阅读说明：本技术 一种促进番茄抗坏血酸合成的基因及应用 (Gene for promoting synthesis of tomato ascorbic acid and application thereof ) 是由 张余洋 郑伟 叶志彪 李汉霞 叶杰 张俊红 张廷艳 于 2020-06-12 设计创作，主要内容包括：本发明属于基因合成技术领域,公开了一种促进番茄抗坏血酸合成的基因及应用,抗坏血酸合成的基因DNA序列为SEQ ID NO：1；本发明还提供一种利用所述促进番茄抗坏血酸合成的基因构建的表达载体,促进番茄抗坏血酸合成的MIOX基因在鉴定MIOX功能上的具有实际的应用意义。本发明从遗传转化方面证明肌醇途径,并初步鉴定MIOX的功能；通过调控抗坏血酸生物合成相关酶基因的表达,改变相关酶的活性,调控植物体内AsA含量,增加植物对氧逆境的抗性；通过调控抗坏血酸生成量是提高植物抗逆性的又一条可行途径,在植物抗逆育种中将会发挥重要作用。(The invention belongs to the technical field of gene synthesis, and discloses a gene for promoting synthesis of ascorbic acid in tomato and application thereof, wherein the DNA sequence of the gene for synthesizing the ascorbic acid is SEQ ID NO: 1; the invention also provides an expression vector constructed by using the gene for promoting the synthesis of the tomato ascorbic acid, and the MIOX gene for promoting the synthesis of the tomato ascorbic acid has practical application significance in identifying the MIOX function. The invention proves the inositol pathway from the aspect of genetic transformation, and preliminarily identifies the function of MIOX; through regulating and controlling the expression of ascorbic acid biosynthesis related enzyme genes, the activity of the related enzymes is changed, the AsA content in the plant body is regulated and controlled, and the resistance of the plant to oxygen stress is increased; the method is another feasible way for improving the stress resistance of plants by regulating the generation amount of the ascorbic acid, and plays an important role in the stress resistance breeding of the plants.)

1. The gene for promoting the synthesis of the tomato ascorbic acid is characterized in that the DNA sequence of the gene for promoting the synthesis of the tomato ascorbic acid is SEQ ID NO: 1.

2. an expression vector constructed using the gene for promoting the synthesis of tomato ascorbic acid according to claim 1.

3. A method for constructing the expression vector of claim 2, wherein the method comprises:

firstly, designing a full-length gene Primer by using Primer 5, and amplifying in a PCR (polymerase chain reaction) instrument by using cDNA (complementary deoxyribonucleic acid) of tomato AC (alternating current) as a template to obtain a target fragment; linking the PCR product to a pEASY-B vector (product of Beijing holotype gold company), converting escherichia coli by a connecting product heat shock method, screening positive clones by a 50mg/L Km resistant plate, selecting the positive clones, carrying out shake culture on a shaker at 37 ℃ at 200r/min overnight, selecting the positive clones after PCR detection by using a gene specific primer, and carrying out sequencing verification on the selected positive clones;

secondly, selecting clones for sequencing, and selecting correct clone to shake bacteria and extract plasmids; xbal and KpnI double enzyme digestion recombinant plasmid for 1.5 h;

thirdly, cutting the gel, recovering the gel by using a gel recovery kit to obtain a target fragment, and connecting the target fragment to a pMV2 vector subjected to double enzyme digestion by Xbal and Kpn I by using T4 ligase; connecting the products, transforming escherichia coli by a heat shock method, selecting positive clones, carrying out PCR positive detection on the single clones by using a 35S binding gene reverse specific primer, selecting the positive clones, shaking the bacteria to extract plasmids, carrying out double enzyme digestion verification, and transferring the plasmids into agrobacterium-induced C58 cells by an electric shock method after the verification is correct;

fourthly, selecting positive clones, carrying out shake culture at 150r/min on a shaking table at 28 ℃ overnight, and carrying out PCR positive detection on the bacterial liquid by using 35S plus gene reverse specific primers on the carrier; adding glycerol into the positive clone, mixing uniformly, and storing in a low-temperature refrigerator at-70 ℃.

