阅读说明：本技术 基于CRISPR/Cas9的香蕉MaACO1基因编辑载体及其构建方法和应用 (Banana MaACO1 gene editing vector based on CRISPR/Cas9 and construction method and application thereof ) 是由 毕方铖 胡春华 杨乔松 窦同心 盛鸥 邓贵明 董涛 易干军 于 2020-06-01 设计创作，主要内容包括：本发明提供了一种基于CRISPR/Cas9的香蕉MaACO1基因编辑载体及其构建方法和应用,针对香蕉基因序列,设计基因编辑靶位点及其引物,以及在构建香蕉MaACO1基因编辑载体时结合特定的扩增程序,构建得到合理的sgRNA表达框,提供了一种有效且稳定的定向突变香蕉ACO1基因的编辑方法,从而使得香蕉的ACO1表达缺失或减弱。延缓了香蕉的后熟过程,可以明显的延长香蕉的保鲜期的效果,解决了采后香蕉在常温下成熟过快、不耐贮的问题。(The invention provides a banana MaACO1 gene editing vector based on CRISPR/Cas9, a construction method and application thereof, wherein a gene editing target site and a primer thereof are designed aiming at a banana gene sequence, and a reasonable sgRNA expression frame is constructed by combining a specific amplification program when the banana MaACO1 gene editing vector is constructed, so that an effective and stable editing method for directionally mutating banana ACO1 gene is provided, and the banana ACO1 expression is deleted or weakened. The postripeness process of the bananas is delayed, the effect of prolonging the fresh-keeping period of the bananas can be obvious, and the problems that the picked bananas are too fast to mature and not storable at normal temperature are solved.)

1. An sgRNA expression cassette, characterized in that the nucleotide sequence is shown in SEQ ID NO. 12.

2. A construction method of a banana MaACO1 gene editing vector based on CRISPR/Cas9 is characterized by comprising the following steps:

s1, taking pYLgRNA-OsU6b plasmid as a template, and amplifying primers with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3 to obtain a OsU6b promoter; taking pYLgRNA-OsU6b plasmid as a template, and amplifying primers with nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 to obtain a gRNA fragment containing a target site with the nucleotide sequence shown as SEQ ID NO. 1;

s2, amplifying primers with nucleotide sequences shown as SEQ ID No.6 and SEQ ID No.7 by using the OsU6b promoter and the gRNA fragment as templates to obtain an sgRNA expression cassette;

s3, inserting the sgRNA expression cassette into pYLCRISPR/Cas9 plasmid.

3. The method for constructing banana MaACO1 gene editing vector based on CRISPR/Cas9 according to claim 2, wherein the amplification procedure for obtaining OsU6b-gRNA expression cassette by amplification in step S1 is as follows: 94 plus or minus 3 ℃ for 10 plus or minus 1s, 58 plus or minus 2 ℃ for 15 plus or minus 1s, 68 plus or minus 2 ℃ for 20 plus or minus 1s, and 20-30 cycles; and/or

The amplification procedure for obtaining the sgRNA expression cassette by amplification described in step S2 includes: 10 plus or minus 1s at 95 plus or minus 3 ℃, 15 plus or minus 1s at 58 plus or minus 2 ℃, 20 plus or minus 1s at 68 plus or minus 2 ℃ and 15-25 cycles.

4. A banana MaACO1 gene editing vector, which contains the sgRNA expression cassette of claim 1, or is prepared by the construction method of the banana MaACO1 gene editing vector based on CRISPR/Cas9 of claim 2 or 3.

5. An engineered bacterium comprising the sgRNA expression cassette of claim 1 or the banana MaACO1 gene editing vector of claim 4.

6. The engineered bacterium of claim 5, wherein the engineered bacterium is an Agrobacterium EHA105 comprising the sgRNA expression cassette of claim 1 or an Agrobacterium EHA105 comprising the banana MaACO1 gene editing vector of claim 4.

