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

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

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CN114438104A
CN114438104A CN202210281557.1A CN202210281557A CN114438104A CN 114438104 A CN114438104 A CN 114438104A CN 202210281557 A CN202210281557 A CN 202210281557A CN 114438104 A CN114438104 A CN 114438104A
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李正国
时源
刘豫东
梁琴
成玉林
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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.
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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:
Figure BDA0003557132110000041
and U26-target-R1:
Figure BDA0003557132110000042
(vector linker sequence underlined) and primer pair 2: U26-target-F2:
Figure BDA0003557132110000043
and U26-NheI-R:
Figure BDA0003557132110000044
(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
Figure BDA0003557132110000045
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
Figure BDA0003557132110000051
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
<110> university of Chongqing
<120> SlGRAS9 gene for regulating sugar content of tomato fruits and application of SlGRAS9 gene in cultivation of tomatoes with high sugar content
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<213> Artificial Sequence (Artificial Sequence)
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atgtcctcgg atttctccgg cggagttcca gactttcacg gcggcgccgg tagatccagc 60
ttgattccga tgaacaactc ccagccacaa attcaattaa cccaacgccc tgacggagtt 120
tctcagaatc tccaccggag acctatgttc atcgggaaac ggtcactcgc agcctttcaa 180
cagcagcaac agtttcagtt tctacaacaa caacagcagc aacaaggttt gggattttat 240
ctacgtaacg tgaagcctag aaattaccag caggcatctc caatttctcc tctagattac 300
tctgtttctt cctcgttgat ttcatctgaa ttttctccga tgactccacg ccatcctctc 360
ccgatttcca cggcgaacac gaacggggtt ttatcttctg gtaacccgaa ttgttcagca 420
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atgaatcgac tgcatgagtt agagaaacag ctcctagaag acaacaatga ggaggaagaa 540
gatacagtct ctgttgtgac taacaacgac gagtggtcgg aaacaataaa gaatctgatt 600
actccgacta gtaaccactt atccccggca tcatctacgt cttcatgttc ttcttctatg 660
gaatctccgc cagtatcttc tcccaggcag tctattgtcg aagctgctac cgcaataatc 720
gacggaaaaa ccaatgttgc agtacagatc ctcacgcgcc tcgcacaggt agctgatgtt 780
agagggtctt ccgaacagcg gctgacggcg tacatggttt cagcactccg atcgcgcgtg 840
aactccacgg agtacccacc gcccgtgatg gagctgcgta gcaaagagca cgcagtttca 900
gctcaaaacc tctacgagat atccccgtgt ttcaagctag gatttatggc agctaatttc 960
gccattgttg aagctgtagc tgatcatccc tcaaacaaaa ttcacgtcat tgatttcgac 1020
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tatcccatca gcttaagaat cacggcgatc acaacagagt tcacggtcag agctgatcac 1140
agtttaaaat ccattgaaga tgatctgaga agtctagcaa acaaaatcgg tatttccttg 1200
attttcaaag taatttcacg tacaatcacc gatttgagta ggggaaaatt agggattgaa 1260
cacgacgaag ctttagcagt gaatttcgca tacagattat atagattacc cgacgagagc 1320
gtaacaacag agaatctaag agacgagctt ctccggcgag tgaaggggtt atcaccaaag 1380
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aacgaggcgt gtggatatta cggagcattg ttggattcac tggatgcaac tgtatcaaga 1500
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gcgtgtgaag gtagggatcg tttggagaga tgcgaggtgt ttggtaaatg gagggcccga 1620
atgagtatgg ctgggttcgg gccgaggccc atgagtcaac aaattgctga ttcactgctt 1680
aagaggctta actcgggccc acgtggcaat ccaggattca atgtgaatga acaaagtggg 1740
ggtattaggt ttggatggat gggaaaaacc ctcaccgttg cttctgcttg gtgttaa 1797
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Val Ala Asp Val Arg Gly Ser Ser Glu Gln Arg Leu Thr Ala Tyr Met
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Val Met Glu Leu Arg Ser Lys Glu His Ala Val Ser Ala Gln Asn Leu
290 295 300
Tyr Glu Ile Ser Pro Cys Phe Lys Leu Gly Phe Met Ala Ala Asn Phe
305 310 315 320
Ala Ile Val Glu Ala Val Ala Asp His Pro Ser Asn Lys Ile His Val
325 330 335
Ile Asp Phe Asp Ile Gly Gln Gly Gly Gln Tyr Leu His Leu Leu His
340 345 350
Ala Leu Ala Ser Lys Lys Thr Asp Tyr Pro Ile Ser Leu Arg Ile Thr
355 360 365
Ala Ile Thr Thr Glu Phe Thr Val Arg Ala Asp His Ser Leu Lys Ser
370 375 380
Ile Glu Asp Asp Leu Arg Ser Leu Ala Asn Lys Ile Gly Ile Ser Leu
385 390 395 400
Ile Phe Lys Val Ile Ser Arg Thr Ile Thr Asp Leu Ser Arg Gly Lys
405 410 415
Leu Gly Ile Glu His Asp Glu Ala Leu Ala Val Asn Phe Ala Tyr Arg
420 425 430
Leu Tyr Arg Leu Pro Asp Glu Ser Val Thr Thr Glu Asn Leu Arg Asp
435 440 445
