CN114438104A

CN114438104A - SlGRAS9 gene for regulating sugar content of tomato fruits and application of SlGRAS9 gene in cultivation of tomatoes with high sugar content

Info

Publication number: CN114438104A
Application number: CN202210281557.1A
Authority: CN
Inventors: 李正国; 时源; 刘豫东; 梁琴; 成玉林
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-05-06
Anticipated expiration: 2042-03-21
Also published as: CN114438104B

Abstract

The invention discloses a SlGRAS9 gene for regulating and controlling sugar content of tomato fruits and application thereof in cultivating tomatoes with high sugar content. The nucleotide sequence of the SlGRAS9 gene is shown in SEQ ID NO. 1. According to the invention, by using a CRISPR/cas9 gene editing technology, an endogenous gene SlGRAS9 of a tomato is knocked out from the tomato, the contents of glucose, fructose and sucrose in the knocked-out tomato fruit are obviously increased, and the contents of malic acid and citric acid are obviously reduced.

Description

SlGRAS9 gene for regulating sugar content of tomato fruits and application of SlGRAS9 gene in cultivation of tomatoes with high sugar content

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a SlGRAS9 gene for regulating and controlling sugar content of tomato fruits and application thereof in cultivation of tomatoes with high sugar content.

Background

Fruits and vegetables are used as an important component of dietary balance and are rich in various nutrients beneficial to human health, including sugars, vitamins, minerals, antioxidants and the like. The quality of the fruits and vegetables is closely related to the economic value of the fruits and vegetables, and mainly comprises aspects of appearance, mouthfeel, flavor, nutrient components, texture and the like, so the research on the quality improvement of the fruits and vegetables has important application value.

The carbohydrate is an important energy source substance and is a main source of heat energy required by all organisms for maintaining life activities, and the energy supplied by the carbohydrate in the diet accounts for about 60-70% of the total heat energy required by the human body. The carbohydrate is a main factor influencing the flavor and the nutritional value of the fruits and the vegetables, and is one of important indexes for measuring the quality and the economic value of the fruits and the vegetables.

Tomatoes are important economic crops, are deeply favored by consumers due to fresh fruit flesh, rich nutrition and unique flavor, and are widely planted as fresh fruits and vegetables in the world. The quality of tomato fruits is mainly divided into two aspects of appearance quality and intrinsic flavor. The appearance quality includes fruit shape, size, color, etc., while the intrinsic flavor is mainly determined by sweetness, acidity, pulp hardness and juice content of the fruit. The contents of sugar and organic acid components are closely related to the flavor quality of the tomato fruits, and the unique flavor of the tomato fruits is created by the proper sugar-acid ratio. For tomato breeders, the key to improving the flavor of tomato fruits lies in increasing the sweetness of the fruits in taste. At present, the traditional breeding method is long in time consumption and unstable in effect, and usually has great blindness and unpredictability.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a SlGRAS9 gene for regulating and controlling sugar content of tomato fruits and application thereof in cultivating tomatoes with high sugar content, endogenous genes SlGRAS9 of the tomatoes are knocked out in the tomatoes by a CRISPR/cas9 gene editing technology, so that the tomato fruits with high sugar content are obtained, the flavor of the fruits is obviously improved, and the quality and the economic value of the tomato fruits are greatly improved.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a SlGRAS9 gene for regulating and controlling the sugar content of tomato fruits, wherein the nucleotide sequence of the SlGRAS9 gene is shown as SEQ ID No. 1.

Furthermore, the SlGRAS9 gene can also be a nucleotide sequence which has more than 80 percent of homology with the sequence shown as SEQ ID NO.1 and encodes the same functional protein.

The amino acid sequence of the protein coded by the SlGRAS9 gene is shown in SEQ ID NO. 2.

A CRISPR/cas9 base editor comprising a vector and a sgRNA that recognizes a SlGRAS9 gene target sequence.

Further, the nucleotide sequence of the vector is shown as SEQ ID NO. 3.

A recombinant agrobacterium comprising the CRISPR/cas9 base editor described above.

A preparation for increasing sugar content of tomato fruit comprises active ingredients capable of inhibiting SlGRAS9 gene.

The SlGRAS9 gene, a base editor or recombinant agrobacterium is applied to the improvement of the sugar content of tomato fruits.

The SlGRAS9 gene, the base editor or the recombinant agrobacterium can be applied to cultivation of high-sugar tomato varieties or improvement of tomato germplasm resources.

