Application of tomato galactoside synthase gene SlGolS2 in regulation and control of fruit coloring and ripening
阅读说明：本技术 一种番茄肌醇半乳糖苷合成酶基因SlGolS2在调控果实着色与成熟中的应用 (Application of tomato galactoside synthase gene SlGolS2 in regulation and control of fruit coloring and ripening ) 是由 齐明芳 张汇东 孟思达 许涛 王峰 刘玉凤 李天来 于 2021-07-27 设计创作，主要内容包括：本发明涉及番茄肌醇半乳糖苷合成酶基因SlGolS2在调控果实着色与成熟中的应用。所述SlGolS2基因属于糖基转移酶家族8,通过限速调节棉子糖系列寡糖的生物合成,参与调节植物生长发育,抵御逆境胁迫。在番茄子房中特异性表达,利用CRISPR-Cas9基因编辑技术获得的slgols2突变番茄不具有绿果肩,并且果实提前转色成熟。基于此研究结果,可为番茄果实绿果肩形成和果实成熟的分子机制研究,加快番茄早熟品种的选育提供新的应用基础。(The invention relates to a tomato galactinol synthetase gene SlGolS2 The application in regulating and controlling fruit coloring and ripening. The above-mentioned SlGolS2 The gene belongs to glycosyltransferase family 8, and participates in regulating plant growth and development by speed-limited regulation of biosynthesis of raffinose series oligosaccharides, so as to resist adversity stress. Specifically expressed in tomato ovary and obtained by using CRISPR-Cas9 gene editing technology slgols2 The mutant tomato has no green shoulder and the fruit turns color and ripens in advance. Based on the research result, a new application basis can be provided for the molecular mechanism research of tomato fruit green shoulder formation and fruit ripening and accelerating the breeding of tomato early-maturing varieties.)
1. Tomato galactoside synthetase geneSlGolS2The application in regulating and controlling fruit coloring and ripening.
2. Tomato galactinol synthase gene according to claim 1SlGolS2The application in regulating and controlling fruit coloring and ripening is characterized in that: the geneSlGolS2The nucleotide sequence is shown as SEQ ID No. 1.
3. Tomato galactinol synthase gene according to claim 1SlGolS2The application in regulating and controlling fruit coloring and ripening is characterized in that: geneSlGolS2The expression level was high in the ovary of the bud and in the ovary of the flower, and the expression level showed a low level in the fruit developed from the ovary.
4. Tomato galactinol synthase gene according to claim 1SlGolS2The application in regulating and controlling fruit coloring and ripening is characterized in that: the tomato is a wild tomato.
5. Tomato galactinol synthase gene according to claim 1SlGolS2The application in regulating and controlling fruit coloring and ripening is characterized in that: construction of gene by using CRISPR-Cas9 technologySlGolS2Knocking out the mutant to obtain two homozygous mutant linesslgols2#23Andslgols2#24；slgols2#23in the strain, 4 bases TTTA are inserted in front of the target position to replace 1 base T as G, and 5 bases ATATA are deleted from the target position;slgols2#24in the strain, 4 bases of TTTA are inserted in front of the target position, and 12 bases of GATGATATA are deleted from the target position.
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a tomato galactinol synthase geneSlGolS2The application in regulating and controlling fruit coloring and ripening.
Background
The tomato fruit has unique flavor, can provide various nutrient elements for human beings, such as various vitamins, carbohydrates, carotenoids, phenolic compounds and the like, and can play roles in resisting oxidation, thrombus, allergy, inflammation and the like (Raiola et al, 2014). Can be eaten fresh, cooked and made into tomato paste and other eating modes (Li et al, 2018), and is one of the essential fruits and vegetables for people to eat every day. Tomato yield and planting area are always at an increasing level worldwide (Quinet et al, 2019).
