CN110004173B

CN110004173B - Method for obtaining non-transgenic storable fresh-eating Chinese wolfberry

Info

Publication number: CN110004173B
Application number: CN201910276123.0A
Authority: CN
Inventors: 张康; 高崑; 冉毅东
Original assignee: Tianjin Genovo Biotechnology Co ltd
Current assignee: Tianjin Genovo Biotechnology Co ltd
Priority date: 2019-04-08
Filing date: 2019-04-08
Publication date: 2023-03-14
Anticipated expiration: 2039-04-08
Also published as: CN110004173A

Abstract

The invention discloses a method for obtaining non-transgenic storable fresh-eating medlar. The invention provides a method for cultivating storage-resistant fresh-eating medlar, which comprises the following steps: inhibiting the expression of pectin lyase gene in the target medlar, thereby ensuring that the medlar is storable; or inhibiting the content or activity of pectin lyase in the fructus Lycii, thereby making fructus Lycii storage-stable. The nutritive value of fresh wolfberry fruit is obviously higher than that of dry fruit, but the short shelf life is the main reason for limiting the sale of fresh fruit. Compared with the traditional breeding technology, the method can quickly obtain a new variety with storage resistance.

Description

Method for obtaining non-transgenic storable fresh-eating Chinese wolfberry

Technical Field

The invention belongs to the field of biotechnology breeding, relates to a method for obtaining non-transgenic storable fresh-eating Chinese wolfberry, and particularly relates to a method for obtaining a Chinese wolfberry storage-resistant breeding material by site-specific mutagenesis of related genes of Ningxia Chinese wolfberry pectin lyase by using a genome editing technology.

Background

Lycium barbarum L is an important medicinal plant resource in China. Contains lycium barbarum polysaccharide, betaine, lycium barbarum pigment and the like, and modern medicine proves that the lycium barbarum has wide medicinal value. In addition, the medlar is drought-resistant and suitable for sand land growth, so that the medlar can be used as shrub for water and soil conservation, and becomes a pioneer for saline-alkali soil tree opening due to salt and alkali resistance.

The fresh Chinese wolfberry fruits have higher nutrient contents than the dry fruits and gradually become popular current fruits for consumers. The fresh medlar has large fruit grain, thin skin, thick meat, less seeds and sweet taste. However, the fresh wolfberry fruit is berry, the moisture content is up to 80%, the fresh wolfberry fruit is rich in sugar, the deterioration speed of the fruit after harvesting is high, the fruit is easy to mildew, the supply period and the shelf life of the fruit are short, and the market demand is difficult to meet. The thin skin and thick meat improve the taste, but increase the difficulty in storage and transportation. The selling cost of the fresh wolfberry fruits is very high, and the risk is very high.

Disclosure of Invention

In order to obtain non-transgenic storage-tolerant fresh-eating medlar, the invention provides the following technical scheme:

the invention provides a method for cultivating storage-resistant fresh-eating medlar, which comprises the following steps:

inhibiting the expression of pectin lyase gene in the target medlar, thereby ensuring that the medlar is storable;

or inhibiting the content or activity of pectin lyase in fructus Lycii, thereby making fructus Lycii storage-stable.

In the above method, the pectin lyase is (a 1), (a 2) or (a 3):

(a1) Protein shown in a sequence 3 in a sequence table;

(a2) A protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a 1) and having the same function;

(a3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to (a 1) and having the same function.

In the above method, the pectin lyase gene is any one of the following (b 1) to (b 4):

(b1) The coding region is a DNA molecule shown as a sequence 1 in a sequence table;

(b2) The coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b3) A DNA molecule which hybridizes with the DNA molecule defined in (b 1) or (b 2) under stringent conditions and encodes a pectin lyase;

(b 4) a DNA molecule encoding a pectin lyase having an identity of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more to (b 1) or (b 2).

In the method, the inhibition of the expression of the pectin lyase gene in the lycium barbarum or the inhibition of the content or the activity of the pectin lyase in the lycium barbarum is realized by a CRISPR/Cas9 system.