The invention has the beneficial effects that:

according to the invention, by using a CRISPR/cas9 gene editing technology, an endogenous gene SlGRAS9 of a tomato is knocked out from the tomato, the contents of glucose, fructose and sucrose in the knocked-out tomato fruit are obviously increased, and the contents of malic acid and citric acid are obviously reduced. In addition, the invention adopts CRISPR/cas9 gene editing technology, efficiently and stably improves the contents of glucose, fructose and sucrose in tomato fruits in a genetic manner, greatly enhances the sweet taste of the tomato fruits in taste, and further improves the fruit quality. Provides gene resources and strategies for tomato germplasm resource innovation and aims to better meet the requirements of consumers.

Drawings

FIG. 1 is a constructed Cas9/pORE CRISPR/Cas9 expression vector;

FIG. 2 shows the positive identification result of the CRISPR/cas9 gene knockout plant of SlGRAS9 gene;

FIG. 3 shows editing of SlGRAS9 knockout lines;

FIG. 4 is a detection result of the contents of sucrose, fructose, glucose, malic acid and citric acid in fruits of wild tomatoes and tomatoes in a red-ripe stage of a SlGRAS9 gene knockout line;

FIG. 5 shows the results of the detection of soluble solid content, acidity and sugar-acid ratio in the red-ripe fruits of wild tomatoes and the tomato 9 knockout line tomato.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1 construction of CRISPR/cas9 Gene knockout System for SlGRAS9 Gene

1. Design target sequence of SlGRAS9 gene

Target site design was performed using the http:// cas9.cbi. pku. edu. cn/index. jsp website. The sequences, positions, GC contents, potential off-target sites and other relevant information of all candidate targets can be evaluated in the target design result, and targets with GC contents in the range of 45% -70%, positions in the first 2/3 region (after ATG but not on the last exon) of the CDS of the gene and at least three base mismatching with any other positions in the genome are selected.

2. Construction of sgRNA expression cassette of SlGRAS9 Gene

(1) An expression cassette of the sgRNA of the SlGRAS9 gene is obtained by using an Overlapping PCR method. First, a first round of PCR was performed using gRNA expression vector U26 as template, using primertstar hi-fi enzyme and primer pair 1: U26-HindIII-F:

and U26-target-R1:

(vector linker sequence underlined) and primer pair 2: U26-target-F2:

and U26-NheI-R:

(vector linker sequence is underlined) PCR amplification was performed to introduce the target sequence downstream of the U6-26 promoter and upstream of the sgRNA sequence, the amplification scheme of which is shown in table 1.

TABLE 1 first round PCR amplification System

The amplification procedure is as follows: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 5 min.

(2) And then carrying out second round PCR, and amplifying by using two PCR products of the first round as templates and U26-HindIII-F and U26-target-R1 to obtain a complete sgRNA expression cassette containing a promoter, a target and sgRNA.

The second round of PCR was performed using the two PCR products of the first round as templates, using PrimerSTAR Hi Fidelity enzyme and U26-HindIII-F and U26-target-R1 amplification primers, and the reaction system is shown in Table 2:

TABLE 2 second round PCR amplification System

The PCR amplification procedure is as follows: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 5 min.

3. Cloning of sgRNA expression cassette into Cas9/pORE vector

The Cas9/pORE vector is subjected to double enzyme digestion by HindIII and NheI, then the sgRNA expression cassette is connected to a Cas9/pORE final vector by adopting a homologous recombination technology, wherein the nucleotide sequence of the Cas9/pORE final vector is shown as SEQ ID No.3, and the constructed vector map is shown as figure 1.

Example 2 Effect of knockout of SlGRAS9 Gene on sugar content in tomato fruit

1. Screening of engineering bacteria

(1) The SlGRAS9 gene CRISPR/cas9 knockout system constructed in the example 1 is transformed into escherichia coli DH5 alpha, screening is carried out on a plate containing 50 mug/mL kanamycin, a monoclonal strain is selected, then PCR amplification identification is carried out on a colony of the monoclonal strain, and a positive strain is screened by sequencing;

(2) shaking the positive strain to extract plasmid, transforming Agrobacterium tumefaciens GV3101, screening on a plate culture medium containing 50 ug/mL kanamycin and 100 ug/mL rifampicin, selecting single clone to perform colony PCR verification, and obtaining the successfully verified strain as the engineering bacterium of the recombinant expression vector.