The development of tomato fruits starts in the floral meristem, where the tissue and structure determine the state of fruit growth. Fruit growth is the longest stage in fruit development, and can reach 5-8 weeks according to different genotypes. 1 to 2 weeks after flowering is the initial stage of fruit growth when cells undergo strong mitosis with an increase in the number of pericarp cells layers, accompanied by cell expansion (Chenicet et al, 2005). Subsequently, cell division gradually slows down, entering the cell expansion phase, where the fruit increases significantly in volume and weight and reaches a final size, entering the full green (MG) phase (Pesaresi et al, 2014). After several days in the MG phase, the fruit metabolites begin to recombine largely, marking the beginning of the fruit ripening process. Mainly comprises a Breaker (BR) stage and a ripening (RR) stage. After the fruit has entered the break phase, chlorophyll is degraded, carotenoids accumulate in large amounts, the fruit gradually softens and peaks with a sharp increase in respiration and ethylene release, which is a distinctive feature of respiratory catastrophe fruits (Alexander and Grierson, 2002). The accumulation of nutrients and the change of flavor, texture, color and aroma finally endow the tomatoes with unique quality and nutritional value.
The formation of the flavor and color of tomato fruits and the accumulation of nutrients are closely related to the ripening stage, so that the molecular mechanism of fruit ripening can be determined to provide a theoretical basis for improving the fruit quality, and the research related to the fruit ripening of tomato is always attractive to the eyes of a plurality of scholars (Yuan et al, 2018; Ji et al, 2020; Wu et al, 2020). The color change is most pronounced during the gradual ripening of tomato fruits, and is inherently due to chlorophyll degradation and carotenoid accumulation in the fruit.
Synthesis and accumulation of carotenoids is done in plastids (Sun et al, 2018). There are a variety of plastids in plants, including: chloroplasts, chromoplasts, proplastids, amyloplasts, etc. (Lopez-Juez and Pyke, 2005; Jarvis and Lopez-Juez, 2013). Among them, a proplastid is a plastid that has not yet differentiated, and is a precursor structure of other plastids. Carotenoids are present in large amounts in chloroplasts and chromoplasts (Egea et al, 2010). However, the color of carotenoids is usually masked by chlorophyll in chloroplasts, and is only fully revealed in chromoplasts. The color bodies in tomato fruits are usually transformed from chloroplasts, and during the ripening process of tomato fruits, the degradation of chlorophyll in pericarp cells causes the disruption of the thylakoid structure of chloroplasts, thereby gradually transforming into color bodies while accumulating a large amount of carotenoids (Min and Chieri, 2008; Egea et al, 2011). A large number of studies have shown that the chlorophyll content and chloroplast structure in tomato fruits often influence the carotenoid content of mature tomato fruits.DE-ETIOLATED1（DET1) The gene is a light signal transduction negative regulator, tomatohigh pigment 2（hp2) The mutant showed a dark green color during the MG phase, which isDET1The result of the mutation of the gene. Period of MGhp2The chloroplasts in the fruits are increased in volume and quantity, the chlorophyll content reaches 2.9 times of that of the wild type tomatoes, and the carotenoid level is remarkably improved after the fruits are fully ripe (Mustalli et al, 1999; Bino et al, 2005).high pigment 1（hp1) Mutants andhp2the phenotype is similar to thatUV-damaged DNA-binding protein 1（DDB1) The result of a mutation in a gene (Liu et al, 2004; Bernhardt et al, 2006). Two tomato species are presentGOLDEN2-LIKE（GLK) Genes of whichGLK2The gene is proved to be related to fruit chlorophyll accumulation and chloroplast development, and is over-expressedSlGLK2The gene increases the chlorophyll content, chloroplast volume and thylakoid number in the fruit, thereby increasing the carotenoid content in the fruit at the mature period (Powell et al, 2012; Nguyen et al, 2014).ARABIDOPSIS PSEUDO RESPONSE REGULATOR2-like（APRR2-like) AndGLKthe gene structure is similar and the overexpressionSlAPRR2-likeThe gene is capable of increasing the number of plastids, the area and the carotenoid content of tomato fruits (Pan et al, 2013). TKN2 and TKN4 are two transcription factors in the KNOX family, and research shows that the transcription factors act onSlGLKAndSlAPRR2- likeupstream, indirectly affecting plastid development, carotenoid accumulation (Nadakuduti et al, 2014).
