CN111518185B - Transcription factor for regulating and controlling tomato fruit quality and application thereof - Google Patents
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Abstract
The invention relates to the technical field of plant biology, in particular to a transcription factor for regulating and controlling tomato fruit quality and application thereof. The research of the invention finds that the EIL2 transcription factor can be directly combined with the promoter of the LCYB to inhibit the expression of the LCYB transcription factor and reduce the conversion of lycopene; the WHY1 transcription factor can be combined with a promoter of EIL2 to inhibit the expression of the transcription factor, so that the transcription of LCYB is promoted, and the conversion of lycopene to beta-carotene is promoted; the EIL2 can also inhibit the expression of genes of inositol monophosphatase (IMP, inositol synthesis rate-limiting enzyme) and inositol oxygenase (MIOX, rate-limiting enzyme for synthesizing ascorbic acid by inositol pathway) by directly combining with a promoter element, and reduce the synthesis of ascorbic acid. WHY1 can promote ascorbic acid synthesis by inhibiting EIL 2. The WHY1-EIL2 mediated regulation mechanism has important significance on the accumulation of lycopene and ascorbic acid in tomato fruits.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a transcription factor for regulating and controlling tomato fruit quality and application thereof.
Background
Tomatoes are popular with people all the time due to unique flavor and rich nutritional value, and are widely planted in northern areas of China. The tomato fruit growth and development is a process of massive carotenoid accumulation and massive starch and sugar hydrolysis, and the metabolic products are rich. Among them, lycopene and ascorbic acid (AsA) are two very important antioxidant substances required by human beings, and are particularly important for maintaining the health of human beings. Therefore, the molecular regulation and control mechanism for anabolism of lycopene and AsA in tomato fruits is clear, and the molecular regulation and control mechanism has important significance for improving the tomato fruit quality.
Lycopene is an intermediate in the carotenoid metabolic pathway. The key to influencing lycopene accumulation in the lycopene synthesis and metabolic pathways lies in PSY 1-mediated biosynthesis and LCYB-mediated degradation (Shewmaker et al, 1999; Ronen et al 2000). Furthermore, Phytoene Desaturase (PDS) and zeta-carotene desaturase (ZDS) and carotene isomerase (CRTISO) also play important roles therein (Cunningham and Gantt, 1998; Isaacson et al, 2002). With the clarification of lycopene metabolic pathway and the cloning of related genes of biosynthesis, the improvement of lycopene yield by using genetic engineering technology becomes possible. Constitutive mass expression of PSY1 gene from CaMV 35S promoter can promote the accumulation of lycopene in tomato fruit (Fray et al, 1995). On the other hand, inhibition of LYCB expression by RNAi interference techniques can increase lycopene levels in tomato fruits by reducing lycopene degradation (Rosati et al, 2000). At present, a great deal of research has been conducted on major genes in the metabolic pathway of plant lycopene, however, detailed mechanisms of gene expression regulation and pigment accumulation are not clear. Therefore, the analysis of the main effect mechanism of the expression regulation of the lycopene-related gene has important significance for improving the content of the lycopene.
Ascorbic acid (vitamin C) has an important antioxidant effect in organisms and is an essential substance in the growth, development and reproduction of plants and animals (Gilbert et al, 2009). Humans and some primates cannot synthesize ascorbic acid themselves due to the deletion of the last key enzyme of the ascorbic acid synthesis pathway (L-guluronic acid-1, 4-lactone oxidase) gene, and must be ingested from food, particularly fresh fruits and vegetables (Watanabe et al, 2006). In view of the important function of ascorbic acid in the body, research on increasing the content of ascorbic acid in vegetables and fruits is very important. The tomato is more beneficial to the stability of the ascorbic acid due to the acidic condition that the fruits of the tomato are rich in the citric acid and the malic acid. The L-galactose pathway is the major pathway for the synthesis of ascorbic acid in tomato (Smirnoff et al, 1996). However, the feeding experiment shows that: the galacturonic acid pathway and the inositol pathway may also play a role in tomato ascorbic acid synthesis (Badejo et al, 2012; Lorence et al, 2004). These different synthetic pathways may play different roles during different growth and development stages of tomato. Most of the current research focuses on the analysis of the functions of the major genes of the L-galactose pathway and the molecular regulation, while the role and the regulation mechanism of the inositol pathway are unclear. Therefore, the analysis of the inositol pathway control mechanism for synthesizing the ascorbic acid in the tomato fruits can provide a new visual angle for improving the ascorbic acid content of the tomato fruits.