2. Transformation of tomato explants by leaf disc method with Agrobacterium containing recombinant plasmid

(1) Seed disinfection

Putting wild tomato seeds into a sterile container, pouring a proper amount of ethanol water solution with the volume percentage of 70% to soak the seeds for 30s, and disinfecting the surfaces of the seeds; pouring out the ethanol water solution, washing the seeds twice with sterile water, pouring in a sodium hypochlorite solution with 5% of available chlorine, soaking for 15min, and continuously shaking during the soaking period to ensure that each seed is sufficiently sterilized; washing the seeds with sterile water for 3-4 times, then completely draining the water in the seeds, transferring the seeds to a seed culture medium, and putting the seeds into an illumination incubator, wherein the culture conditions are as follows: illuminating for 14 hours at the temperature of 25 ℃; dark 10 hours, temperature 20 ℃; illumination intensity, 250. mu. mol. m^-2·s^-1(ii) a Relative humidity, 80%; culturing for 8-10 days.

(2) Explant preculture

After about 9-10 days of seed germination, when the true leaves of tomato seedlings are about to grow out, in order to obtain the optimum period of explants, the seedlings are placed on sterile filter paper, cotyledons and hypocotyls are cut into sections by using sterilized surgical blades, and the sections are carefully transferred to a KCMS pre-culture medium by using sterile tweezers to culture in dark for one day.

(3) Agrobacterium infection explant

At 20mL LB (containing 20. mu.L of 50. mu.g/mL kanamycin and 40. mu.L of 100. mu.g/mL rifampicin) was added with 100. mu.L of the preserved Agrobacterium glycerol, incubated overnight at 28 ℃ and 230rpm until the absorbance OD₆₀₀About 0.8 to about 1.0. Sucking 1mL of bacterial liquid, centrifuging at 5000rpm for 5min, discarding supernatant, washing precipitate with KCMS liquid culture medium, and diluting the bacterial liquid to OD₆₀₀About 0.1. And (3) dropwise adding a drop of agrobacterium diluent to the wound of the explant by using a pipette gun, sealing the plate, and placing the plate on a KCMS pre-culture medium for dark culture for 2 days without sucking out the bacterial liquid.

(4) Differentiation and rooting of explants

After 2 days of co-culture of explants and Agrobacterium, the explants were carefully transferred from KCMS pre-culture medium to primary screen 2Z medium with sterile forceps and cultured, replacing the primary screen medium every 15 days. When differentiated shoots grew out of the explants, the explants were transferred to 1Z medium for culture, and the medium was changed every 15 days. After two times of culture on a 1Z culture medium, the differentiated callus grows buds with independent main stems, is cut off and inserted into a rooting culture medium ENR, and is transplanted after rooting.

(5) Identification of transgenic Positive plants

Transgenic T0 generation seedlings utilize CRISPR/Cas9 vector detection primer Cas9-F/Cas-R to confirm whether the seedlings are transgenic positive plants (Cas9-F: 5'-TTAGGTTTACCCGCCAATA-3'; Cas-R: 5'-GAGTAGACAAGTGTGTCGTGCT-3'), and the detection result is shown in figure 2.

As shown in FIG. 2, the genomic DNA of T0 seedlings was used as a template for PCR amplification, and the transgenic positive plants were identified as 500bp fragments detected by electrophoresis. And then, taking the genome DNA of T0 generation seedlings as a template, designing a detection primer containing a target site to perform PCR amplification (GRAS9-Cas9check-F: 5'-CTGAATCATTTTATTAATGCCT-3'; GRAS9-Cas9 check-R: 5'-GATAAAACCCCGTTCGTG-3'), recovering a target band after agarose gel electrophoresis, constructing a purified PCR product on a pEASY-Blunt-Zero cloning vector to perform monoclonal sequencing, performing 20 monoclonal sequencing on each corresponding transgenic positive plant, and comparing a sequencing result with a wild type sequence to obtain the editing condition of the T0 generation plant.

The T0 generation seed which is heterozygous in sequence is screened by kanamycin and then is continuously sown, the editing condition of the T1 generation plant is analyzed (the method is the same as the method for editing and detecting the T0 generation plant), 2-3 homozygous lines with different editing forms are selected, the seeds are sown to T2 after screened by kanamycin and are used for subsequent research, and the mutation modes of the selected lines are four base deletion and one base insertion respectively, as shown in figure 3.