Galactinol synthase (GolS) belongs to the family 8 glycosyltransferase (GT 8) which contains a variety of enzymes involved in the biosynthesis of oligosaccharides, polysaccharides and glycoconjugates and plays an important role in the growth and development of plants. RFOs are important soluble carbohydrates in plants, including the trisaccharides raffinose (raffinose), tetrasaccharide stachyose (stachyose) and pentasaccharide verbascose (verbascose) (Senguta et al, 2015; Jorge et al, 2017). Has the functions of storing carbohydrate, stabilizing membrane structure, resisting adversity stress and participating in osmotic regulation (Li et al, 2017a; Weyuxia, 2017). GolS can catalyze the transfer of galactoside, UDP-Galactose (UDP-Galactose, UDP-Gal) is taken as a glycosyl donor, galactoside is transferred to myo-inositol (myo-inositol) to synthesize galactinol (Gol) (Senguta et al, 2012), and important precursor substances are provided for the biosynthesis of RFOs. This reaction is a key step in the synthesis of RFOs, and also a rate-limiting step (sengutta et al, 2015). GolS is used as a key enzyme for RFOs biosynthesis, and is involved in regulating plant growth and development and resisting stress by influencing the content of RFOs. In tomatoAt 4 piecesGolSThe functions of the 4 genes are not reported at present.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a tomato galactinol synthetase geneSlGolS2The application in regulating and controlling the coloring and the ripening of fruits aims at regulating and controlling the formation of green shoulders of tomato fruits and the ripening process of the fruits.
The technical scheme is as follows:
tomato galactoside synthetase geneSlGolS2The application in regulating and controlling fruit coloring and ripening.
The above-mentionedSlGolS2The gene nucleotide sequence is shown as SEQ ID No. 1.
SlGolS2The gene expression level was high in the ovary of the bud and the ovary of the flower, and the expression level showed a low level in the fruit developed from the ovary.
The tomato is a wild type tomato Ailsa Craig.
Construction of gene by using CRISPR-Cas9 technologySlGolS2Knocking out the mutant to obtain two homozygous mutant linesslgols2#23Andslgols2#24；slgols2#23in the strain, 4 bases TTTA are inserted in front of the target position to replace 1 base T → G, and 5 bases ATATA are deleted in the target position;slgols2#24in the strain, 4 bases of TTTA are inserted in front of the target position, and 12 bases of GATGATATA are deleted from the target position.
Has the advantages that:
the result of the gene expression level detection in the invention shows that,SlGolS2the gene is specifically expressed in the tomato ovary, and the tomato fruit development starts in the ovary, which shows that the gene has a potential important role in the tomato fruit development process.
Construction by CRISPR-Cas9 technologyslgols2And (3) knocking out the mutant, wherein the mutant fruit has the appearance that the green fruit has no green shoulder in the green mature period, the color change is advanced and the maturity is improved. Namely the tomatoSlGolS2The gene deletion eliminates the formation of green shoulders of tomato fruits and promotes the fruit ripening.
Based on the above research results, the method can be used forSlGolS2The gene is knocked out by CRISPR-Cas9 gene editing technology and is used for culturing green-ripe-stage green-free fruits of tomatoesEarly-maturing varieties of shoulders.
Drawings
FIG. 1 is a drawing of the present inventionSlGolS2Analyzing the expression patterns of the genes at different tissue parts of the wild tomato Ailsa Craig;
FIG. 2 shows the present inventionSlGolS2A gene mutation mode;
FIG. 3 shows the present inventionslgols2And wild type tomato phenotype;
FIG. 4 shows the present inventionslgols2And the chlorophyll content of wild type tomato, wherein, the graph of fig. 4a shows the part of the tomato peel; FIG. 4b shows chlorophyll content in pericarp of green ripe stage of tomato fruit;
FIG. 5 shows the present inventionslgols2Synthesis of precursor content with wild type tomato chlorophyll, wherein fig. 5a is delta-aminolevulinic acid content, fig. 5b is porphobilinogen content, fig. 5c is uroporphyrinogen III content, fig. 5d is uroporphyrinogen III content, fig. 5e is magnesium protoporphyrin IX content, fig. 5f is protoporphyrin acid ester;
FIG. 6 shows the present inventionslgols2Microscopic observation with wild type tomato, wherein FIGS. 6a-c are chlorophyll fluorescence observations; FIGS. 6d-f are views of plastids; FIGS. 6g-i are views of chloroplast structures; FIG. 6j-l is a view of thylakoid structure;
FIG. 7 shows the present inventionslgols2Ethylene release from wild type tomato fruit;
FIG. 8 is a drawing of the present inventionslgols2Hardness of wild type tomato fruit;
FIG. 9 shows the present inventionslgols2The color of the wild type tomato fruit;
FIG. 10 shows the present inventionslgols2The content of total carotenoids of wild type tomato fruits;
FIG. 11 shows the present inventionslgols2And the content of lycopene in wild tomato fruit.