4. Use of the tomato ascorbic acid synthesis promoting gene of claim 1 for identifying the function of MIOX, the method comprising:

tomato is used as a material, an agrobacterium-mediated transformation method is used for introducing an MIOX overexpression vector into tomato plants, a gulose pathway and an inositol pathway are proved from the aspect of genetic transformation, and the function of MIOX is preliminarily identified; through regulating and controlling the expression of ascorbic acid biosynthesis related enzyme genes, the activity of the related enzymes is changed, the AsA content in the plant body is regulated and controlled, and the resistance of the plant to oxygen stress is increased;

and measuring the AsA content of the transgenic tomato plant, measuring the influence of MI on the SlMIOX enzyme activity, analyzing the gene expression amount under adverse circumstances, and detecting the anti-oxidative stress capability.

5. The use according to claim 4, wherein the method for determining the AsA content of a transgenic tomato plant comprises:

taking 6-8 leaves at the same part of each tomato plant, quickly freezing the leaves by using liquid nitrogen, grinding the leaves into powder by using the liquid nitrogen, weighing the powder, dividing the powder into three parts (about 0.2g of each leaf and about 1g of each fruit), and extracting the three parts by using 0.1% metaphosphoric acid;

centrifuging the sample mixture at 10000r/min for 10min, filtering supernate by a microporous filter membrane, loading 100ul of the supernate to determine the content of AsA, adding equal-volume DTT (draw texturing yarn) to react at room temperature for 15min, determining the content of total AsA, and determining each sample for three times;

step three, adopting an SB-Aq C18 column, using an acetic acid buffer solution with pH value of 4.5 and 0.2mol.l < -1 > as a mobile phase, wherein the flow rate is 1.0ml.min < -1 >, and the measuring wavelength is 254 nm;

and step four, making a standard curve by using the AsA standard sample.

6. The use of claim 4, wherein the method of determining the effect of MI on SlMIOX enzyme activity comprises:

feeding with chemical MI;

MI concentration was 0.25mM, also with distilled water as control; the first three groups were cultured in 0.25mM MI and the second three groups were cultured in distilled water; after 24h feeding, the AsA content of the leaves was measured by liquid chromatography.

7. The use according to claim 4, wherein the method for analyzing the expression level of a gene under stress comprises:

step 1, selecting tomato A57 seeds with consistent plump size, uniformly accelerating germination, sowing the seeds in a hole tray with 72 holes, placing the hole tray in a greenhouse for culture, and treating the seeds when the seeds grow to three leaves and one heart; setting one treatment for every two plug trays with 72 holes and 12 plants in total;

step 2, high-temperature treatment: treating at 40 ℃ for 9 h; low-temperature treatment: treating at 4 ℃ for 12 h; PEG treatment: washing off the matrix on the roots, and treating in 9% PEG solution for 9 h; high-salt treatment: washing off the matrix on the roots, and treating in 150mM NaCl solution for 9 h;

step 3, after the material in the step 1 is processed in the step 2, quickly freezing the material by using liquid nitrogen; extracting total RNA of tomato and carrying out RT-PCR analysis.