In the above method, the CRISPR/Cas9 system comprises sgRNA and Cas9 protein;

or the CRISPR/Cas9 system comprises a plasmid expressing sgRNA and Cas9 proteins;

the target sequence of the sgRNA is (c 1) or (c 2) as follows:

(c1) A nucleic acid molecule shown as a sequence 7 in a sequence table;

(c2) A nucleic acid molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to (c 1).

Or the sgRNA and the Cas9 protein are introduced into the medlar of interest through a CRISPR/Cas9 system.

The method for introducing the genetic material or the non-genetic material into the cells or tissues of the lycium barbarum is a particle gun method, an agrobacterium infection method, a PEG-induced protoplast method or any other introduction method.

In the method, the nucleotide sequence of the sgRNA is RNA or a sequence 10 coded by a sequence 4 in a sequence table;

the sgRNA is (d 1), (d 2) or (d 3) as follows:

(d1) RNA encoded by sequence 4 of the sequence listing;

(d2) A nucleic acid molecule shown as a sequence 10 in a sequence table;

(d3) A nucleic acid molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to (d 1) or (d 2).

Or the amino acid sequence of the Cas9 protein is sequence 11.

The invention also aims to provide a method for preparing the fresh medlar with long storage life.

The method provided by the invention comprises the following steps: introducing Cas9 protein and specific sgRNA into cells or tissues of target medlar to obtain storable fresh medlar;

the target sequence of the sgRNA is (c 1) or (c 2) as follows:

(c1) A nucleic acid molecule shown as a sequence 7 in a sequence table;

In the above method, the sgRNA is (d 1), (d 2), or (d 3):

(d1) RNA encoded by sequence 4 of the sequence listing;

(d2) A nucleic acid molecule shown as a sequence 10 in a sequence table;

The 3 rd object of the present invention is to provide sgRNA or a plasmid expressing the same.

The sgRNA provided by the invention has the following target sequence (c 1) or (c 2):

(c1) A nucleic acid molecule shown as a sequence 7 in a sequence table;

The invention also provides a protein which is (a 1) or (a 2) or (a 3) as follows:

(a1) Protein shown in a sequence 3 in a sequence table;

In the above, the sgRNA is (d 1), (d 2) or (d 3):

(d1) RNA encoded by sequence 4 of the sequence listing;

(d2) A nucleic acid molecule shown as a sequence 10 in a sequence table;

The cell is any cell which can be used as an introduction receptor and can be regenerated into a complete plant through tissue culture; the tissue can be any tissue which can be used as an introduction receptor and can be regenerated into a complete plant through tissue culture; specifically, the cells are protoplast cells or suspension cells; the tissue is leaf disk, stem segment or cotyledon.

The nutritive value of fresh wolfberry fruit is obviously higher than that of dry fruit, but the short shelf life is a main reason for limiting the sale of fresh fruit. Compared with the traditional breeding technology, the method can quickly obtain a new variety with storage resistance. In terms of safety, the method adopts a transient expression method of sequence specific nuclease, so that the artificial nuclease gene is not integrated into a receptor genome, and meanwhile, the site-directed knockout genetic material without transgenic traces is obtained. Site-directed knockout greatly reduces the safety risk due to unintended effects of random insertion and integration as compared to traditional transgenic techniques. Therefore, the technology has higher safety in technical aspect than the traditional transgenic technology. In cost, the method greatly reduces the number of the group samples needing to be screened, only needs a breeding period of 2-3 years, and is obviously shortened compared with the breeding period of 8-10 years of transgenosis. In policy, the method can be used for more quickly and easily obtaining the examination and approval of the policy based on the consideration of safety.

Drawings

FIG. 1 is a diagram of a target site of LbPL gene of Lycium barbarum.

FIG. 2 is a graph showing the comparative effect of wild Ningqi No. 7 and pl mutant fruits stored at room temperature for 15 days after picking.