Detailed Description
The invention is described in more detail below with reference to the accompanying drawings.
In the inventionSlGolS2The gene belongs to glycosyltransferase family 8, and participates in regulating plant growth and development by speed-limited regulation of biosynthesis of raffinose series oligosaccharides, so as to resist adversity stress. Specifically expressed in tomato ovary, the invention utilizes CRIObtained by SPR-Cas9 gene editing technologyslgols2The mutant tomato has no green shoulder and the fruit turns color and ripens in advance. Based on the research result, a new application basis can be provided for the molecular mechanism research of tomato fruit green shoulder formation and fruit ripening and accelerating the breeding of tomato early-maturing varieties.
The invention provides a tomato galactoside synthetase geneSlGolS2SaidSlGolS2The gene has a nucleotide sequence shown in SEQ ID No. 1. The tomato galactoside synthase gene SlGolS2 is applied to regulation and control of fruit coloring and ripening.
The tomato material is wild type tomato Ailsa Craig,slgols2mutant tomato lines (A)slgols2#23Andslgols2#24）；
experimental reagent:
ultrapure RNA extraction Kit (Ultrapure RNA Kit), purchased from health in the century;
real-time fluorescent quantitation kit (TB Green Premix Ex Taq), purchased from TaKaRa;
DNA Marker 2000, 2 xtaqq PCR Master Mix, purchased from kuitai;
other molecular biological reagents, purchased from Sigma;
other chemicals, available from cologne.
Example 1
The particular location or period of gene expression often determines the potential biological function of the gene. Thus, the invention determinesSlGolS2The expression content of the gene in each tissue part of the wild tomato comprises the following experimental steps:
collecting organs (sepals, petals, stamens and ovaries) of wild tomato root, stem, leaf, flower bud, flower bud and flower organ (sepals, petals, stamens and ovaries) of flower, extracting RNA, reverse transcribing into cDNA, diluting to 3 times, and usingSlGolS2Amplifying qRT-PCR primers of the genes;
the qRT-PCR primer sequences were as follows:
Slgols2 F：5’- TTGTCAAGCCGTGCCTATGT -3’ （SEQ ID No.2）；
Slgols2 R：5’- CAGGCAAACAAGCCACAACA -3’ （SEQ ID No.3）；
qRT-PCR reaction System: 2 × Real Master Mix 10 μ L, Primer F (10mM) 0.4 μ L, Primer R (10mM) 0.4 μ L, cDNA 3 μ L, ddH2O 6.2 µL；
qRT-PCR reaction procedure: 30 s at 95 ℃, 5 s at 95 ℃, 30 s at 60 ℃, 45 cycles, Melt 15 s;
SlGolS2the expression of the gene in various tissues of wild tomato is shown in FIG. 1,SlGolS2the gene is expressed in various tissue parts of tomato, in particular,SlGolS2the gene expression level was high in both the ovary of the bud and the ovary of the flower, while the expression level showed a low level in the fruit from which the ovary developed.
