Method for improving tomato storability through gene editing
阅读说明：本技术 一种通过基因编辑提高番茄耐贮藏性的方法 (Method for improving tomato storability through gene editing ) 是由 刘江娜 张爱萍 张西英 李荣霞 白云凤 闫建俊 薛丽萍 刘伟 于 2020-06-09 设计创作，主要内容包括：本发明提供一种通过基因编辑提高番茄耐贮藏性的方法,利用CRISPR/Cas9系统对番茄材料基因组中的催化合成乙烯前体调控基因SlACS2进行编辑,进而使催化合成乙烯前体调控基因SlACS2性能丧失,从而提高番茄耐贮藏性能；该系统包括两个sgRNA靶位点,分别为sgRNA1和sgRNA2；sgRNA1和sgRNA2识别的靶序列均为所述番茄材料基因组中编码SlACS2蛋白的DNA片段。利用上述编辑位点,可以在核酸内切酶Cas9的介导下对番茄自身的SlACS2基因进行编辑,形成SlACS2基因的定点突变。本发明对促进内源基因敲除或外源基因定点整合技术在番茄基因育种中的应用具有十分重要的作用。(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.)
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).
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
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
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.