7. A kit for site-directed mutagenesis of a banana MaACO1 gene is characterized by comprising at least one of the following components:

the sgRNA expression cassette of claim 1;

the primer of claim 2;

the banana MaACO1 gene editing vector of claim 4; and

the engineered bacterium of any one of claims 5 to 6.

8. A site-directed mutagenesis method of banana MaACO1 gene is characterized by comprising the following steps: the engineering bacteria of claim 5 or 6 are used for banana genetic transformation by taking bananas as receptor materials to obtain banana positive plants with site-directed mutation of MaACO1 genes.

9. Use of the banana MaACO1 gene editing vector of claim 4, the engineered bacterium of claim 5, the primer of claim 2, the banana MaACO1 gene site-directed mutagenesis kit of claim 7, or the banana MaACO1 gene site-directed mutagenesis method of claim 8 for delaying banana maturation.

10. A shelf stable banana prepared by site directed mutagenesis of the banana MaACO1 gene of claim 8.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a banana MaACO1 gene editing vector based on CRISPR/Cas9, and a construction method and application thereof.

Background

Genome editing refers to a technique of obtaining a desired change in the genome sequence of an organism by site-specific modification of a genome with a sequence-specific nuclease. The technology is a genome directed editing technology developed in recent years, can carry out site-directed mutation (insertion, deletion, modification and the like), and has the characteristics of low cost, simple operation, high mutation efficiency and the like.

The problem of postharvest loss is one of the major problems faced by the current banana industry, and the postharvest loss of bananas is estimated to reach 20-30%, which is mostly caused by the fact that bananas are too fast to mature at normal temperature, are not resistant to storage and have short shelf life, and the bananas rapidly mature and rot one week after ethylene ripening, causing huge loss. In the past, the function of genes is generally researched by biological means such as expression analysis, RNAi and the like, the expression of ACO genes is inhibited in an RNAi mode, so that the synthesis of ethylene is reduced, and the ripening of fruits is delayed, but the methods cannot completely inhibit the transcription of the ACO genes in bananas, have low inhibition efficiency, have the problem of transgenic biological safety, and cannot well solve the problems that the picked bananas ripen too fast at normal temperature and cannot be stored enduringly. Therefore, a method for reducing the ethylene synthesis of bananas and delaying the banana ripening is needed to be found, so as to solve the problems that the bananas ripen too fast and cannot be stored at normal temperature after being picked.

Disclosure of Invention

Based on the above, the invention aims to provide a banana MaACO1 gene editing vector based on CRISPR/Cas9, and a construction method and application thereof.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

an sgRNA expression cassette, the nucleotide sequence of which is shown in SEQ ID NO. 12.

A construction method of a banana MaACO1 gene editing vector based on CRISPR/Cas9 comprises the following steps:

s1, taking pYLgRNA-OsU6b plasmid as a template, and amplifying primers with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3 to obtain a OsU6b promoter; taking pYLgRNA-OsU6b plasmid as a template, and amplifying primers with nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 to obtain a gRNA fragment containing a target site with the nucleotide sequence shown as SEQ ID NO. 1;

s2, amplifying primers with nucleotide sequences shown as SEQ ID No.6 and SEQ ID No.7 by using the OsU6b promoter and the gRNA fragment as templates to obtain an sgRNA expression cassette;

s3, inserting the sgRNA expression cassette into pYLCRISPR/Cas9 plasmid.

In some embodiments, the amplification procedure described in step S1 to obtain the OsU6b-gRNA expression cassette is: 94 plus or minus 3 ℃ for 10 plus or minus 1s, 58 plus or minus 2 ℃ for 15 plus or minus 1s, 68 plus or minus 2 ℃ for 20 plus or minus 1s, and 20-30 cycles; and/or the amplification procedure for obtaining the sgRNA expression cassette by amplification described in step S2 is as follows: 10 plus or minus 1s at 95 plus or minus 3 ℃, 15 plus or minus 1s at 58 plus or minus 2 ℃, 20 plus or minus 1s at 68 plus or minus 2 ℃ and 15-25 cycles.