Example 2
Constructed by Baige Gene CoSlGolS2The CRISPR-Cas9 knockout vector of the gene is transferred into wild tomato by an agrobacterium-mediated method. The inventor aims atslgols2Purifying T0 mutant, screening homozygous strain with stable inheritance, and the experimental steps are as follows:
extracting with CTAB methodslgols2The method for mutating the genomic DNA of the young and tender leaves of the tomato comprises the following specific steps: grinding tomato leaves in CTAB extracting solution, adding saturated phenol after 65 deg.C water bath, shaking, mixing, centrifuging, transferring supernatant to new centrifuge tube, adding isopropanol, placing at-20 deg.C, centrifuging for 5 min, transferring supernatant to new centrifuge tube, rinsing with 75% ethanol twice, air drying ethanol, adding ddH2O dissolving DNA, and storing at-20 ℃;
the preparation method of the CTAB extracting solution comprises the following steps: tris 1.21 g, EDTA-Na20.74 g, DTT 1 g, CTAB 2 g, NaCl 4.1 g, distilled water to 100 mL. Filtering with a water system filter membrane, and storing at room temperature;
amplifying the extracted DNA for subsequent sequencing analysisSlGolS2Amplifying PCR primers of the genes;
the PCR primer sequences were as follows:
SlGolS2 F：5’- ATGTTTTTGGTCTTGCAACTAAGGC -3’（ SEQ ID No.4）；
SlGolS2 R：5’- ACAATTAGAGTCCAGAAAAAGGGCT -3’ (SEQ ID No.5)；
and (3) PCR reaction system: 2XTaq PCR Master 12.5 muL, Primer F (10mM) 1 muL, Primer R (10mM) 1 muL, DNA 1 muL, ddH2O 9.5.5 muL;
PCR reaction procedure: 5 min at 95 ℃, 30 s at 57 ℃, 1 min at 72 ℃ for 35 cycles, 5 min at 72 ℃ and 10 min at 16 ℃;
the obtained PCR product was sent to Beijing Nonsula genome research center, Inc. for DNA sequencing. And analyzing the sequencing result by using DNAMAN software, and aligning the base sequences of the target positions, as shown in figure 2. Obtaining two homozygous mutant linesslgols2#23Andslgols2#24。slgols2#23in the strain, 4 bases (TTTA) are inserted in front of the target position to replace 1 base (T → G), and 5 bases (ATATA) are deleted at the target position;slgols2#24in the strain, 4 bases (TTTA) are inserted before the target position, and 12 bases (GATGATATA) are deleted at the target position.
Example 3
To pairslgols2The observation and comparison of the phenotype of the mutant and the wild tomato show that,SlGolS2the gene can influence the accumulation of chlorophyll, the development of chloroplast and the maturity of tomato fruits, and the related experimental steps are as follows:
tomato fruit phenotype observation and maturation time statistics: to calibrate the tomato fruit ripening process uniformly, the flowers were marked on the day of full expansion of the tomato petals, and the time thereafter was recorded as days post anthesis (dpa). Fruits with the same dpa are selected for phenotype observation, physiological index determination and molecular biological experiments. Marking the tomato fruits as a color breaking period when the tomato fruits show visible yellowing and recording the growing days at the time as a basis for counting the ripening time of the tomato fruits;
measuring the chlorophyll content: taking 0.1 g of pericarp of each part of tomato, adding 5 mL of extract (V acetone: V anhydrous ethanol =2: 1), placing in dark for 24 h, and after all samples are decolorized and whitened, measuring absorbance at 663 nm and 645 nm. 3 replicates per assay;
measuring the content of a chlorophyll synthesis precursor:
measuring the content of delta-aminolevulinic acid (ALA): weighing tomato peel, adding 4% trichloroacetic acid, grinding, centrifuging the ground solution, transferring supernatant to a new centrifuge tube, adding sodium acetate and acetylacetone, reacting in boiling water bath for 10 min, cooling, adding Ehrhch-Hg reagent, developing color in dark for 15 min, and measuring light absorption value at 553 nm;
determination of Porphobilinogen (PBG) content: weighing tomato peel, adding extractive solution (0.6 mol. L)-1 Tris，0.1 mol·L-1EDTA, pH 8.2), centrifuging the ground solution, transferring the supernatant to a new centrifuge tube, adding an Ehrhch-Hg reagent, developing color in the dark for 15 min, and measuring the light absorption value at 553 nm;
uroporphyrinogen iii (urogene iii) assay: weighing tomato peel, adding extractive solution (0.067 mol. L)-1Phosphate buffer, pH 6.8) trituration extraction. The milled solution was centrifuged, the supernatant transferred to a new centrifuge tube and 1% Na added2S2O3Irradiating the solution under strong light for 20 min, and adding glacial acetic acid (1 mol. L)-1) Adjusting pH to 3.5, extracting with diethyl ether for 3 times, mixing, and measuring the absorbance of water phase at 405.5 nm;
content determination of protoporphyrin IX (Proto IX), magnesium protoporphyrin IX (Mg-Proto IX) and protoporphyrin ester (Pchlide a): weighing tomato peel, adding 80% alkaline acetone, grinding on ice, centrifuging the ground solution, transferring supernatant to a new centrifuge tube, fixing the volume with 80% alkaline acetone, and measuring the absorbance values of OD575, OD590 and OD 628;