8. The use according to claim 4, wherein the method for detecting the resistance to oxidative stress comprises a conductivity measurement, in particular:

a. and (3) paraquat treatment: selecting full transgenic plants T1 generation and control A57 seeds with consistent size, uniformly accelerating germination, and then sowing in a 72-hole plug tray, wherein two lines are sowed in each line; culturing in a greenhouse, treating after one month, detecting positive plants of transgenic offspring by using PCR before treatment, and pulling out negative plants; the method comprises the following steps of (1) soaking tomato seedlings which are tidy and grow consistently into a paraquat solution, and continuously illuminating for 36 hours; two paraquat concentrations were set: 0.15mM and 0.30 mM;

b. selecting the same part of leaves of the treated tomato seedlings, weighing 0.5g of leaves, adding 20ml of distilled water to completely immerse the leaves, and repeating the steps for three times; shaking on a shaker for 24h, and measuring the conductivity of the solution by using a conductivity meter at the constant temperature of 20-25 ℃; after the conductivity is measured, the plant tissue is killed in 100 deg.C boiling water bath for 15min, and the plant tissue is naturally cooled and measured with conductivity meter at constant temperature of 20-25 deg.C.

9. The use of claim 4, wherein the method for detecting oxidative stress resistance further comprises DAB staining, in particular comprising:

a. soaking the tomato in-vitro leaves treated by MV and water in DAB solution with the concentration of 1mg/mL, and placing the material in a dark environment at normal temperature for 24h for treatment;

b. taking out the leaf, transferring to 96% ethanol triangular flask, and placing in boiling water bath for 10min to remove chlorophyll;

c. removing the ethanol solution, adding a proper amount of clean 96% ethanol into a triangular flask to soak the leaves until the floating color is removed, and storing the decolored leaves in the 96% ethanol solution;

d. the active oxygen burst on the leaves after decolorization was observed.

10. The use of claim 4, wherein the method for detecting antioxidant stress capacity further comprises tomato leaves H₂O₂The fluorescent staining identification specifically comprises the following steps:

in vivo H of plant₂O₂The generation of (A) can be identified by staining with 2 ', 7 ' -dichlorodihydrofluorescein acetoacetate or 3,3 ' -diaminobenzidine;

collecting tomato leaves subjected to MV treatment for one week, placing the tomato leaves in a 50mL centrifuge tube, adding 25 mu M H2DCFDA for soaking, placing the tomato leaves in a dark environment for 15min, and then washing the tomato leaves with 20mM potassium phosphate buffer solution with the pH value of 6.0; making temporary sections of different tomato plant leaves respectively.

Technical Field

The invention belongs to the technical field of gene synthesis, and particularly relates to a gene for promoting synthesis of tomato ascorbic acid and application thereof.

Background

At present, AsA has important antioxidant and metabolic functions in organisms. In plants, AsA is associated with stress resistance in plants. Generally, the AsA content in the plant body is increased, and the capability of the plant in resisting cold, high temperature, drought, salt and alkali and other adverse circumstances can be enhanced. In animals, AsA is a necessary nutrient for human and animal maintenance, reproduction, and health. AsA can prevent and treat scurvy; can promote the growth of teeth and bones and prevent gum bleeding; has antioxidant effect, and can block the generation of nitrosamine, prevent cancer cell diffusion, and reduce the activity of external carcinogen in vivo, thereby preventing and treating cancer; can improve the metabolism of fat and lipid, especially cholesterol, and prevent cardiovascular diseases; has antiaging effect, and can promote metabolism of subcutaneous tissue, improve blood supply of skin, and prevent premature wrinkle of skin under combined action of vitamin A, E and vitamin C; can also enhance the anti-stress capability and immunity of the body to the external environment. However, humans and some other mammals have lost their own synthetic capacity (Chatterjee, 1973; Victoriano and Miguel,2004), must be ingested from food, and fresh fruits and vegetables are rich in ascorbic acid. The molecular structure of AsA was identified as early as 1933, and three pathways for AsA biosynthesis have been proposed: the mannose/L-galactose pathway (Wheeler et al, 1998), the galacturonic acid pathway (Agius et al, 2003), the gulose pathway (Wolucka and Montagu, 2003).