Disclosure of Invention
In view of the above prior art, the present invention aims to provide a transcription factor for regulating and controlling tomato fruit quality and applications thereof. The research of the invention finds out a new mechanism for inhibiting the metabolism of the lycopene and regulating the pathway of inositol-ascorbic acid metabolism, and has very important significance for improving the contents of the lycopene and the ascorbic acid in tomato fruits.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a use of the transcription factor described in the following 1) or 2) for regulating tomato fruit quality:
1) the amino acid sequence of the transcription factor EIL2 is shown in SEQ ID NO. 1;
2) the amino acid sequence of the transcription factor WHY1 is shown in SEQ ID NO. 2.
In the application, the transcription factor WHY1 is directly combined with the EIL2 promoter to inhibit the expression of EIL2 so as to regulate the quality of tomato fruits.
In the above applications, the regulating tomato fruit quality comprises: regulating and controlling the content of lycopene and ascorbic acid in tomato fruits.
In a second aspect of the invention, the use of a gene encoding the above transcription factor for regulating tomato fruit quality is provided; the genes include:
the nucleotide sequence of the gene for coding the transcription factor EIL2 is shown in SEQ ID NO. 3; or
The nucleotide sequence of the gene for coding the transcription factor WHY1 is shown as SEQ ID NO. 4.
In the above applications, the regulating tomato fruit quality comprises: regulating and controlling the contents of lycopene, ascorbic acid, abscisic acid, ethylene synthesis precursor and ethylene in tomato fruits.
In a third aspect of the present invention, there is provided a method for regulating lycopene content in tomato fruit, comprising the steps of:
up-regulating the expression of EIL2 or down-regulating the expression of WHY1 so as to improve the content of lycopene in tomato fruits;
or, the expression of EIL2 is down-regulated or the expression of WHY1 is up-regulated, so as to reduce the content of lycopene in tomato fruits.
In a fourth aspect of the invention, there is provided a method of modulating the ascorbic acid content in tomato fruit comprising the steps of:
down-regulating the expression of EIL2 or up-regulating the expression of WHY1 to increase the content of ascorbic acid in tomato fruits;
or up-regulating the expression of EIL2 or down-regulating the expression of white 1 to reduce the level of ascorbic acid in tomato fruit.
In the above method, expression of Inositol Monophosphatase (IMP) and inositol oxygenase (MIOX) is promoted by inhibiting expression of EIL2, so that inositol pathway of ascorbic acid synthesis is activated and content of ascorbic acid is increased.
In the above method, the up-regulation or down-regulation of the expression of EIL2 and the up-regulation or down-regulation of the expression of white 1 are realized by conventional genetic engineering techniques, such as RNAi interference vector, cripper Cas9 vector or overexpression vector.
In a fifth aspect of the invention, there is provided the use of the above transcription factor or a gene encoding a transcription factor in breeding transgenic tomato.
In the application, compared with the original tomato plant, the color and the quality of the tomato fruit are changed.
The invention has the beneficial effects that:
the invention discovers and confirms a new gene regulation path related to fruit color and ascorbic acid (AsA) metabolism in tomato by utilizing reverse genetics for the first time. Lycopene Cyclase (LCYB) mediates the conversion of lycopene to beta-carotene, thereby affecting the coloration of tomato fruits. The EIL2 transcription factor can be combined with the promoter of the LCYB to inhibit the expression of the LCYB, so that the conversion of lycopene is reduced; the WHY1 transcription factor can be combined with a promoter of EIL2 to inhibit the expression of the transcription factor, thereby promoting the transcription of LCYB and promoting the conversion of lycopene to beta-carotene. This provides a new molecular mechanism for controlling the degradation of lycopene. On the other hand, AsA is an important antioxidant substance in tomato fruits and is very important for human health. EIL2 is also capable of inhibiting the expression of myo-inositol monophosphatase (IMP, myo-inositol synthesis rate-limiting enzyme) and myo-inositol oxygenase (MIOX, AsA myo-inositol pathway-limiting enzyme) by direct incorporation of promoter elements, reducing the synthesis of AsA. WHY1 can promote AsA synthesis by inhibiting EIL 2. The WHY1-EIL2 mediated regulation mechanism discovered by the invention has important significance on the accumulation of lycopene and ascorbic acid in tomato fruits.