and (3) observation by a laser confocal microscope: tomato fruits that bloom 30 days later are taken, a thin layer of tomato peel is quickly cut by a clean scalpel, and the tomato peel is immediately placed on a glass slide. PBS buffer was added dropwise and the coverslip was gently covered. Observing under a laser confocal microscope;
and (3) plastid observation: taking tomato fruits 30 days after blooming, cutting peel parts into 2 mm × 1 mm × 1 mm cubic small blocks with a clean scalpel, quickly placing in 3.5% glutaraldehyde solution, completely immersing the sample in the solution, and reacting in the dark for 1 h. The sample was transferred to 0.1 mol. L-1 EDTA-Na2(pH 9.0) and placing in a water bath at 65 deg.C for 20-30 min. (the sample is placed at 0.1 mol. L-1 EDTA-Na2(pH 9.0) after the solution is stored at 4 ℃ for half a year. ) Separating the incompletely dispersed cells by using a liquid transfer device, sucking a small amount of tissues on a glass slide, and slightly covering a cover glass to completely disperse the cells into single cells. Observing and photographing under a microscope;
observation by a transmission electron microscope: tomato fruits 30 days after flowering are taken, the peel parts are cut into small cubic blocks of 2 mm multiplied by 1 mm by a clean scalpel, and the small cubic blocks are quickly placed in a penicillin bottle filled with glutaraldehyde solution. The air in the penicillin bottle was evacuated with a 50 mL syringe to completely immerse the sample in the solution and stored at 4 ℃ protected from light. Subsequent rinsing, fixing, dehydrating, embedding, sectioning and staining steps were performed by the analytical testing center of Shenyang university of agriculture. Observing the chloroplast structure under a Hitachi HT7700 transmission electron microscope and taking a picture;
determination of total carotenoid content: taking 1 g of pericarp at equator of tomato fruit, adding extractive solution (V n-hexane: V acetone =6: 4), grinding thoroughly, vortex shaking for 2 min, and centrifuging at 4000 g for 5 min. Transferring the supernatant to a new centrifuge tube, adding the extract into the precipitate, grinding, vortex oscillating, centrifuging, and mixing the supernatants. Until the pigment was completely eluted, the precipitate was white. Measuring the light absorption value at 450 nm, and calculating the total carotenoid concentration according to the formula: total carotenoid concentration (mg. mL)-1）=OD450/0.25；
And (3) determination of lycopene content: taking 1 g of tomato fruit equatorial pericarp, adding 10mL of extractive solution (V n-hexane: V anhydrous ethanol: V acetone =2:1:1, and adding 0.05% BHT antioxidant into acetone), grinding thoroughly, and vortex and oscillating for 15 min. After 3 mL of deionized water was added, the mixture was shaken for 2 min and centrifuged at 1000 g for 1 min until the solvent was separated. The upper layer solution (n-hexane) was taken in, adjusted to zero with n-hexane, and the absorbance at 503 nm was measured. The lycopene calculation formula is as follows: lycopene concentration (mg. mL)-1）=A503×3.12；
The hardness of tomato fruits was measured using a TMS-PILOT texture analyzer. Setting parameters: initial force0.75N, input speed 30 mm min-1Puncturing distance of 8 mm, output speed of 30 mm.min-1And the return distance is 50 mm. Placing the equator of the tomato fruit under a probe, recording the maximum pressure value in the puncturing process, and measuring at least 3 points of each fruit along the equator;
and (3) ethylene release amount determination: the picked tomatoes are placed at room temperature for 2 h to fully release the ethylene. The volume and mass of the fruit were measured. The fruits are sealed in a 300 mL fresh-keeping box and are placed in an incubator at 25 ℃ for 4 h. Extracting 1 mL of gas by using an injector, detecting the ethylene peak area in each sample by using a high performance gas chromatograph, calculating the ethylene concentration according to an ethylene standard curve, and determining the ethylene release amount of the fruits according to the volume and the mass of the fruits;
and (3) measuring the color: and (5) measuring the chromaticity of the tomato fruits by using a color difference meter. The colour of the tomato fruit was measured along the equator and L, a and b values were recorded, with at least 3 points measured along the equator for each fruit. Tomato Color Index (TCI) was calculated according to the formula (Pan et al, 2013): TCI =）；
By passingslgols2Phenotypic observations of the mutant lines (FIG. 3) showed that, during the green stage of ripening of the fruits,slgols2mutant fruits appeared in a lighter green than WT fruits, while there were no dark green fruit shoulders of wild type tomatoes.slgols2The mutant tomato fruits are broken color 4-6 days ahead.