MIOX does not contain ferrous atoms, catalyzing four electrons to oxidize only one electron to the product D-glucuronic acid. The completion of the arabidopsis genome sequencing project enabled researchers to find sequences in plants that are homologous to the pig myoinositol oxygenase gene. Through searching, 4 open reading frames homologous with the gene are found on the 1 st, 2 nd, 4 th and 5 th chromosomes of arabidopsis thaliana. Rice inositol oxygenase genes are researched by Wang Haikang and the like in 2007, real-time quantitative PCR shows that OsMIOX genes are up-regulated and expressed under water stress, and the expression quantity in upland rice is obviously higher than that in rice. It is thought that the rice inositoxygenase gene plays a role in molecular responses in plant water stress, and it is presumed that ABA may induce the expression of OsMIOX (royal sea light, 2007). In 2008, Endres over-expressed MIOX, increased its enzymatic activity by more than 30 times, and after over-expressed MIOX, MI was always in a low state, feeding MI experiments also showed that over-expressed MIOX increased MIOX enzymatic activity less than control accumulated MI, due to over-expressed MIOX. Since inositol is a substrate for ascorbic acid synthesis, whether MIOX has a promoting effect on ascorbic acid synthesis is to be further investigated.

The tomato (Solanum lycopersicon.) is an annual or perennial herbaceous plant of the genus tomato of the family solanaceae, originates from peru, ecuador, borlivia and the like in south america, is an edible crop with high nutrition, high value and good flavor, and is also one of main vegetables planted in the world. Tomatoes contain abundant citric acid and malic acid, so the tomatoes are in an acidic environment, AsA has strong reducibility and unstable property, is easily oxidized by oxygen in the air, is easily influenced by factors such as temperature, pH value and light, is easily damaged under an alkaline condition, and is stable under an acidic condition. Thus, under the same conditions, the retention rate of AsA in tomato is much higher than that of other vegetables (von yangbo, 2002). Most of the tomato varieties studied at present have AsA content of 10-30mg/100 g.

In view of the important function of ascorbic acid in organisms, there is a great need to define the role of MIOX in ascorbic acid biosynthesis, and this patent identifies the function of tomato MIOX genes in ascorbic acid biosynthesis.

In summary, the problems of the prior art are as follows: ascorbic acid biosynthesis in tomato is controlled by multiple genes, is MIOX involved in ascorbic acid synthesis? The role and function of MIOX in other plant species is not consistent, what is the specific function in tomato? Is there an inositol pathway for ascorbic acid biosynthesis in tomato? These are unknown problems.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a MIOX gene for promoting the synthesis of tomato ascorbic acid and application thereof.

The invention is realized by that, a gene for promoting the synthesis of tomato ascorbic acid, the gene is a MIOX gene; the DNA sequence of the MIOX gene for promoting the synthesis of the tomato ascorbic acid is SEQ ID NO: 1.

GAGAAAATGACTATTCTCATTGAGCAGCCTGAATTTGGATCACAAGTGGAGGAGAAAAAAGTCTCATTCAATGCCAATGAACTTATTTTGGATGGTGGATTTATGGTACCAAAGACATTGTCTTCTCAAGATGAAATATTTGAAGTGCCAGACATAAATGCATTTGGTCAATCATTTAGGGATTATAATGTAGAAAGTGAGAGACAAAAATCAGTGGAAGAATTTTATAGGGTTCAACACATTAATCAAACATATGACTATGTGAAAAAAATGAGAAAAGAATATGGAAAATTGAACAAAATTGAAATGAGTATTTGGGATTGTTGTGAACTTTTGAATGATGTAGTTGATGATAGTGATCCTGATTTGGATGAACCACAAATTGAGCATTTGTTACAAACTGCTGAAGCTATTAGAAAAGATTATCCAAATGAAGATTGGCTTCATTTGACCGGCCTCATTCACGACCTAGGTAAAGTACTTCTTCATCCAAGTTTTGGAGGGCTTCCTCAATGGGCTGTTGTTGGAGACACATTTCCTCTTGGTTGTGCTTTTGATGAATCAATTGTTCACCACAAGTATTTTAAGGAAAATCCAGACATCAACAACAATATTTATAATACAAAAAATGGTGTATATGAAGAAGGTTGTGGACTTGACAAAGTTGTTATGTCATGGGGACATGATGATTATATGTATTTAATTGCAAAGGAAAATAAAACTACTCTTCCTTCTGCTGCTTTATTTGTCATACGTTACCACTCTTTCTATGCATTACATAGATCAGGAGCATATACACACTTGATGAATGAGGAGGACAAAGAGAACATGAAGTGGCTCAACATTTTTAATAAATATGATTTATATAGCAAGAGTAAAGTTCGAATTGATGTGGAAAAAGTCAAGCCATACTATCTCTCTCTTATCGAAAAGTATTTTCCAACAAAGCTGAGGTGGTAATTGAATCCATGG