Drawings
FIG. 1: panel A is the identification of the expression level of WHY1 in fruits of WHY1 RNAi tomato line (WR 4); FIG. B shows the identification of the expression level of EIL2 in fruits of EIL2RNAi tomato lines (ER1, ER2, ER 3); panel C is the identification of EIL2 and WHY1 expression levels in fruits of WHY1 and EIL2RNAi hybrid lines (W × E1, W × E2, W × E3); panel D shows the fruit color phenotype of each transgenic tomato line. Panel E shows the fruit color phenotype of each transgenic tomato line. Panel E is a determination of lycopene content in the fruits of each transgenic tomato line. Panel F is a determination of the ascorbic acid content in the fruits of each transgenic tomato line.
FIG. 2: the results of the EIL2 on the influence of the biosynthesis of the tomato abscisic acid; in the figure, WT-Y: fruit in the wild-type color transition stage, ER-Y: fruits of EIL2RNAi strain at the color-changing stage; WT-R: fruit of wild type maturity; ER-R: fruits in mature stage of EIL2RNAi strain.
FIG. 3: the results of the EIL2 on the regulation of ethylene synthesis precursor (ACC) and ethylene content in different developmental stages of tomato fruits; in the figure, WT-Y: fruit in the wild-type color transition stage, ER-Y: fruits of EIL2RNAi strain at the color-changing stage; WT-R is fruit of wild type mature period; ER-Y: EIL2RNAi line mature fruit.
FIG. 4: panel A is a yeast single-hybridization experiment of WHY1-GAD with EIL2 pro-LacZ; WHY1-GAD + -, LacZ and-GAD + P1, LacZ is negative control group. FIG. B is a yeast single-hybridization experiment of EIL2-GAD with LCYBpro-LacZ; EIL2-GAD + -LacZ and-GAD + P1, LacZ is negative control group. FIG. C is a yeast single-hybrid experiment of EIL2-GAD and IMPpro-LacZ; EIL2-GAD + -LacZ and-GAD + P1, LacZ is negative control group. FIG. D is a yeast single-hybrid experiment of EIL2-GAD and MIOXpro-LacZ; EIL2-GAD + -LacZ and-GAD + P1, LacZ is negative control group. FIG. E shows LUC activation experiments of WHY1-FLAG and EIL2 promoters; FLAG + EIL2-pro-LUC as a negative control. FIG. F is an LUC activation experiment for EIL2-FLAG and LCYB promoters; EIL2 is capable of inhibiting the expression of LCYB; FLAG + LCYB-pro-LUC as negative control. FIG. G is a LUC activation experiment of EIL2-FLAG and IMP promoters; EIL2 is capable of inhibiting the expression of IMP; FLAG + IMP-pro-LUC as a negative control. FIG. H is a LUC activation experiment of EIL2-FLAG and MIOX promoters; EIL2 is capable of inhibiting expression of MIOX; FLAG + MIOX-pro-LUC as negative control.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, lycopene and ascorbic acid have strong antioxidant capacity and are beneficial to human health in both vegetables and fruits. Compared with industrial and biological fermentation synthesized lycopene and ascorbic acid products, the lycopene and ascorbic acid product is more easily accepted by people by directly obtaining the lycopene and ascorbic acid from fruits and vegetables. Therefore, the analysis of the molecular regulation mechanism of the synthesis and metabolism of lycopene and vitamin C has important significance for the endogenous improvement of the content of lycopene and vitamin C. However, at present, people have less research on the gene expression regulation mechanism in the lycopene metabolic pathway in plants and the function and regulation mechanism of the ascorbic acid inositol synthesis pathway.
