CN114438104B

CN114438104B - SlGRAS9 gene for regulating and controlling sugar content of tomato fruits and application of SlGRAS9 gene in cultivation of tomatoes with high sugar content

Info

Publication number: CN114438104B
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: 2023-06-20
Anticipated expiration: 2042-03-21
Also published as: CN114438104A

Abstract

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

Description

SlGRAS9 gene for regulating and controlling 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 the 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 saccharides, vitamins, minerals, antioxidants and the like. The quality of the fruits and vegetables is closely related to the economic value thereof, mainly comprising aspects of appearance, mouthfeel, flavor, nutritional ingredients, texture and the like, so that research on improving the quality of the fruits and vegetables is of great application value.

Sugar compounds are important energy source substances, are main sources of heat energy required by all organisms to sustain life activities, and the energy supplied by sugar in diet accounts for 60% -70% of the total heat energy required by human body. Sugar compounds are main factors influencing the flavor and the nutritional value of fruits and vegetables, and are one of important indexes for measuring the quality and the economic value of fruits and vegetables.

Tomato is an important commercial crop, and is widely planted worldwide as a fresh fruit and vegetable because of delicious fruit quality, rich nutrition and unique flavor. The quality of tomato fruits is largely divided into two aspects, namely appearance quality and intrinsic flavor. Appearance quality includes fruit shape, size, color, etc., while intrinsic flavor is primarily determined by the sweetness, acidity, pulp hardness, and juice content of the fruit. The content of sugar and organic acid components is closely related to the flavor quality of the tomato fruits, and the proper sugar-acid ratio brings about the unique flavor of the tomato fruits. For tomato breeders, the key to improving tomato fruit flavor is how to increase the sweetness on the fruit mouthfeel. At present, the traditional breeding method is long in time consumption and unstable in effect, and quite large blindness and unpredictability often exist.

Disclosure of Invention

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

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

a nucleotide sequence of the SlGRAS9 gene for regulating and controlling the sugar content of tomato fruits 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 codes the same functional protein.

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

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

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

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

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

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

The SlGRAS9 gene, the base editor or the recombinant agrobacterium tumefaciens is applied to cultivation of tomato varieties with high sugar content in fruits or improvement of tomato germplasm resources.

The invention has the beneficial effects that:

according to the invention, through CRISPR/cas9 gene editing technology, the endogenous gene SlGRAS9 of the tomato is knocked out in the tomato, the glucose, fructose and sucrose content in the knocked-out tomato fruit is obviously increased, and the malic acid and citric acid content is obviously reduced. In addition, the CRISPR/cas9 gene editing technology is adopted, so that the content of glucose, fructose and sucrose in the tomato fruits can be improved efficiently and stably, and the sweet taste of the tomato fruits is greatly enhanced, thereby improving the quality of the fruits. Provides genetic resources and strategies for innovation of tomato germplasm resources, and aims to better meet the demands of consumers.

Drawings

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

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

FIG. 3 is an edit of a SlGRAS9 knockout line;

FIG. 4 shows the results of detection of sucrose, fructose, glucose, malic acid and citric acid content in fruits of wild type tomato and SlGRAS9 knockout strain in red ripe stage;

FIG. 5 shows the results of detection of the content of soluble solids, acidity and sugar acid ratio in fruits of wild type tomato and the SlGRAS9 knockout strain in red ripe stage.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate 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 all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

EXAMPLE 1 construction of a SlGRAS9 Gene CRISPR/cas9 Gene knockout System

1. Design of target sequence of SlGRAS9 Gene

Target site design was performed using the http:// cas9.Cbi. Pku. Edu. Cn/index. Jsp website. The sequence, position, GC content, potential off-target position and other relevant information of all candidate targets can be evaluated in the target design result, and the target with GC content in the range of 45% -70% and position in the front 2/3 region of the gene CDS (after ATG but not on the last exon) which is not matched with at least three bases in any other position in the genome can be selected.

2. Construction of sgRNA expression cassette of SlGRAS9 Gene

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

and U26-target-R1:

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

and U26-NheI-R: />

PCR amplification was performed (underlined as vector linker sequence) to introduce the target sequence downstream of the U6-26 promoter and upstream of the sgRNA sequence, the amplification system 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 1min; denaturation at 98℃for 10s, annealing at 55℃for 15s, elongation at 72℃for 1min,35 cycles; extending at 72℃for 5min.

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

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

TABLE 2 second round PCR amplification System

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

3. Cloning of the sgRNA expression cassette into Cas 9/port vector

The Cas9/pORE vector is cut by double enzymes of HindIII and NheI, and then the sgRNA expression cassette is connected to the 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 the 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 flat plate containing 50 mug/mL of kanamicin, monoclonal strains are selected, then colony of the monoclonal strains is subjected to PCR amplification identification, and positive strains are screened by sequencing;

(2) And (3) extracting plasmids from positive strains by shaking, transforming agrobacterium tumefaciens GV3101, screening on a flat-plate culture medium containing 50 mug/mL of kanamycin and 100 mug/mL of rifampicin, and selecting monoclonal to perform colony PCR verification, wherein the strains after verification are engineering bacteria of the recombinant expression vector.