slgols2The chlorophyll content in the mutant lines was reduced, especially at the fruit shoulders. Originally, the wild type has a dark green fruit shoulderslgols2No longer present in the tomato fruit (fig. 4);
slgols2the content of 6 precursor substances during chlorophyll synthesis was also reduced in the mutant lines (FIG. 5);
by multiple microscope pairsslgols2Observation of the internal structure of mutant lines and wild-type tomato pericarp cells revealed (fig. 6):slgols2the chlorophyll fluorescence level in the mutant line is reduced, the intracellular plastid is light green to white, and the chloroplast is simultaneouslyThe number of the castellations of the bag-like body stack is reduced;
ethylene release results show (figure 7): the ethylene release amount of the wild tomato is increased sharply at 42 dpa,slgols2#24andslgols2#23the tomato release amount is increased sharply at 38 dpa and 36 dpa respectively, and is earlier than that of the wild tomato. The ethylene release of wild type tomato during peak period is about 12.32. mu.L ∙ kg-1∙h-1，slgols2#24Andslgols2#23the released amount of (A) was about 7.65. mu.L ∙ kg-1∙h-1And 8.50. mu.L of ∙ kg-1∙h-1All are lower than wild tomato. These results showSlGolS2After gene knockout, the ethylene release amount is increased rapidly in advance, and the ethylene release amount is reduced, which shows thatSlGolS2The gene can influence the generation time and the release amount of ethylene surge;
hardness measurement results showed (fig. 8): WT,slgols2#24Andslgols2#23the tomato hardness starts to be greatly reduced after 40dpa, 36 dpa and 34 dpa respectively, tends to be stable after 44 dpa, 42 dpa and 40dpa, and has no obvious difference after 48 dpa. These results showSlGolS2The hardness of the fruit is reduced in advance after gene knockout, but the hardness of the fruit after complete maturity is not influenced;
the colorimetric results show (fig. 9): WT,slgols2#24Andslgols2#23the tomato fruit color values are greatly reduced after 40dpa, 36 dpa and 34 dpa respectively, the tomato fruit color values tend to be stable after 46 dpa, 42 dpa and 40dpa, and the TCI values of the tomato fruit color values are not obviously different after 48 dpa. These results showSlGolS2After gene knockout, the fruit is changed color in advance, but the color of the fruit after complete maturity is not influenced;
the results of the carotenoid content measurement (FIGS. 10 and 11) showed a value of 42 dpaslgols2#24Andslgols2#23the total carotenoid and lycopene content is significantly higher than that of wild tomato at 48 dpaslgols2#24Andslgols2#23the total carotenoid content and the lycopene content in the peel of the wild tomato are not obviously different;
in view of the above, it is desirable to provide,SlGolS2the gene deletion can eliminate the green shoulder of tomato fruits and promote the early ripening of the fruits.
