectively, and double distilled water added to 20 uL; the PCR amplification conditions were: 5min at 94 ℃; 30s at 94 ℃; 30s at 58 ℃; 30s at 72 ℃; 35 cycles; the size of the PCR product was obtained by electrophoresis on a 1.0% agarose gel at 72 ℃ for 5 min.
Example 4 detection of storage-resistant Properties of Gene-edited tomato
The tomato fruit respiration transition is a characteristic of the fruit entering complete maturity, and the fruit pressure resistance is an important quality character closely related to storage and transportation resistance. For processing tomatoes, the important purpose is to delay the ripening of the tomatoes in the ripening harvesting, storage and transportation process, which is how to improve the pressure resistance of the tomato fruits and prolong the branch hanging time because the ripening is too concentrated. The gene is modified by a CRISPR-Cas9 genome editing system, and ethylene overexpression of a regulation system II is regulated so as to delay over-ripening and rotting of tomatoes. The following is a pressure resistance measurement test of the tomato mutant line New tomato No. 72.
4.1 design of the experiment
The new tomato No. 72T 0 seeds A and B obtained by the method of the embodiment 1-3 and the control wild type parents are sown and cultivated at the same time and then transferred to the field for observing and researching the main agronomic characters of the T1 processed tomatoes. In the field test, the wide-narrow double-row drip irrigation cultivation mode is adopted for planting, the width row is 100cm, the narrow row is 50cm, the plant spacing is 30cm, 20 plants are planted in each plot, three biological repeated tests are carried out randomly, the field planting is carried out for 3 days in 5 months, and the field management is carried out on the same field in the whole growth period.
4.2 test results
Selecting and marking tomatoes in the color transition stage of a wild parent and two T1 mutant plants in 7-10 months in 2019, marking 60 fruits on each test material, marking the initial maturity stage of the material in a test cell when the test cell reaches 50% of mature fruits, sampling once every 5 days after the initial maturity stage, selecting 10 fruits with uniform shapes and sizes, and measuring the pressure resistance of the fruits. The pressure resistance is determined by changing the electronic platform scale, and the specific determination method comprises the following steps: the tomato is placed on the pressing plate to be tested, the handle is rotated to gradually pressurize the upper pressing plate until the tomato is broken, and meanwhile, the pressure kilogram number displayed on the electronic platform scale is read. The results show that the pressure resistance of the two mutant types after gene editing is higher than that of wild plants, and particularly after 20 days of field branch hanging, the pressure resistance of the contrast is obviously reduced faster than that of mutant strains. See table below:
TABLE 1 Gene editing data for testing influence of new tomato No. 72 mutant strain on fruit pressure resistance
Example 5 obtaining tomato germplasm with improved Gene editing storage tolerance
After self-pollination is carried out on the T1 generation SlACS2 gene editing plants obtained in the example 4, selfed seeds are harvested and planted, the method in the step 3 of the example 3 is applied, T-DNA sequences are screened from T2 generation plants, so that two SlACS2 mutant plants of a mutant (A) with 2 base deletions at 197-215 and a mutant (B) with 7 base deletions at 197-215 are obtained, and the new cas-free 9 tomato plant material with improved storage resistance is obtained through selfing.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for improving the storability of tomato by gene editing, comprising: editing a catalytic synthesis ethylene precursor regulatory gene SlACS2 in a tomato material genome by using a CRISPR/Cas9 system, and further losing the performance of the catalytic synthesis ethylene precursor regulatory gene SlACS2 so as to improve the tomato storage resistance;
the CRISPR/Cas9 system includes two sgRNA target sites, designated sgRNA1 and sgRNA2, respectively;
the sgRNA1 and the sgRNA2 recognize target sequences which are DNA fragments encoding SlACS2 proteins in the tomato material genome.
2. The method of claim 1, wherein:
the sequence of the sgRNA1 target site is as follows: 5 '-GGATTAAGAGAAACCCAAAAGG-3';
the sequence of the sgRNA2 target site is as follows: 5 '-TAATCTTGAAAGTTGGCAATGG-3'.
3. The method according to claim 1 or 2, characterized in that: the editing method is to introduce a tomato genome editing vector into the tomato material;
the tomato genome editing vector contains the sgRNA1 target site sequence, the sgRNA2 target site sequence and a Cas9 protein encoding gene.
4. The method according to claim 1 or 2, characterized in that: the method further comprises the step of screening for homozygous mutants of slocs 2.
5. A method for obtaining a shelf-stable tomato material, characterized in that: the method comprises the following steps: selfing the tomato material obtained by the method of any one of claims 1 to 4 to obtain selfed progeny, and selecting selfed progeny which are homozygous mutation of SlACS2 and do not carry exogenous DNA fragments, namely the storage-resistant tomato material.
6. Use of the method according to claim 5 for growing storage-resistant tomato material.
7. A biomaterial as described in any one of (1) to (3) below:
(1) a target site sequence as set forth in claim 1;
(2) a tomato genome editing vector as claimed in claim 3;
(3) a microbial transformant comprising the tomato genome editing vector of claim 3.
8. Use of the vector or microbial transformant or target site sequence of claim 7 for improving the storage-tolerant performance of tomato;
or, the use of the vector or microbial transformant or target site sequence of claim 7 in the cultivation of storage-tolerant tomato;
or, the use of the vector or microbial transformant or target site sequence of claim 7 in tomato breeding.
9. A method for identifying a test tomato as a storage-tolerant tomato obtained by the method of any one of claims 1-4 or progeny thereof, comprising: the method comprises the following steps:
respectively extracting genomic DNAs of a tomato wild plant and a tomato transformant to be detected, carrying out PCR amplification on the genomic DNA of the tomato to be detected by using an upstream primer and a downstream primer to respectively obtain PCR amplification products, judging whether the tomato to be detected is the storage-resistant tomato obtained by the method or the progeny thereof according to the sequencing result of the PCR amplification products, wherein the judgment method comprises the steps of comparing the sequencing result of the transformant plant with the sequencing result of the wild plant, and judging whether the tomato to be detected is the storage-resistant tomato obtained by the method or the progeny thereof if the deletion or insertion of a base occurs near the sgRNA1 or between the sgRNA 2;
the upstream primer is as follows: CTCTTACACCATAACACAAC, respectively;
the downstream primer is as follows: CCAGCCATAACAACTCTTTC are provided.
10. A method for identifying or identifying whether a tomato to be tested is a product of a storage-tolerant tomato obtained by the method of any one of claims 1 to 4 or progeny thereof, which is any one of the following (1) to (3):
(1) the forward primer and the reverse primer as set forth in claim 9;
(2) PCR reagents comprising the forward primer and the reverse primer of (1);
(3) a kit comprising the forward primer and the reverse primer described in (1) or the PCR reagent described in (2).
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