FIG. 3 is a diagram of PHSE401-LbPL1 structure.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 cloning of LbPL Gene of Lycium barbarum and determination of target sequence

1. Cloning of Lbpl Gene from Lycium barbarum

And (3) amplifying the full-length cDNA of the LbPL gene of the lycium barbarum pectin lyase by using cDNA obtained by reverse transcription of total RNA of lycium barbarum leaves as a template and adopting an RT-PCR method. Conserved sequence design primer based on solanaceae plant tomato PL gene

F:ATGGG(G/C)CACTTCCTCTGTTTT

R:TT(T/C)AGCAACGAGAACCCTTTTTAC

The primer combination F/R is used for amplification, and a 1212bp fragment (figure 2) is obtained by sequencing the RT-PCR result, and is a complete open reading frame and codes 403 amino acids. The genes represented by the sequences were designated as LbPL by Blast alignment based on homology.

The sequence of the genomic DNA of the LbPL gene is sequence 1, and the sequence of cDNA is sequence 2; the amino acid sequence of the protein LbPL encoded by the gene is sequence 3.

2. Selection of target site to be knocked out of lycium barbarum LbPL gene

The LbPL gene locus number is Solyc03g111690, is located on chromosome 3 of Lycium barbarum, comprises 4 exons and 3 introns, and totally encodes 403 amino acids, and a target site for constructing knockout vector selection is located in exon 2 (FIG. 1).

The sequence of the target site and the sequence of sgRNA thereof may be 3 kinds as follows:

the coding sequence of sgRNA-1 is: GGGTATTTATCGTGTGGTACCGG (seq id No. 4), target sequence 5'-GGGTATTTATCGTGTGGTACCGG-3' (seq id No. 7);

the coding sequence of sgRNA-2 is: CGGTTAGCTGACTGTGCAATTGG (seq. No. 5), target sequence 5' -CGGTTAGCTGACTGTGCAATTGG-3 (seq. No. 8);

the coding sequence of sgRNA-3 is: ACTGTGGATTATTTTCGCGAGGG (SEQ ID NO: 6) and the target sequence 5' -ACTGTGGATTATTTTCGCGAGGG-3 (SEQ ID NO: 9).

The sgRNA-1 is analyzed to have the highest mutation efficiency, so the sgRNA-1 is selected for subsequent work.

Example 2 knocking out LbPL Gene

1. Preparation of Cas9 protein and sgRNA transcribed in vitro

1. Preparation of Cas9 protein

The protein containing the purified Cas9 protein (with the amino acid sequence of sequence 11) was obtained from the institute of genetics and developmental biology, academy of Chinese sciences.

2. In vitro transcription of sgRNA at target sites

The Target sequence of sgRNA-1 is Target-LbPL1:5'-GGGTATTTATCGTGTGGTACCGG-3' (series 7)

1) Construction of recombinant plasmid

The following single-stranded primers with sticky ends (underlined) were synthesized: lbPL1F:5'-ATTGGGGTATTTATCGTGTGGTAC-3'; the LbPL1R is 5'-AAACGTACCACACGATAAATACCC-3', and double-stranded DNA with sticky ends is formed through a primer annealing procedure;

then inserting the double-stranded DNA with sticky ends into a pHSE401 vector (described in the following documents: A CRISPR/Cas9toolkit for multiplex genome editing in plants. Xing HL, dong L, wang ZP, zhang HY, han CY, liu B, wang XC, chen QJ. BMC Plant biol.2014Nov 14 (1): 327) to obtain a plasmid containing target-LbPL 1.

The plasmid was sequenced to verify a positive plasmid having a DNA molecule represented by sequence 4 (the coding DNA molecule corresponding to sgRNA) and expressing the sgRNA encoded by sequence 4, and the positive plasmid was named PHSE401-LbPL1 (fig. 3).

2. In vitro transcription of sgRNA containing a target site for LbPL

Synthesizing a primer:

T7-LbPL-sgRNA-F：TAATACGACTCACTATAGGGGGTATTTATCGTGTGGTAC，

gRNA-Sc-R：AAAAGCACCGACTCGGTGCCA；

performing PCR amplification by using PHSE401-LbPL1 as a template, purifying an obtained PCR product by using a PCR purification kit (full-formula gold EP 101-02), wherein the PCR product is a DNA molecule containing a T7 promoter and an LbPL target sgRNA1, and synthesizing RNA (sgRNA-LbPL 1) containing the T7 promoter and the LbPL target sgRNA1 in vitro by using the full-formula gold T7 in-vitro transcription kit: ggguauuuaucgugugguacguuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu (seq id No. 10), and a PolyA tail was added to the 3' end of the sgRNA to improve mRNA stability.