the invention also aims to provide an expression vector constructed by using the MIOX gene for promoting the synthesis of the tomato ascorbic acid, wherein the vector construction is based on the pMV2 vector to construct an excessive expression vector (the pMV2 vector and the construction method refer to the Proc. of Ph. Va.tonkinensis, 2014, university of agriculture in Huazhong).

The invention also aims to provide a construction method of the expression vector, which comprises the following steps:

firstly, designing a full-length gene Primer by using Primer 5, and amplifying in a PCR (polymerase chain reaction) instrument by using cDNA (complementary deoxyribonucleic acid) of tomato AC (alternating current) as a template to obtain a target fragment; linking the PCR product to a pEASY-B vector (product of Beijing holotype gold company), converting escherichia coli by a connecting product heat shock method, screening positive clones by a 50mg/L Km resistant plate, selecting the positive clones, carrying out shake culture on a shaker at 37 ℃ at 200r/min overnight, selecting the positive clones after PCR detection by using a gene specific primer, and carrying out sequencing verification on the selected positive clones;

secondly, selecting clones for sequencing, and selecting correct clone to shake bacteria and extract plasmids; xbal and KpnI double enzyme digestion recombinant plasmid for 1.5 h;

thirdly, cutting the gel, recovering the gel by using a gel recovery kit to obtain a target fragment, and connecting the target fragment to a pMV2 vector subjected to double enzyme digestion by Xbal and Kpn I by using T4 ligase; connecting the products, transforming escherichia coli by a heat shock method, selecting positive clones, carrying out PCR positive detection on the single clones by using a 35S binding gene reverse specific primer, selecting the positive clones, shaking the bacteria to extract plasmids, carrying out double enzyme digestion verification, and transferring the plasmids into agrobacterium-induced C58 cells by an electric shock method after the verification is correct;

fourthly, selecting positive clones, carrying out shake culture at 150r/min on a shaking table at 28 ℃ overnight, and carrying out PCR positive detection on the bacterial liquid by using 35S plus gene reverse specific primers on the carrier; adding glycerol into the positive clone, mixing uniformly, and storing in a low-temperature refrigerator at-70 ℃.

The invention also aims to provide an application of the tomato ascorbic acid synthesis promoting MIOX gene in identification of the MIOX function, and the application method comprises the following steps:

tomato is used as a material, an agrobacterium-mediated transformation method is used for introducing an MIOX overexpression vector into tomato plants, a gulose pathway and an inositol pathway are proved from the aspect of genetic transformation, and the function of MIOX is preliminarily identified; through regulating and controlling the expression of ascorbic acid biosynthesis related enzyme genes, the activity of the related enzymes is changed, the AsA content in the plant body is regulated and controlled, and the resistance of the plant to oxygen stress is increased;

and measuring the AsA content of the transgenic tomato plant, measuring the influence of MI on the SlMIOX enzyme activity, analyzing the gene expression amount under adverse circumstances, and detecting the anti-oxidative stress capability.