Sequence listing
<110> Shenyang agriculture university
<120> application of tomato galactoside synthase gene SlGoS 2 in regulation and control of fruit coloring and ripening
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aaattataga aagcaacaaa aaaatctgaa ccgttggatc ttacctgacc acaacacgta 120
cagtccgatc cttgtggtgc aaaatataat ttttaagatt tatggggccc aaatgctcta 180
cttcaaagaa acttacacag acaataaaag taaaacgacg tcgtattgcg taaacgccat 240
gcaatctaat tttttatttt tttttacata tttttaattc ccacccaagg cctatataaa 300
caacgtcaaa aacactcaaa tctcatcaac aaacaaacag caatcttctc aaaaaacaat 360
cttttcttat tcaactactc tttactagct tctttttttc tcattttaaa tctcacaaaa 420
aacactcatt tctaatttac caattataaa aaaaaatggc acctaatgtt tttggtcttg 480
caactaaggc aactggttta gccaaggcaa aaagtttgtc aagccgtgcc tatgttacat 540
ttttagctgg aaatggtgat tattggaaag gcgttgttgg tttggttaaa ggtttgcgaa 600
aggcgaaatc cgcttatcca cttgttgtgg cttgtttgcc tgatgtcccg gaggaacatc 660
gacgtatact cattaaccaa ggttgcatcg ttcgagagat cgagcctgtt tatcctcctc 720
acaatcaaac tcaatttgct atggcttatt atgttatcaa ctattcaaag cttcgtattt 780
gggaggtaca actgtccttt ctgtttcagt aattataatg tttatttatt ttgtatatat 840
gctaaaagtg tttttttttt tttgggatgc agtttgtgga gtatagcaag atgatatact 900
tggacggtga tattcaggtg tttgataaca tagaccactt gtttgacttg ccagatggct 960
atttttatgc tgtgatggat tgtttttgtg agaaaacatg gagtcacacc ccacaatata 1020
aagttggcta ctgtcaacag tgtcctgata aggtccagtg gactgaagac ttgggcccta 1080
agccatcact ctattttaat gctggcatgt ttgtgtatga gccaagtctc tccacttatg 1140
atgatctctt gaaaacactc aaagttaccc ctcctacccc atttgctgaa caggtaacaa 1200
tctttttttt aagccctttt tctggactct aattgttgaa atgattaaaa atttttatgt 1260
taactttttg caggattttt tgaacatgta tttcagagat gtctacaagc caattccgaa 1320
cgattataac ttagttttag ctatgttgtg gcgtcaccct gagaatgtgg atcttgagaa 1380
agtaaaagtt gttcactact gtgcggcggg gtcgaagcca tggaggtaca ctggcaagga 1440
agagaacatg gacagagaag acattaagat gctgataaaa aaatggtggg atatttatga 1500
tgacgagtca ttggattaca agaattccaa cgttgttatg aatgccgtag atggagaagt 1560
tgaagctcaa aaaattatgg aagcgttatc agaggctggt gttgtgcact acataactgc 1620
tccatcggcc gcttagaata ttataggaag ctttcaaatt gttttttatc tttaaaaaaa 1680
caacaacttt gggatacata gtcttaaagc aaattatact agctttacag agtgcttttg 1740
ttatttattt ttttacaaaa ggattataaa ttgtctgtat ataatagggg agcaggcaat 1800
ttaagttctt ctataagtta aagagtgttt ttgtttgttt gtttgtggat attggaagaa 1860
cacagaaatg aaaaaaagta cgtatttatg tttatactga atcttgagat acgtatttat 1920
gttatggctc gagtaatctt ttgtcttttg ttaattgcac taaatatttt gagtaaatcc 1980
gtaggtggat aatttattca gaaagttaaa taattcatgt gattgtttgc aataactata 2040
agctcctaaa ttaactataa gaatccaaaa aagtagtaac tgtctcagtc tatgtgacaa 2100
caagatagct atagatgtga aagagttgaa ggggaaatgg ttgaatattc attatattgc 2160
ttgtgtattt gaaacctgtt ggtgttcgtg gataaggact acttttacta aacttggaat 2220
ttttttgtgt taattcgatt c 2241
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttgtcaagcc gtgcctatgt 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
caggcaaaca agccacaaca 20
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgtttttgg tcttgcaact aaggc 25
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acaattagag tccagaaaaa gggct 25