2. Transformation of tomato explants by leaf disc method using agrobacterium containing recombinant plasmid

(1) Seed disinfection

Placing the wild tomato seeds into a sterile vessel, pouring an appropriate amount of 70% ethanol water solution by volume percent into the vessel, soaking for 30s, and sterilizing the surfaces of the seeds; pouring out ethanol water solution, washing the seeds twice with sterile water, pouring sodium hypochlorite solution with 5% of available chlorine, soaking for 15min, and continuously shaking during the soaking to ensure that each seed is sufficiently disinfected; washing 3-4 times with sterile water, pouring water from the seeds, transferring the seeds to a seed culture medium, and placing the seeds into an illumination incubator under the culture conditions that: illuminating for 14 hours at 25 ℃; dark for 10 hours at 20 ℃; the light intensity was 250. Mu. Mol.m ^-2 ·s ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Relative humidity, 80%; culturing for 8-10 days.

(2) Pre-culture of explants

After germination of the seeds for about 9-10 days, the seedlings were placed on sterile filter paper for optimal period of explant growth immediately before the true leaves of tomato seedlings were grown, the cotyledons and hypocotyls were cut with sterilized surgical blades and carefully transferred to KCMS preculture medium with sterile forceps for dark culture for one day.

(3) Agrobacterium infection explant

100. Mu.L of stored Agrobacterium were added to 20mL of LB (containing 20. Mu.L of 50. Mu.g/mL kanamycin and 40. Mu.L of 100. Mu.g/mL rifampicin), and incubated overnight at 28℃at 230rpm until the absorbance OD was reached ₆₀₀ About 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 once, and diluting bacterial liquid to OD with KCMS liquid culture medium ₆₀₀ About 0.1. A drop of agrobacterium dilution is dripped at the wound of the explant by using a pipetting gun, the bacterial liquid is not required to be sucked out, and the plate is sealed and then placed on a KCMS preculture medium for dark culture for 2 days.

(4) Differentiation and rooting of explants

After 2 days of co-cultivation of the explants with Agrobacterium, the explants were carefully transferred from KCMS preculture medium to primary screening 2Z medium with sterile forceps and the primary screening medium was changed every 15 days. When shoots to be differentiated were grown from the explants, the explants were transferred to 1Z medium for culture, with medium changed every 15 days. After culturing twice on 1Z medium, the differentiated callus grows buds with independent main stems, and is cut and inserted into rooting medium ENR for transplanting after rooting.

(5) Identification of transgenic positive plants

The transgenic T0 generation seedlings are confirmed whether to be transgenic positive plants (Cas 9-F:5'-TTAGGTTTACCCGCCAATA-3'; cas-R: 5'-GAGTAGACAAGTGTGTCGTGCT-3') by using CRISPR/Cas9 vector detection primers Cas9-F/Cas-R, and the detection results are shown in figure 2.

As shown in FIG. 2, the genomic DNA of T0 generation seedlings was used as a template for PCR amplification, and the fragment of 500bp size detected by electrophoresis was determined as a transgenic positive plant. And then, taking genomic DNA of the T0 generation seedling as a template, designing a detection primer containing a target site for PCR amplification (GRAS 9-Cas9check-F:5'-CTGAATCATTTTATTAATGCCT-3'; GRAS9-Cas9 check-R: 5'-GATAAAACCCCGTTCGTG-3'), recovering a target strip after agarose gel electrophoresis, constructing a purified PCR product on a pEASY-Blunt-Zero cloning vector for monoclonal sequencing, carrying out 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.

Sequencing heterozygous T0 generation seeds, screening by kanamycin, sowing continuously, analyzing the editing condition of T1 generation plants (the same as the editing detection method of T0 generation plants), selecting 2-3 homozygous lines with different editing forms, collecting seeds, screening by kanamycin, sowing to T2 generation for subsequent research, wherein the mutation modes of the selected lines are respectively that four bases are deleted and one base is inserted, as shown in figure 3.

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

After the homozygous mutant is obtained, fruit samples of wild type and gene knockout plants in the red ripe stage are collected for sugar content determination. 0.1g of fruit sample is weighed, and the tomato fruit soluble sugar and the organic acid are extracted by taking chromatographic pure (Ar) methanol as an extracting agent, and 3 biological repeats are set.

The specific process is as follows: samples were derivatized with methoxyamine hydrochloride and BSTFA. The measurement was performed using Shimadzu GC 2010 pro. The chromatographic parameters are SPL temperature 250 ℃, split ratio 10:1, a step of; the chromatographic column flow rate is 1mL/min, the temperature is raised to the initial temperature of 130 ℃, the temperature is raised to 185 ℃ at 5 ℃/min, the temperature is raised to 190 ℃ at 0.5 ℃/min, the temperature is raised to 300 ℃ at 8 ℃/min, and the temperature is kept for 10min. The detector temperature was 320℃with a hydrogen flow of 40mL/min, a dry air flow of 400mL/min, and a nitrogen flow of 40mL/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, CR#1 and CR#2 are two SlGRAS9 knockout tomatoes. According to the test results of fig. 4, after knocking out the SlGRAS9 gene, the Sucrose (Sucrose), fructose (Fructose) and Glucose (Glucose) contents in the tomato fruits are significantly increased, while the Malic acid (Malic acid) and Citric acid (Citric acid) contents are significantly decreased.

4. Determination of soluble solid content of SlGRAS9 knocked-out fruit

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

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

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

Sequence listing

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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.SlGRAS9Application of gene in regulating and controlling sugar content of tomato fruitsSlGRAS9The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. A formulation for increasing the sugar content of tomato fruits, said formulation comprising an inhibitorSlGRAS9An active ingredient of gene expression, saidSlGRAS9The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

3. The method of claim 1SlGRAS9Use of gene knockout in increasing sugar content in tomato fruits.

4. The method of claim 1SlGRAS9The gene knockout is applied to cultivation of high-sugar tomato fruit varieties or improvement of tomato germplasm resources.