3. Determination of sugar and organic acid content of SlGRAS9 knockout plant fruit

After obtaining the homozygous mutant, collecting fruit samples of wild type and gene knockout plants in the red mature period for sugar content determination. A 0.1g sample of the fruit was weighed and the tomato fruit soluble sugars and organic acids were extracted with chromatographically pure (Ar) methanol as extractant, setting 3 biological replicates.

The specific process is as follows: samples were derivatized using methoxyamine hydrochloride and BSTFA. The determination was performed using shimadzu GC 2010 pro. The chromatographic parameters are SPL temperature of 250 ℃, the split ratio of 10: 1; the flow rate of the chromatographic column is 1mL/min, the temperature rise program is that the initial temperature is 130 ℃, the temperature rises to 185 ℃ at 5 ℃/min, the temperature rises to 190 ℃ at 0.5 ℃/min, the temperature rises to 300 ℃ at 8 ℃/min, and the temperature is kept for 10 min. The detector temperature was 320 ℃, the hydrogen flow rate was 40mL/min, the dry air flow rate was 400mL/min, and the nitrogen flow rate was 40 mL/min. The results of the glucose, fructose, sucrose, malic acid and citric acid content measurements are shown in FIG. 4.

In fig. 4, WT is wild type tomato and CR #1 and CR #2 are two SlGRAS9 knockout lines tomato. According to the detection results of fig. 4, after the SlGRAS9 gene is knocked out, the Sucrose (Sucrose), Fructose (Fructose) and Glucose (Glucose) contents in tomato fruits are remarkably increased, and the Malic acid (Malic acid) and Citric acid (Citric acid) contents are remarkably reduced.

4. Determination of soluble solid content of SlGRAS9 knockout fruit

Picking fruits in red ripe period of wild type and SlGRAS9 knockout plants, setting 3 biological repetitions, cleaning the surfaces of the fruits by distilled water, wiping the fruits dry, putting the tomatoes into gauze, forcibly extruding, extruding juice into a clean beaker to obtain crude extract of soluble solids, and measuring the crude extract by using a handheld refractometer, wherein the measurement results of the content, acidity and sugar-acid ratio of the soluble solids are shown in figure 5.

In FIG. 5, WT is a wild type tomato, and CR #1 is a tomato of SlGRAS9 knockout line. As shown in fig. 5, the soluble solid content (Total soluble solid) of tomato fruits of SlGRAS9 knockout line is significantly increased, the acidity (Acid) is decreased, and the sugar-Acid Ratio (Ratio) is significantly increased.

In conclusion, the SlGRAS9 gene in the tomato is knocked out by using the gene editing technology, so that the sugar content of the tomato fruit can be obviously improved, the acidity of the tomato fruit can be reduced, the sweet taste of the tomato fruit in taste is greatly enhanced, and the fruit quality is improved. Provides good reference and reference for tomato germplasm resource innovation and aims to better meet the requirements of consumers.

Sequence listing

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ccgtcggctc gatggtccag caagctacgg ccaagatcga gcgcgacagc gtgcaactgg 2640

ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg ttcgcgtcgt ctcgaacagg 2700

aggcggcagg tttggcgaag tcgatgacca tcgacacgcg aggaactatg acgaccaaga 2760

agcgaaaaac cgccggcgag gacctggcaa aacaggtcag cgaggccaag caagccgcgt 2820

tgctgaaaca cacgaagcag cagatcaagg aaatgcagct ttccttgttc gatattgcgc 2880

cgtggccgga cacgatgcga gcgatgccaa acgacacggc ccgctctgcc ctgttcacca 2940

cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa ggtcattttc cacgtcaaca 3000

aggacgtgaa gatcacctac accggcgtcg agctgcgggc cgacgatgac gaactggtgt 3060

ggcagcaggt gttggagtac gcgaagcgca cccctatcgg cgagccgatc accttcacgt 3120

tctacgagct ttgccaggac ctgggctggt cgatcaatgg ccggtattac acgaaggccg 3180

aggaatgcct gtcgcgccta caggcgacgg cgatgggctt cacgtccgac cgcgttgggc 3240

acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct ggaccgtggc aagaaaacgt 3300

cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct gtttgctggc gaccactaca 3360