The invention also provides a gene editing vector, and the specific technical scheme is as follows:

the gene editing vector is a vector containing the sgRNA expression cassette;

or the gene editing vector is the banana MaACO1 gene editing vector prepared by the construction method of the banana MaACO1 gene editing vector based on CRISPR/Cas 9.

The invention also provides an engineering bacterium, and the specific technical scheme is as follows:

an engineering bacterium, which is a bacterium containing the sgRNA expression cassette or a bacterium containing the banana MaACO1 gene editing vector.

In some of these embodiments, the bacterium is agrobacterium EHA 105.

The invention also provides a kit, which has the following specific technical scheme:

a gene site-directed mutagenesis kit comprising at least one of the following components: the sgRNA expression cassette, the primer, the banana MaACO1 gene editing vector and the engineering bacteria are described above.

The invention also provides a site-directed mutagenesis method of the banana MaACO1 gene, which comprises the following specific technical scheme:

a site-directed mutagenesis method of banana MaACO1 gene comprises the following steps: the engineering bacteria are used, bananas are used as receptor materials, banana genetic transformation is carried out, and banana positive plants with MaACO1 gene site-directed mutation are obtained.

The invention also provides a new application, and the specific technical scheme is as follows:

the sgRNA expression cassette, the banana MaACO1 gene editing vector, the primers, the engineering bacteria, the gene site-directed mutagenesis kit or the banana MaACO1 gene site-directed mutagenesis method are applied to delaying banana maturation.

The invention also provides a banana, and the specific technical scheme is as follows:

a shelf stable banana prepared by site directed mutagenesis of the banana MaACO1 gene as described above.

Based on the technical scheme, the invention has the following beneficial effects:

the invention designs a gene editing target site and a primer thereof aiming at a banana gene sequence, constructs a reasonable sgRNA expression frame by combining a specific amplification program when constructing a banana MaACO1 gene editing vector, and provides an effective and stable editing method for directionally mutating banana ACO1 gene, thereby enabling the banana ACO1 expression to be deleted or weakened.

Furthermore, the gene editing vector is constructed and applied to site-directed mutation of the MaACO1 gene of bananas, so that the genetic banana mutant plants can be stabilized, key enzymes for ethylene biosynthesis of the banana mutant plants are not expressed or are low expressed, the banana ethylene synthesis path is inhibited/blocked, the ethylene synthesis is greatly reduced, the fruit ripening is obviously slowed down, the after-ripening process of the bananas is delayed, the fresh-keeping period of the bananas can be obviously prolonged, and the problems that the picked bananas are ripened too fast at normal temperature and cannot be stored are solved.

Drawings

FIG. 1 shows the structure of the MaACO1 gene and the selection of target sites;

FIG. 2 is a plasmid map of pYLgRNA-OsU6 b;

FIG. 3 is a map of pYLCRISPR/Cas9 plasmid;

FIG. 4 is a schematic diagram of a constructed gene editing vector;

FIG. 5 is a graph comparing the mature phenotype of gene-edited bananas and wild bananas at room temperature;

FIG. 6 is a graph comparing the ripening phenotypes of gene-edited bananas and wild bananas under ethephon ripening conditions.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It will be appreciated that the experimental procedures for the following examples, where specific conditions are not indicated, are generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention discloses a construction method of a banana MaACO1 gene editing vector based on CRISPR/Cas9, which comprises the following steps:

s1, taking pYLgRNA-OsU6b plasmid as a template, and amplifying primers with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3 to obtain a OsU6b promoter; taking pYLgRNA-OsU6b plasmid as a template, and amplifying primers with nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5 to obtain a gRNA fragment containing a target site with the nucleotide sequence shown as SEQ ID NO. 1;

s2, amplifying primers with nucleotide sequences shown as SEQ ID No.6 and SEQ ID No.7 by using the OsU6b promoter and the gRNA fragment as templates to obtain an sgRNA expression cassette;

s3, inserting the sgRNA expression cassette into pYLCRISPR/Cas9 plasmid.