The sgRNA-LbPL1 exists in the form of solution, the solute is the sgRNA-LbPL1, the solvent is water, and the concentration is 250 ng/mu L.

2. Site-specific editing of lycium barbarum LbPL gene by transforming purified Cas9 protein and in vitro transcribed sgRNA through particle gun method

The Cas9 protein and the sgRNA-LbPL1 obtained in the previous step are introduced into Ningxia wolfberry No. 7 by a gene gun transformation method. Taking a leaf disc of Ningqi No. 7 as a transformation receptor, obtaining a complete regeneration plant through tissue culture after transformation, carrying out molecular detection, and screening out mutants, wherein the specific steps are as follows:

1. silica-loaded Cas9 protein and sgRNA-LbPL1

Silica Au-MSN with a pore diameter of 10nm is selected as a medium, 20mg of Au-MSN is added into 5mL of Phosphate Buffer Solution (PBS) with pH7.4 for ultrasonic treatment, and then 7mg of purified Cas9 protein is added. The mixture was stirred at 22 ℃ for 24 hours, centrifuged at 12000rpm, the supernatant was discarded, and the pellet was spun up with PBS buffer to give a Cas9 protein-Au-MSN (10. Mu.g/. Mu.L) medium. mu.L of in vitro transcribed sgRNA-LbPL1 (250 ng/. Mu.L) was added to 10. Mu.L of Cas9 protein-Au-MSN (10. Mu.g/. Mu.L) medium, followed by 12.5. Mu.L of 2.5M CaCl ₂ And 5 μ L of 0.1M spermidine, centrifuged again at 5000rpm for 15s, the supernatant discarded, the pellet washed twice with 100% ethanol, and mRNA-coated Cas9 protein-loaded Au-MSN suspended with 5 μ L of 100% ethanol to form sgRNA-Cas9-Au-MSN complex.

2. Gene gun transformed wolfberry leaf disc

1) Taking Ningqi No. 7 seeds, after 3 percent NaClO sterilization, placing the seeds on an MS culture medium for germination, then cutting 2cm stem segments every two weeks, and cutting the stem segments to a 1/2MS culture medium for propagation.

2) Covering two layers of neutral filter paper on an induction culture medium (MS +1.0mg/L NAA +0.5 mg/L6-BA), cutting a sterile leaf of the medlar with the far axis end facing upwards, placing on the induction culture medium, and pre-culturing for 1 day.

3) Bombarding leaves by using a Bio-Rad PDS-1000/He gene gun, and loading 5 mu L of sgRNA-Cas9-Au-MSN complex on a sample loading membrane; bombarding by using a gene gun, wherein the bombardment distance of each bombardment is 6cm, the bombardment pressure is 1100psi, and the bombardment diameter is 2cm.

4) The bombarded leaf is placed at 25 deg.C for 2 days, and then cut into 9mm ² The squares of (4) are respectively inoculated on an induction medium, and are cultured for 2-4 weeks at 25 ℃ under illumination, and subcultured every two weeks.

5) The induced callus was subcultured to a differentiation medium (MS +0.1mg/L NAA +0.5 mg/L6-BA) to induce seedling formation.

6) The plantlets over 2cm were subcultured to rooting medium (MS +0.2mg/L NAA +0.01 mg/L6-BA). And obtaining the T0 generation gene edited Chinese wolfberry plants after 14-28 days.

3. Molecular detection

Extracting genomic DNA from the medlar plant seedlings with the edited T0 generation gene, and carrying out PCR/RE (Polymerase Chain Reaction/recovery diagnosis) experimental analysis by taking the DNA as a template. Meanwhile, DNA of wild medlar is set as a control. The PCR/RE analysis method refers to Shan, qi, et al, rapid and effective gene modification in rice and Brachypodium using genes molecular Plant (2013), and the restriction enzyme (PstI) was used in the PCR/RE analysis experiment because the recognition sequence (5 '-GGTACC-3') of restriction enzyme exists on the target fragment of the LbPL endogenous gene of Lycium barbarum.