cgaaattcat atgggagaag taccgcaagc tgtcgccgac ggcccgacgg atgttcgact 3420

atttcagctc gcaccgggag ccgtacccgc tcaagctgga aaccttccgc ctcatgtgcg 3480

gatcggattc cacccgcgtg aagaagtggc gcgagcaggt cggcgaagcc tgcgaagagt 3540

tgcgaggcag cggcctggtg gaacacgcct gggtcaatga tgacctggtg cattgcaaac 3600

gctagggcct tgtggggtca gttccggctg ggggttcagc agccagcgct ttactgagat 3660

cctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 3720

tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 3780

agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 3840

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 3900

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 3960

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 4020

gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 4080

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 4140

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 4200

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 4260

ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 4320

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 4380

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 4440

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 4500

tggtcatgag attatcaaaa aggatcttca cctagatcct tttggatctc ctgtggttgg 4560

catgcacata caaatggacg aacggataaa ccttttcacg cccttttaaa tatccgatta 4620

ttctaataaa cgctcttttc tcttaggttt acccgccaat atatcctgtc aaacactgat 4680

agtttaaact gaaggcggga aacgacaatc tgctagtgga tctcccagtc acgacgttgt 4740

aaaacgggcg ccccgcggaa agcttgtaat acgactcact atagggagac ccaagctggc 4800

tagcgtttaa acttaagctg atccactagt cctgcaggtc aacatggtgg agcacgacac 4860

acttgtctac tccaaaaata tcaaagatac agtctcagaa gaccaaaggg caattgagac 4920

ttttcaacaa agggtaatat ccggaaacct cctcggattc cattgcccag ctatctgtca 4980

ctttattgtg aagatagtgg aaaaggaagg tggctcctac aaatgccatc attgcgataa 5040

aggaaaggcc atcgttgaag atgcctctgc cgacagtggt cccaaagatg gacccccacc 5100

cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg 5160

atgtgataac atggtggagc acgacacact tgtctactcc aaaaatatca aagatacagt 5220

ctcagaagac caaagggcaa ttgagacttt tcaacaaagg gtaatatccg gaaacctcct 5280

cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa aggaaggtgg 5340

ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg cctctgccga 5400

cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 5460

aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc 5520

acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 5580

gaggacctcg acctcaacac aacatataca aaacaaacga atctcaagca atcaagcatt 5640

ctacttctat tgcagcaatt taaatcattt cttttaaagc aaaagcaatt ttctgaaaat 5700

tttcaccatt tacgaacgat agaattcgcc atggccccaa agaaaaagag aaaggttgat 5760

tacaaagacc acgacggaga ctacaaagac cacgacattg attataaaga tgatgatgat 5820

aaaggaacga tggacaaaaa gtatagcatc ggtctggata ttggaactaa ctccgtcggc 5880

tgggctgtaa tcaccgacga atacaaggtc ccgtcaaaaa agttcaaggt attgggtaac 5940

acagatcgtc actctatcaa aaagaatctc attggagctc tgttgttcga cagcggcgaa 6000

acagctgagg ccactagact gaagcgcacc gccagacgcc gttacacgag gagaaagaac 6060

agaatctgct acttgcaaga aatattctca aacgagatgg ccaaagtgga cgattcgttc 6120

tttcataggt tagaagagag tttccttgtt gaagaggata aaaagcacga aagacatccg 6180

atatttggaa acatcgtgga cgaagttgct tatcacgaga agtaccccac gatctatcat 6240

ctgcgtaaaa agttggtgga ctcgacagat aaggccgacc tcaggttaat ataccttgca 6300

ctggcgcaca tgatcaaatt cagaggccat tttctgattg aaggtgacct gaaccctgac 6360

aatagtgatg tggacaaact cttcattcaa ttagttcaga cctacaatca actgtttgaa 6420

gagaacccta tcaacgcttc aggagttgac gctaaggcca tccttagtgc gagactgagc 6480

aaatcccgcc gtctcgaaaa cttaatcgca cagttgcctg gagagaaaaa gaacggtttg 6540

ttcggaaatc tcattgcgtt gtcactcgga ctcacgccaa acttcaagtc taacttcgat 6600