Preferably, the OsU6b-gRNA expression cassette contains a target site with a nucleotide sequence shown as SEQ ID No. 1.

In some embodiments, the amplification procedure described in step S1 to obtain the OsU6b-gRNA expression cassette is: 94 plus or minus 3 ℃ for 10 plus or minus 1s, 58 plus or minus 2 ℃ for 15 plus or minus 1s, 68 plus or minus 2 ℃ for 20 plus or minus 1s, and 20-30 cycles. Preferably 94 + -2 deg.C 10 + -0.5 s, 58 + -1 deg.C 15 + -0.5 s, 68 + -1 deg.C 20 + -0.5 s, 22-28 cycles. More preferably 94 ℃ for 10s, 58 ℃ for 15s, 68 ℃ for 20s, and 25-28 cycles.

More preferably, in the amplification described in step S1, the pYLgRNA-OsU6b plasmid is 1-6 ng; preferably, the primers with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.4 are 0.15-0.25 μ g each; preferably, the primers with the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.5 are 0.05-0.15. mu.g each.

In some embodiments, the amplification procedure described in step S2 to obtain the sgRNA expression cassette is: 10 plus or minus 1s at 95 plus or minus 3 ℃, 15 plus or minus 1s at 58 plus or minus 2 ℃, 20 plus or minus 1s at 68 plus or minus 2 ℃ and 15-25 cycles. Preferably 95 + -2 deg.C 10 + -0.5 s, 58 + -1 deg.C 15 + -0.5 s, 68 + -1 deg.C 20 + -0.5 s, 15-22 cycles. More preferably 95 ℃ for 10s, 58 ℃ for 15s, 68 ℃ for 20s,17-20 cycles.

Preferably, when the sgRNA expression cassette is inserted into the pYLCRISPR/Cas9 plasmid as described in step S3, the sgRNA expression cassette is cleaved with the pYLCRISPR/Cas9 plasmid with Bsa I-HF and then ligated with T4 DNA ligase.

The banana MaACO1 gene editing vector is prepared by a construction method of a banana MaACO1 gene editing vector based on CRISPR/Cas 9.

The engineering bacteria are bacteria containing the banana MaACO1 gene editing vector.

Preferably, the engineering bacteria are agrobacterium EHA105 containing the banana MaACO1 gene editing vector as described above.

Preferably, the preparation method of the engineering bacteria comprises the following steps: inserting the sgRNA expression cassette into pYLCRISPR/Cas9 plasmid, thermally shocking to transform E.coli DH10B competent cells, screening positive clones by using a culture medium containing Kan and IPTG after culture, extracting plasmids from the positive clones and sequencing, and transforming agrobacterium tumefaciens EHA105 by using a plasmid electrode with correct sequencing.

Preferably, the nucleotide sequence of the primer used in sequencing is shown as SEQ ID NO.8 and SEQ ID NO. 9.

The invention relates to a site-directed mutagenesis method of a banana MaACO1 gene, which comprises the following steps: the engineering bacteria are used, bananas are used as receptor materials, banana genetic transformation is carried out, and banana positive plants with MaACO1 gene site-directed mutation are obtained.

Preferably, the banana is triploid Brazil banana Musa acuminata (AAA group, cv.

Preferably, said banana is genetically transformed, comprising: hygromycin is taken as a screening marker, and agrobacterium tumefaciens co-culture, liquid screening and plant regeneration are carried out to obtain a hygromycin-resistant regeneration plant.

The invention also provides a banana prepared by the site-directed mutagenesis method of the banana MaACO1 gene.

Example 1 construction of Banana MaACO1 Gene editing vector

1. According to the gene sequence of banana, 5'-CCTCATGGATGAAGTGGAGAAGG-3' (SEQ ID NO.1) on the second exon of the MaACO1 gene is selected as a target site, as shown in FIG. 1.