Wherein, the primers used for PCR amplification are as follows:

LbPL-F：5’-CCTCCTTGCCTCCTCTAATCC-3’；

LbPL-R：5’-ACAGTTCTCCACCCGTAATGC-3’。

because the target sgRNA-1 is selected at the pstI enzyme cutting site, if the number of the gel running strips is two, the PCR amplification strip is cut, and the fact that the plant genome has no variation at the target point is suggested, the plant is not a knockout mutant; if the rubber running strip is one strip, the PCR amplification strip is not cut, the variation of the plant genome at the target point is suggested, and the plant is a homozygous mutant; if there are three running bands, it shows that one PCR amplified band is cut and one PCR amplified band is not cut, which implies that one strand of the plant genome is mutated at the target point, and one strand is not mutated, and the plant is heterozygous mutant.

The analysis result of the PCR/RE experiment shows that the LbPL gene target site in the medlar plant seedling is mutated after the gene is edited. Recovering and sequencing the band in the map, wherein the sequencing result shows that the target sites of the LbPL gene are mutated in a base insertion/deletion type;

the following is the result of a mutation, deleting a base according to the first site:

AATTTAGGGTATTTATCGTGTGGTACCGGAAATCC WT

AATTTAGGGTATTTATCGTGTG.TACCGGAAATCC pl。

the plant with the mutation is named as T0 generation medlar mutant plant pl.

506 leaf discs are bombarded to obtain 3662 medlar plants, and all the medlar plants are detected by a PCR/RE experiment, so that 52 mutants are found, and 36 mutants are homozygous mutants.

4. Identification of fruit maturity of pl homozygous mutant

And (3) selecting a homozygous mutant from the T0 generation medlar mutant plants pl obtained by the three screening steps to obtain the T0 generation medlar homozygous mutant plants pl.

The T0 generation medlar homozygous mutant plant pl and the wild type Ningqi No. 7 with the same size are planted in the Ningxia Ningning New castle town Song Yingcun field, all the plants are divided into two groups, and each group comprises 100 plants. The T0 generation medlar homozygous mutant plant pl and the wild type No. 7 are in the same mode of fertilization, watering, pruning and other farmland management, and fresh fruits are harvested at the same time in the 3 rd year. By comparing the weight of each fresh fruit, the yield of each plant, the color, the soluble solid matter, the lycium barbarum polysaccharide and the lycium barbarum betaine, no obvious difference is found.

But the T0 generation homozygous mutant plant pl was harvested from fresh fruit of wild type No. 7 (WT) under the same environmental conditions: after being placed in an incubator with 24 degrees and relative humidity of 20% for 15 days, the wild type is obviously more rotten than the mutant (as shown in figure 2).

Sequence listing

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gacaatgccg tcactccgaa gccggggact ctccggcacg cagtgattca gactgagcca 360

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ggtgatggag tgtccatatt cggtgggagc catgtttggg tggatcattg ttctttgtcc 660

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gataaaaaca tgcaagtaac tatagctttt aatcactttg gtgaaggtct tgtccaaaga 840

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Glu Val His Arg Ser Ile Asn Gly Ser Arg Arg Asn Leu Gly Tyr Leu

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His Ala Val Ile Gln Thr Glu Pro Leu Trp Ile Ile Phe Ala Arg Asp

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Met Val Ile Gln Leu Lys Glu Glu Leu Ile Met Asn Ser Phe Lys Thr

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Ile Asp Gly Arg Gly Ala Ser Val His Ile Ala Gly Gly Pro Cys Ile

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Asp Cys Lys Gln Gly Gly Asn Ala Met Val Arg Ser Ser Pro Ser His

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Ala Pro Asp Ile Arg Phe Ser Lys Glu Val Thr Lys His Glu Asp Ala