Glu Leu Leu Arg Arg Val Lys Gly Leu Ser Pro Lys Val Val Thr Leu
450 455 460
Val Glu Gln Glu Leu Asn Gly Asn Thr Ala Ala Phe Val Ala Arg Val
465 470 475 480
Asn Glu Ala Cys Gly Tyr Tyr Gly Ala Leu Leu Asp Ser Leu Asp Ala
485 490 495
Thr Val Ser Arg Glu Glu Thr Gly Arg Val Lys Ile Glu Glu Gly Leu
500 505 510
Ser Arg Lys Leu Thr Asn Ser Val Ala Cys Glu Gly Arg Asp Arg Leu
515 520 525
Glu Arg Cys Glu Val Phe Gly Lys Trp Arg Ala Arg Met Ser Met Ala
530 535 540
Gly Phe Gly Pro Arg Pro Met Ser Gln Gln Ile Ala Asp Ser Leu Leu
545 550 555 560
Lys Arg Leu Asn Ser Gly Pro Arg Gly Asn Pro Gly Phe Asn Val Asn
565 570 575
Glu Gln Ser Gly Gly Ile Arg Phe Gly Trp Met Gly Lys Thr Leu Thr
580 585 590
Val Ala Ser Ala Trp Cys
595
<210> 3
<211> 11649
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240
atgttactag atccctaggg aagttcctat tccgaagttc ctattctctg aaaagtatag 300
gaacttcttt gcgtattggg cgctcttggc ctttttggcc accggtcgta cggttaaaac 360
caccccagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt 420
ttacaccaca atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca 480
caaaatcacc actcgataca ggcagcccat cagtccacta gacgctcacc gggctggttg 540
ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa cgcgccagaa acgccgtcga 600
agccgtgtgc gagacaccgc agccgccggc gttgtggata cctcgcggaa aacttggccc 660
tcactgacag atgaggggcg gacgttgaca cttgaggggc cgactcaccc ggcgcggcgt 720
tgacagatga ggggcaggct cgatttcggc cggcgacgtg gagctggcca gcctcgcaaa 780
tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat gatgtggaca agcctgggga 840
taagtgccct gcggtattga cacttgaggg gcgcgactac tgacagatga ggggcgcgat 900
ccttgacact tgaggggcag agtgctgaca gatgaggggc gcacctattg acatttgagg 960
ggctgtccac aggcagaaaa tccagcattt gcaagggttt ccgcccgttt ttcggccacc 1020
gctaacctgt cttttaacct gcttttaaac caatatttat aaaccttgtt tttaaccagg 1080
gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg tgccccccct tctcgaaccc 1140
tcccggcccg ctctcgcgtt ggcagcatca cccataattg tggtttcaaa atcggctccg 1200
tcgatactat gttatacgcc aactttgaaa acaactttga aaaagctgtt ttctggtatt 1260
taaggtttta gaatgcaagg aacagtgaat tggagttcgt cttgttataa ttagcttctt 1320
ggggtatctt taaatactgt agaaaagagg aaggaaataa taaatggcta aaatgagaat 1380
atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc gtaaaagata cggaaggaat 1440
gtctcctgct aaggtatata agctggtggg agaaaatgaa aacctatatt taaaaatgac 1500
ggacagccgg tataaaggga ccacctatga tgtggaacgg gaaaaggaca tgatgctatg 1560
gctggaagga aagctgcctg ttccaaaggt cctgcacttt gaacggcatg atggctggag 1620
caatctgctc atgagtgagg ccgatggcgt cctttgctcg gaagagtatg aagatgaaca 1680
aagccctgaa aagattatcg agctgtatgc ggagtgcatc aggctctttc actccatcga 1740
catatcggat tgtccctata cgaatagctt agacagccgc ttagccgaat tggattactt 1800
actgaataac gatctggccg atgtggattg cgaaaactgg gaagaagaca ctccatttaa 1860
agatccgcgc gagctgtatg attttttaaa gacggaaaag cccgaagagg aacttgtctt 1920
ttcccacggc gacctgggag acagcaacat ctttgtgaaa gatggcaaag taagtggctt 1980
tattgatctt gggagaagcg gcagggcgga caagtggtat gacattgcct tctgcgtccg 2040
gtcgatcagg gaggatattg gggaagaaca gtatgtcgag ctattttttg acttactggg 2100
gatcaagcct gattgggaga aaataaaata ttatatttta ctggatgaat tgttttagta 2160
cctagatgtg gcgcaacgat gccggcgaca agcaggagcg caccgacttc ttccgcatca 2220
agtgttttgg ctctcaggcc gaggcccacg gcaagtattt gggcaagggg tcgctggtat 2280
tcgtgcaggg caagattcgg aataccaagt acgagaagga cggccagacg gtctacggga 2340
ccgacttcat tgccgataag gtggattatc tggacaccaa ggcaccaggc gggtcaaatc 2400
aggaataagg gcacattgcc ccggcgtgag tcggggcaat cccgcaagga gggtgaatga 2460
atcggacgtt tgaccggaag gcatacaggc aagaactgat cgacgcgggg ttttccgccg 2520
aggatgccga aaccatcgca agccgcaccg tcatgcgtgc gccccgcgaa accttccagt 2580
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 (9)

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.
CN202210281557.1A 2022-03-21 2022-03-21 SlGRAS9 gene for regulating and controlling sugar content of tomato fruits and application of SlGRAS9 gene in cultivation of tomatoes with high sugar content Active CN114438104B (en)

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CN202210281557.1A CN114438104B (en) 2022-03-21 2022-03-21 SlGRAS9 gene for regulating and controlling sugar content of tomato fruits and application of SlGRAS9 gene in cultivation of tomatoes with high sugar content

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CN114438104A true CN114438104A (en) 2022-05-06
CN114438104B CN114438104B (en) 2023-06-20

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