Further, the method for measuring the AsA content of the transgenic tomato plant comprises the following steps:

taking 6-8 leaves at the same part of each tomato plant, quickly freezing the leaves by using liquid nitrogen, grinding the leaves into powder by using the liquid nitrogen, weighing the powder, dividing the powder into three parts (about 0.2g of each leaf and about 1g of each fruit), and extracting the three parts by using 0.1% metaphosphoric acid;

centrifuging the sample mixture at 10000r/min for 10min, filtering supernate by a microporous filter membrane, loading 100ul of the supernate to determine the content of AsA, adding equal-volume DTT (draw texturing yarn) to react at room temperature for 15min, determining the content of total AsA, and determining each sample for three times;

step three, adopting an SB-Aq C18 column, using an acetic acid buffer solution with pH value of 4.5 and 0.2mol.l < -1 > as a mobile phase, wherein the flow rate is 1.0ml.min < -1 >, and the measuring wavelength is 254 nm;

and step four, making a standard curve by using the AsA standard sample.

Further, the method of determining the effect of MI on SlMIOX enzyme activity comprises:

feeding with chemical MI;

MI concentration was 0.25mM, also with distilled water as control; the first three groups were cultured in 0.25mM MI and the second three groups were cultured in distilled water; after 24h feeding, the AsA content of the leaves was measured by liquid chromatography.

Further, the method for analyzing the gene expression level under stress comprises the following steps:

step 1, selecting tomato A57 seeds with consistent plump size, uniformly accelerating germination, sowing the seeds in a hole tray with 72 holes, placing the hole tray in a greenhouse for culture, and treating the seeds when the seeds grow to three leaves and one heart; setting one treatment for every two plug trays with 72 holes and 12 plants in total;

step 2, high-temperature treatment: treating at 40 ℃ for 9 h; low-temperature treatment: treating at 4 ℃ for 12 h; PEG treatment: washing off the matrix on the roots, and treating in 9% PEG solution for 9 h; high-salt treatment: washing off the matrix on the roots, and treating in 150mM NaCl solution for 9 h;

step 3, after the material in the step 1 is processed in the step 2, quickly freezing the material by using liquid nitrogen; extracting total RNA of tomato and carrying out RT-PCR analysis.

Further, the method for detecting the anti-oxidative stress capacity comprises the following steps:

(1) and (3) conductivity measurement:

a. paraquat (MV) treatment: selecting full transgenic plants T1 generation and control A57 seeds with consistent size, uniformly accelerating germination, and then sowing in a 72-hole plug tray, wherein two lines are sowed in each line; culturing in a greenhouse, treating after one month, detecting positive plants of transgenic offspring by using PCR before treatment, and pulling out negative plants; the method comprises the following steps of (1) soaking tomato seedlings which are tidy and grow consistently into a paraquat solution, and continuously illuminating for 36 hours; two paraquat concentrations were set: 0.15mM and 0.30 mM;

b. selecting the same part of leaves of the treated tomato seedlings, weighing 0.5g of leaves, adding 20ml of distilled water to completely immerse the leaves, and repeating the steps for three times; shaking on a shaker for 24h, and measuring the conductivity of the solution by using a conductivity meter at the constant temperature of 20-25 ℃; after the conductivity is measured, putting the plant tissue into a 100 ℃ boiling water bath for 15min to kill the plant tissue, taking out the plant tissue for natural cooling, and measuring the boiling conductivity by using a conductivity meter at the constant temperature of 20-25 ℃;

(2) DAB dyeing:

a. soaking the tomato in-vitro leaves treated by MV and water in DAB solution (pH3.8) with the concentration of 1mg/mL, and treating the material in a dark environment at normal temperature for 24 h;

b. taking out the leaf, transferring to 96% ethanol triangular flask, and placing in boiling water bath for 10min to remove chlorophyll;

c. removing the ethanol solution, adding a proper amount of clean 96% ethanol into a triangular flask to soak the leaves until the floating color is removed, and storing the decolored leaves in the 96% ethanol solution;

d. observing active oxygen outbreak on the decolored leaves;