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Pro Glu Ser Glu Trp Lys Asn Trp Asn Trp Arg Thr Asp Gly Asp Leu

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Met Leu Asn Gly Ala Phe Phe Thr Arg Ser Gly Val Arg Thr Gly Ser

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Ser Ser Tyr Ala Lys Ala Ser Ser Leu Ser Ala Arg Pro Ser Ser Leu

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<211> 23

<212> DNA

<213> Artificial sequence

<400> 8

cggttagctg actgtgcaat tgg 23

<210> 9

<211> 23

<212> DNA

<213> Artificial sequence

<400> 9

actgtggatt attttcgcga ggg 23

<210> 10

<211> 103

<212> RNA

<213> Artificial sequence

<400> 10

ggguauuuau cgugugguac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60

cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103

<210> 11

<211> 1368

<212> PRT

<213> Artificial sequence

<400> 11

Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp

1365

Claims

1. A method for preparing storable fresh food Ningqi No. 7 Chinese wolfberry comprises the following steps: introducing Cas9 protein and specific sgRNA-LbPL1 into cells or tissues of the number 7 of the target medlar ningwolfberry to obtain storable fresh medlar;

the target sequence of the sgRNA-LbPL1 is a nucleic acid molecule shown as a sequence 7 in a sequence table;

the sgRNA-LbPL1 is (d 1) or (d 2) as follows:

(d1) RNA encoded by sequence 4 of the sequence listing;

(d2) A nucleic acid molecule shown as a sequence 10 in a sequence table;

the Cas9 protein and sgRNA-LbPL1 were introduced into the ningqi No. 7 by gene gun transformation as follows:

1) Preparation of silica-loaded Cas9 protein and sgRNA-LbPL1

Selecting 10 nm-aperture silicon dioxide Au-MSN as a medium, adding 20mg of Au-MSN into 5mL of Au-MSN with pH of 7.4Performing ultrasonic treatment in the phosphate buffer solution, and then adding 7mg of purified Cas9 protein; stirring the mixture at 22 ℃ for 24 hours, centrifuging at 12000rpm, removing supernatant, and spinning up the precipitate with PBS buffer solution to obtain Cas9 protein-Au-MSN medium; mu.L of in vitro transcribed sgRNA-LbPL1 was added to 10. Mu.L of Cas9 protein-Au-MSN medium, followed by 12.5. Mu.L of 2.5M CaCl ₂ And 5 μ L of 0.1M spermidine, centrifuging at 5000rpm for 15s, discarding the supernatant, washing the precipitate twice with 100% alcohol, and suspending the mRNA-coated Au-MSN loaded with Cas9 protein with 5 μ L of 100% alcohol to form sgRNA-Cas9-Au-MSN complex;

2) Gene gun transformed wolfberry leaf disc

1) Taking Ningqi No. 7 seeds, after 3 percent NaClO sterilization, placing the seeds on an MS culture medium for germination, then cutting 2cm stem sections every two weeks, and cutting the stem sections to a 1/2MS culture medium for propagation;

2) Covering two layers of neutral filter paper on an induction culture medium, shearing a sterile leaf of the Chinese wolfberry with the far-axis end upward, placing on the induction culture medium, and pre-culturing for 1 day;

3) Bombarding leaves by using a Bio-Rad PDS-1000/He gene gun, and loading 5 mu L of sgRNA-Cas9-Au-MSN complex on a sample loading membrane; bombarding by using a gene gun, wherein the bombardment distance of each bombardment is 6cm, the bombardment pressure is 1100psi, and the bombardment diameter is 2cm;

4) The bombarded leaf is placed at 25 deg.C for 2 days, and then cut into 9mm ² Respectively inoculating the square blocks on an induction culture medium, and culturing for 2-4 weeks at the temperature of 25 ℃ under illumination, wherein subculture is carried out once every two weeks;

5) Subculturing the induced callus on a differentiation culture medium, and inducing the callus into seedlings;

6) And (3) subculturing the plantlets with the length of more than 2cm to a rooting culture medium, and obtaining the Chinese wolfberry plants with the edited T0 generation genes after 14-28 days.