Based on this, the invention has conducted intensive research on the molecular mechanisms controlling lycopene metabolism and ascorbic acid synthesis in tomato. The research of the invention finds that: ETHYLENE INSENSITIVE 3-LIKE2(EIL2) is a "switch" that controls lycopene degradation and ascorbic acid synthesis. On one hand, the lycopene inhibitor can reduce the conversion of lycopene to beta-carotene by inhibiting the expression of lycopene cyclase (LYCB), and increase the accumulation of lycopene in fruits; on the other hand, the expression of Inositol Monophosphatase (IMP) and inositol oxygenase (MIOX) can be promoted by inhibiting the expression of the enzyme, so that an inositol pathway for ascorbic acid synthesis is activated, and the content of ascorbic acid is increased. While WHIRLY1 transcription factor can be used as a "key" in this process to control the expression of EIL 2.
Thus, the present invention provides a novel mechanism for inhibiting lycopene metabolism and regulating inositol-ascorbic acid (AsA, Vitamin C) metabolic pathway. During the tomato fruit ripening process, the WHIRLY1(WHY1) transcription factor in the cell nucleus can relieve the inhibition effect of EIL2 on the expression of lycopene cyclase (LYCB) by inhibiting the expression of EIN3-like2(EIL2), promote the conversion of lycopene to beta-carotene and present the phenotype of yellow fruits. On the other hand, after the EIL2 is inhibited, the inhibition of inositol monophosphatase (IMP, inositol synthesis rate-limiting enzyme) and inositol oxygenase (MIOX) is also relieved, the process of synthesizing vitamin C by an inositol pathway is promoted, and the content of the vitamin C in the fruit is increased. The method provides a feasible and theoretical basis for improving the endogenous lycopene and ascorbic acid in the tomato fruits through biological engineering.
Further research shows that WHY1 can be directly bonded to the promoter of EIL2 to inhibit the expression of the EIL 2. While EIL2 was able to bind directly to and repress the expression of the LCYB, IMP and MIOX promoters.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier.
Example 1: acquisition and phenotypic analysis of individual transgenic lines
1. The experimental method comprises the following steps:
1.1, WHY1 and EIL2RNAi strains
Sequences specific for the WHY1 and EIL2 coding regions were obtained by BLAST analysis: (https:// phytozome.jgi.doe.gov/pz/portal.htmlDatabase, WHY1 numbered Solyc05g007100 and EIL2 numbered Solyc01g009170) and this sequence was amplified into a PC336 plant RNAi interference vector (supplied by Shandong university of agriculture). Through https:// crispr. cos. uni-heidelberg. de/designed Crisper Cas9 target sequence and construct it to PYAO expression vector (institute of genetics and developmental biology, Chinese academy of sciences). The vector is transformed into an agrobacterium strain LBA4404 by a freeze-thaw method, and then is transformed into tomato (Micro-TOM) by an agrobacterium-mediated leaf disc method to obtain RNAi silencing strains of WHY1 and EIL2 and mutant strains of WHY 1. The expression level of the obtained strain is detected by qRT-PCR.
1.2 obtaining of WHY1 and EIL2RNAi hybrid lines
The hybridization of tomato is carried out by taking EIL2RNAi strain as female parent and WHY1 RNAi strain as male parent. Stamens were removed before petals of the EIL2RNAi strain were unfolded, and pollen of the WHY1 RNAi strain was applied to the stigma of the EIL2RNAi strain and bagged. The expression level of the obtained strain is detected by qRT-PCR.
1.3 phenotypic observations and determination of physiological indices of the strains
The ascorbic acid content was determined using a kit from Beijing Soilebao. Wuhanmaiwei Metabolic corporation provides an analysis of the tomato fruit metabolome.
2. The planting mode is as follows: planting tomatoes (Micro-TOM) in a greenhouse, wherein the sunshine time is 16 hours, and the dark culture time is 8 hours, the culture temperature is 25 ℃ at normal temperature, and the air humidity is 60-70%; greenhouse locations were in the crop biology national focus laboratory of Shandong university of agriculture.
3. Results of the experiment
The results are shown in FIG. 1, panel A showing that the expression level of WHY1 in fruits of WHY1 RNAi tomato strain (WR4) is much lower than that of wild type strain (WT); panel B shows that EIL2RNAi tomato lines (ER1, ER2, ER3) expressed much less EIL2 in fruits than WT; panel C shows that the expression levels of EIL2 and WHY1 in fruits of WHY1 and EIL2RNAi hybrid lines (W × E1, W × E2, W × E3) were lower than WT; panel D shows the fruit color phenotype of each transgenic tomato line, with the sequential red color increasing for the EIL2RNAi line, WHY1 and EIL2RNAi hybrid line, WT, WHY1 RNAi line. Panel E shows the fruit color phenotype of each transgenic tomato line. Panel E is a determination of lycopene content in the fruits of each transgenic tomato line. The results of the assay were consistent with the phenotypic trends described above. And the graph F is the determination of the content of the ascorbic acid in the fruits of each transgenic tomato line, and the trend of the determination result is opposite to the trend of the change of the lycopene. WC9 and WC10 in the figure represent mutant strains of WHY 1.