ttggcagaag acgcgaaact gcaactgagc aaagacacat atgacgatga cctcgataac 6660

ctcttagctc agatcggcga tcaatacgcc gacttgttcc tcgctgccaa aaatctgtcg 6720

gacgctatac ttctgagtga tatcttgcgc gtcaacacag aaattactaa ggctcctctg 6780

tcggccagta tgataaaacg ctatgacgaa caccatcagg atttgacatt gctcaaagcc 6840

ctcgtgcgtc aacagctccc agaaaagtac aaggagattt tctttgatca gtccaagaat 6900

ggctacgcag gttatataga cggtggagcg tcgcaagaag agttctacaa gttcatcaag 6960

ccaatattag aaaagatgga cggcacggaa gagttacttg ttaagctgaa tcgtgaggac 7020

ctgttgcgta aacagaggac attcgataac ggatcaattc cgcaccaaat acatcttggc 7080

gaactgcacg ctatcctcag gagacaagag gacttctacc cctttttaaa ggataaccgt 7140

gaaaagatcg agaaaatcct gactttcagg attccttact atgtcggccc actggctcgt 7200

ggtaatagca ggtttgcctg gatgaccagg aagtccgaag agacaattac tccgtggaac 7260

ttcgaagagg tggttgataa aggagcatca gcgcagtctt tcatagaacg catgacaaat 7320

tttgacaaga acttaccgaa tgagaaggtc cttcccaaac actcactcct ctacgaatac 7380

ttcacagtat acaacgagct cactaaagtc aagtacgtaa ccgagggtat gcgcaaaccc 7440

gctttcctgt ctggagagca gaaaaaggcc atcgtggacc ttctgttcaa gacaaaccgt 7500

aaggtcactg taaagcaact caaggaagac tacttcaaaa agatagagtg tttcgattca 7560

gtggaaatct ctggcgttga ggacagattt aacgcttcct tgggtactta ccacgatttg 7620

ctcaagatca ttaaagataa ggacttcctc gacaacgaag agaacgaaga tatcttagag 7680

gacatagttc tcacccttac gctgtttgaa gatagagaga tgattgaaga gcgcctgaag 7740

acttatgctc atttgttcga tgacaaagtc atgaagcaac tgaaacgccg taggtacacc 7800

ggctggggta gattatcgcg caaacttatt aatggtataa gggacaagca gtcgggaaaa 7860

acgatattgg actttctcaa gagtgatggt ttcgccaaca gaaattttat gcaactcata 7920

cacgatgaca gcttaacatt caaggaagat atccaaaaag cacaggtgtc gggacagggc 7980

gacagtttgc acgaacatat tgctaacctc gccggctccc cggcgataaa aaagggtatc 8040

cttcagactg tgaaagtcgt agatgaactg gtgaaggtta tgggtcgtca taaacccgag 8100

aacatagtta tcgaaatggc tagggagaat caaacaactc agaagggaca gaaaaactca 8160

agagaacgca tgaagcgcat tgaagagggt atcaaagagc ttggcagtca aatcctgaag 8220

gaacaccctg tcgagaacac gcaacttcag aacgaaaaat tgtacctcta ctatctgcag 8280

aatggtagag atatgtacgt agaccaagaa ttggatatta accgcctctc agattacgac 8340

gtggatcata tagttccgca gtcattcttg aaggatgact ctatcgacaa caaagtcctc 8400

acaagatcag acaagaaccg cggaaaatca gataatgtac cctctgaaga ggtggttaaa 8460

aagatgaaaa actactggag acagttactt aacgctaagt tgatcacgca aagaaagttc 8520

gataacctca caaaggctga acgcggcggt ttaagcgagc ttgacaaggc cggtttcata 8580

aaacgtcagt tagtcgaaac caggcaaatt acgaaacacg tagcccaaat attggattcc 8640

cgcatgaaca ctaaatacga tgaaaatgac aagctcatcc gtgaggtcaa agtaattacc 8700

ctgaaaagca agttggtgtc cgacttcaga aaggatttcc agttctacaa agttcgcgaa 8760

atcaacaact accaccatgc acatgacgct tacctgaacg cagtcgtagg cactgcgtta 8820

attaaaaagt accctaaact ggaatctgag ttcgtgtacg gtgactataa agtgtacgat 8880

gttagaaaga tgatcgctaa aagcgaacag gagattggaa aggctaccgc caagtatttc 8940

ttttactcca acatcatgaa tttctttaag accgaaatca cgttagcaaa tggcgagata 9000

cgtaaaaggc cacttatcga aacaaacgga gaaactggcg agatagtgtg ggacaagggt 9060

agagattttg ccactgtccg caaagtactg tcgatgccgc aagtgaatat cgttaaaaag 9120

accgaagttc aaacgggagg cttcagcaaa gagtccatcc tgcccaagcg taacagtgat 9180

aaattgatag ctaggaaaaa ggactgggat cctaaaaagt atggtggatt cgacagccca 9240

actgtcgcat actccgtatt ggtggttgcg aaagtcgaaa aaggaaagag caaaaagctc 9300

aagtccgtaa aagagctgtt gggcattacc ataatggaaa gatcatcttt cgagaagaat 9360

cctatcgatt ttctggaagc caagggatat aaagaggtca aaaaggacct cataatcaag 9420

ttaccaaaat acagtctgtt cgaattggag aacggcagaa aacgcatgct tgcatcagcg 9480

ggtgaactgc aaaagggaaa tgagttagca cttccttcta aatacgtcaa cttcctgtat 9540

ttggcgtcac actacgaaaa actgaagggc tctccagaag ataacgagca aaagcagtta 9600