(3) fluorescent staining identification of tomato leaves H2O 2:

the generation of H2O2 in plants can be identified by staining with 2 ', 7 ' -dichlorodihydrofluorescein acetoacetate (H2DCFDA) or 3,3 ' -Diaminobenzidine (DAB);

collecting tomato leaves subjected to MV treatment for one week, placing the tomato leaves in a 50mL centrifuge tube, adding 25 mu M H2DCFDA for soaking, placing the tomato leaves in a dark environment for 15min, and then washing the tomato leaves with 20mM potassium phosphate buffer solution with the pH value of 6.0; temporary sections are respectively made on the leaves of different strains of the tomato, and a Zeiss visual fluorescent signal ApoTome microscope is used.

In summary, the advantages and positive effects of the invention are: the invention provides an MIOX gene for promoting the synthesis of tomato ascorbic acid and application thereof, wherein tomatoes are used as materials, an agrobacterium-mediated transformation method is used for introducing an MIOX overexpression vector into tomato plants, a gulose pathway and an inositol pathway are proved from the aspect of genetic transformation, the function of MIOX is preliminarily identified, and the tomato material with high Vc content and improved stress resistance is obtained. Through regulating and controlling the expression of ascorbic acid biosynthesis related enzyme genes, the activity of the related enzymes is changed, and the AsA content in the plant body is regulated and controlled, so that the resistance of the plant to oxygen stress is increased. The over-expression of the MIOX gene can increase the content of the ascorbic acid in tomato leaves from 490ug/g to 590ug/g, and increase the content of the ascorbic acid in fruits from 220ug/g to 360ug/g, which shows that the MIOX gene plays an important role in the anabolism of the tomato ascorbic acid (see example figure 4). Therefore, the regulation of the generation amount of the ascorbic acid is another feasible way for improving the stress resistance of the plants, and plays an important role in the stress resistance breeding of the plants.

The invention shows that the SlMIOX positive transgenic plant can increase the gene expression level; the overexpression of SlMIOX can improve the ascorbic acid content of tomato plants; can increase the activity of MIOX enzyme in tomato; the content of soluble solid matters in the fruits can be improved; the SlMIOX strain has stronger oxidative stress tolerance.

Drawings

FIG. 1 is a flow chart of the gene assay analysis for promoting the synthesis of tomato ascorbic acid provided by the embodiment of the present invention.

Fig. 2 is a schematic diagram of the expression analysis of SlMIOX in different tissues of tomato provided by the embodiment of the invention;

in the figure: 1. a root; 2. a stem; 3. young leaves; 4. mature leaves; 5. young flowers; 6. mature flowers; 7. young fruits; 8. green ripe fruits; 9. breaking color fruits; 10. and (5) red ripe fruits.

FIG. 3 is a schematic diagram of RT-PCR analysis of SlMIOX sense transgenic plants provided by embodiments of the invention;

in the figure: CK. A57; lines 7-24, transgenic plants.

FIG. 4 is a bar graph of AsA content in leaves and fruits of an over-transgenic line of SlMIOX provided by an embodiment of the invention.

Fig. 5 is a bar graph of AsA content analysis after myo-inositol feeding of the SlMIOX over-transgenic lines provided by the examples of the invention.

FIG. 6 is a schematic diagram of the expression analysis of SlMIOX under various stress conditions provided by the embodiment of the invention;

in the figure: CK. Under normal conditions; HT, high temperature; LT, low temperature; PEG, PEG stress; NaCl, salt stress.

Fig. 7 is a schematic graph of oxidation resistance of a SlMIOX over-transgenic line provided by an embodiment of the invention.

FIG. 8 is a schematic diagram of expression levels of AsA anabolism-related genes of a SlMIOX hypertransgenic line provided by an embodiment of the invention.

FIG. 9 is a diagram of agronomic traits of an over-transgenic line of SlMIOX provided by an embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.