2. 2-5ng of pYLgRNA-OsU6b plasmid (FIG. 2, donated by the Liudazzling teacher's laboratory) was used as template, designed according to target sites and used 4 primers in one reaction: U-F and gRTF + each 0.2. mu.M, U6 bR-and gRTR each 0.1. mu.M. And (2) 25-28 circulation: 94 ℃ for 10s, 58 ℃ for 15s and 68 ℃ for 20s, and is used for amplifying gRNA fragments containing target sites and promoter fragments driving gRNA expression, and later circulation generates 2 fragments combined sgRNA expression frame fragments containing targets, namely OsU 6-6 b-gRNA expression frame, through overlapping PCR.

Promoter amplification primers were as follows:

U-F：5′-CTCCGTTTTACCTGTGGAATCG-3′；SEQ ID NO.2

U6bR-:5′-TCTCCACTTCATCCATGAGGCaacacaagcggcagc-3′；SEQ ID NO.3

gRNA amplification primers were as follows:

gRTF+：5′-CCTCATGGATGAAGTGGAGAgttttagagctagaaat-3′；SEQ ID NO.4

gRTR:5′-CGGAGGAAAATTCCATCCAC-3′；SEQ ID NO.5

3. taking 1 μ l of each of the first round PCR product in step 2, OsU6b promoter and gRNA fragment containing target point, and using H₂After 10-fold O dilution, 1. mu.l of the template was used and amplified with 1/10 amounts of working solutions of primers PpsF and PgsR (final concentration 0.15. mu.M) using the appropriate amount of high fidelity PCR enzyme. Amplifying for 17-20 cycles according to the following conditions: 95 ℃ for 10s, 58 ℃ for 15s and 68 ℃ for 20 s. And (3) taking 2-3 mul of electrophoresis to check whether the length of the product is correct, and further purifying by using a PCR product purification kit to obtain an OsU6b-gRNA expression cassette containing a target point, wherein the expression cassette can be used for further enzyme digestion connection.

The second round PCR primers were as follows:

PpsF SEQ ID NO.6：

5′-TTCAGAggtctcTctcgACTAGTATGGAATCGGCAGCAAAGG-3′；

PgsR SEQ ID NO.7：

5′-AGCGTGggtctcGaccgACGCGTATCCATCCACTCCAAGCTC-3′

OsU6b-gRNA expression cassette sequence (SEQ ID NO.12) is as follows:

TTCAGAGGTCTCTctcgACTAGTATGGAATCGGCAGCAAAGGATGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGAGAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGCCGACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGGAGACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTGCCTCATGGATGAAGTGGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTTGGAGTGGATGGATACGCGTcggtCGAGACCCACGCT

4. the reaction of cleavage and ligation was carried out as follows

Reagent	Addition amount (μ l)	Final concentration	Source/manufacturer
				10×CutSmart Buffer	1.5	1×	New England Biolabs,Inc
10mM ATP	1.5	1mM	TaKaRa
				pYLCRISPR/Cas9 plasmid	60-80ng	4-6ng/μl	Liu dazzling teacher laboratory present (fig. 3)
OsU6b-sgRNA expression cassette	10-15ng		PCR amplification
				Bsa I-HF	10U	0.1-0.2U/μl	New England Biolabs,Inc
T4 DNA ligase	35U	2-3U/μl	New England Biolabs,Inc
				ddH2O	Final 15. mu.l		Self-made

Carrying out enzyme digestion connection by using variable temperature circulation for about 10-15 cycles: 5min at 37 ℃; 5min at 10 ℃ and 5min at 20 ℃; finally 5min at 37 ℃. The OsU6b-sgRNA expression cassette was inserted into pYLCRISPR/Cas9 plasmid to obtain a ligation product.