tttgtggaac agcacaaaca ttaccttgac gaaattatag agcaaatctc ggagttcagt 9660

aagagagtga ttttggctga cgccaatctt gataaagttc tgtctgctta caacaagcac 9720

cgtgataaac cgattaggga acaggccgag aacatcatac atctcttcac actcactaac 9780

cttggtgcac ccgcagcgtt caaatatttt gacaccacga tagatcgtaa gaggtacacc 9840

agcacgaaag aagttttgga cgcgacactc atccatcaat caatcacggg cctgtacgag 9900

accagaatcg acctgtccca gctcggtggc gactagcggc cgcatcgata ctgcaggagc 9960

tcggtacctt ttactagtga tatccctgtg tgaaattgtt atccgctacg cgtgatcgtt 10020

caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta 10080

tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt 10140

tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag 10200

aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac 10260

tagatcccat gggaagttcc tattccgaag ttcctattct ctgaaaagta taggaacttc 10320

agcgatcgca gacgtcggga tcttctgcaa gcatctctat ttcctgaagg tctaacctcg 10380

aagatttaag atttaattac gtttataatt acaaaattga ttctagtatc tttaatttaa 10440

tgcttataca ttattaatta atttagtact ttcaatttgt tttcagaaat tattttacta 10500

ttttttataa aataaaaggg agaaaatggc tatttaaata ctagcctatt ttatttcaat 10560

tttagcttaa aatcagcccc aattagcccc aatttcaaat tcaaatggtc cagcccaatt 10620

cctaaataac ccacccctaa cccgcccggt ttcccctttt gatccatgca gtcaacgccc 10680

agaatttccc tatataattt tttaattccc aaacacccct aactctatcc catttctcac 10740

caaccgccac atagatctat cctcttatct ctcaaactct ctcgaacctt cccctaaccc 10800

tagcagcctc tcatcatcct cacctcaaaa cccaccgggg ccggccatga ttgaacaaga 10860

tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 10920

acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggaggcc 10980

ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaacttcaag acgaggcagc 11040

gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 11100

tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 11160

tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 11220

gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 11280

tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 11340

cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt 11400

cgtgactcat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 11460

attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 11520

ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 11580

tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 11640

aggcgcgcc 11649

Claims

1. A SlGRAS9 gene for regulating sugar content of tomato fruits is characterized in that the nucleotide sequence of the SlGRAS9 gene is shown as SEQ ID No. 1.

2. The SlGRAS9 gene for regulating sugar content of tomato fruits as claimed in claim 1, wherein the SlGRAS9 gene is a nucleotide sequence which has more than 80% homology with the sequence shown as SEQ ID No.1 and encodes the same functional protein.

3. The protein encoded by the SlGRAS9 gene of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 2.

4. A base editor, comprising a vector and a sgRNA that recognizes a target sequence of a SlGRAS9 gene.

5. The base editor of claim 4, wherein the vector has a nucleotide sequence as shown in SEQ ID No. 3.

6. A recombinant Agrobacterium comprising the base editor of claim 4.

7. A preparation for increasing sugar content of tomato fruits, which comprises an active ingredient capable of inhibiting expression of SlGRAS9 gene or the base editor of claim 4.

8. Use of the SlGRAS9 gene of claim 1, the base editor of claim 4, or the recombinant agrobacterium of claim 6 to increase sugar content of tomato fruits.

9. Use of the SlGRAS9 gene of claim 1, the base editor of claim 4, or the recombinant agrobacterium of claim 6 for breeding high sugar tomato variety or improvement of germplasm resources of tomato.