5. And (3) taking 1-1.5 mu l of the ligation product prepared in the step 4, thermally shocking the E.coli DH10B competent cells, adding 1ml of LB culture medium after thermal shock, and culturing at 37 ℃ for 1-1.5 h. The plating medium is LB +50 mug/ml Kan, 0.3-0.5 mM IPTG. Wherein ccdBs is a lethal gene in pYLCRISPR/Cas9 plasmid, and only strains transformed with plasmids in which ccdB was successfully excised and the target fragment ligated can grow: when the sgRNA expression cassette is successfully inserted into pYLCRISPR/Cas9 plasmid, the ccdBs gene on the plasmid is successfully excised, and the transformed strain can grow; when the sgRNA expression cassette was not successfully inserted into the pYLCRISPR/Cas9 plasmid, the ccdBs gene was not excised, and IPTG induced lethality in ccdBs expression of empty plasmid transformants that did not completely excise ccdBs. While positive clones containing the target insert (OsU6b-gRNA expression cassette) can be grown on antibiotic-containing plates.

6. Plasmid extraction, using sequencing primer F: 5'-GCGGTGTCATCTATGTTACTA-3' (SEQ ID NO.8) and sequencing primer R: 5'-GGCTGTATCTACGTTATTGAAG-3' (SEQ ID NO.9) confirmed the sequence of the OsU6b-gRNA expression cassette.

7. The correct plasmid for sequencing was shocked into Agrobacterium (EHA 105). Plasmids are extracted from agrobacterium clones, about 5-10ng of plasmids are taken as a template, PCR confirmation is carried out by using primers for amplifying promoters and gRNA, and the constructed vector is shown in figure 4.

Example 2 obtaining and identification of Banana MaACO1 Gene knockout line

1. Based on the confirmed positive agrobacterium (EHA105) constructed in example 1, an agrobacterium-mediated banana genetic transformation experiment is performed with a triploid Brazilian banana Musa acuminata (AAA group, cv. Brazilian) suspension line preserved in a subject group as a receptor material, hygromycin is used as a screening marker, and a hygromycin-resistant regenerated plant is obtained through the processes of agrobacterium co-culture, liquid screening, plant regeneration and the like.

2. PCR identification of positive strains: after obtaining the regeneration plants, extracting the genome DNA of each regeneration plant, designing a primer to amplify the hygromycin resistance gene, and selecting the positive plants into which the expression cassettes are transferred.

3. Selecting two positive plants, and respectively designing primers at the upstream and downstream of an editing target point, wherein the sequences of the primers are as follows:

MaACO1F：5′-GAAGGGAGAGAGGAGAAG-3′，SEQ ID NO.10

MaACO1R：5′-CAAGAAACGAAACGATGAG-3′，SEQ ID NO.11

the 447bp fragment of the target region of the positive plant is amplified by PCR. The reaction conditions were as follows: 10s at 95 ℃, 30s at 55 ℃, 30s at 72 ℃ and 32-35 cycles.

And (3) delivering the PCR product to a sequencing company for high-throughput amplicon sequencing, wherein according to a sequencing result, the result shows that the target sites of the two positive plants are edited, and the gene editing modes are all multi-allele mutation.

4. Two lines, which were confirmed to be edited by high throughput sequencing, were planted in the experimental greenhouse until the fruit was harvested, and found:

under the condition of normal-temperature non-ripening (22 ℃), the ripening of the fruit of the edited mutant line is obviously slowed down compared with that of the wild type fruit, the wild type fruit is completely ripe and begins to rot in about 20 days, while the MaACO1 mutant fruit begins to ripen in about 80 days (figure 5), which can greatly increase the normal-temperature storage period of the picked bananas.

Under the condition of normal-temperature ethylene ripening (22 ℃), the fruits of the wild type and the mutant line can be completely ripe within 7-10 days, but the ripening process of the fruits of the mutant line is 1-2 days later (figure 6).

This shows that the gene editing vector site-directed mutation MaACO1 gene constructed in example 1 can play a role in obviously delaying fruit ripening, and is an ideal target site for prolonging the postharvest shelf life of fruits by applying gene editing.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.