The test results show that the WHY1-EIL2 regulation pathway participates in lycopene metabolism and ascorbic acid synthesis in tomato fruits.
The tests also show that: EIL2 promotes the biosynthesis of abscisic acid (fig. 2), and regulates the content of ethylene synthesis precursors (ACC) and ethylene in different developmental stages of fruits (fig. 3).
Example 2:
1. the experimental method comprises the following steps:
1.1 Yeast Single hybridization experiments
Respectively constructing full-length sequences of WHY1 and EIL2 coding regions into a GAD vector; mixing EIL2, LCYB, IMP,The promoter sequence of MIOX (about 1900bp before the coding region, divided into two segments; the above genes are all described inhttps:// phytozome.jgi.doe.gov/pz/portal.htmlThe database specifically includes: solyc05g007100(WHY1), Solyc01g009170(EIL2), Solyc04g040190(LCYB), Solyc11g012410(IMP), Solyc06g062430(MIOX)) were ligated into LacZ2u yeast expression vectors. Then the constructed WHY1-GAD + EIL2pro-LacZ, EIL2-GAD + LCYBpro-LacZ, EIL2-GAD + IMPpro-LacZ and EIL2-GAD + MIOXpro-LacZ are respectively transferred into the yeast EGY48 strain. For specific operation, refer to Zhuang et al, (2019).
1.2 Luciferase (LUC) activation assay
Respectively constructing full-length sequences of WHY1 and EIL2 coding regions into a pZP211-FLAG plant expression vector; the promoter sequences (about 1800bp before the coding region) of EIL2, LCYB, IMP and MIOX are connected into a pZP211-LUC plant expression vector. The constructed WHY1-FLAG/-FLAG + EIL2pro-LUC, EIL2-FLAG/-FLAG + LCYBpro-LUC, EIL2-FLAG/-FLAG + IMPpro-LUC and EIL2-FLAG/-FLAG + MIOXpro-LUC are respectively transferred into agrobacterium GV3101 to carry out transient transformation tobacco experiments. For specific operation, refer to Zhuang et al, (2020).
2. The planting mode is as follows: planting tobacco (Ben's tobacco) in a greenhouse at normal temperature of 25 deg.C for 16 hr, and culturing in dark for 8 hr with air humidity of 60-70%; greenhouse locations were in the crop biology national focus laboratory of Shandong university of agriculture.
3. Results of the experiment
The results are shown in FIG. 4, in which Panel A is a yeast single-hybridization experiment with WHY1-GAD and EIL2 pro-LacZ. WHY1 was able to bind to the P2 region of the EIL2 promoter. FIG. B shows a single-hybridization experiment between EIL2-GAD and LCYBpro-LacZ in yeast. EIL2 was able to bind to the P1 and P2 regions of the LCYB promoter. FIG. C shows a single-hybridization experiment with EIL2-GAD and IMPpro-LacZ in yeast. EIL2 was able to bind to the P2 region of the IMP promoter. Panel D shows yeast single-hybridization experiments for EIL2-GAD and MIOXpro-LacZ. EIL2 was able to bind to the P2 region of the MIOX promoter. FIG. E shows LUC activation experiments of WHY1-FLAG and EIL2 promoters. WHY1 was able to inhibit the expression of EIL 2. FIG. F shows LUC activation experiments for EIL2-FLAG and LCYB promoters. EIL2 is capable of inhibiting the expression of LCYB. FIG. G shows LUC activation experiments of EIL2-FLAG and IMP promoters. EIL2 is capable of inhibiting the expression of IMP. FIG. H shows LUC activation experiments for EIL2-FLAG and MIOX promoters. EIL2 is capable of inhibiting expression of MIOX.
The test results show that WHY1 can be directly bonded to the promoter of EIL2 to inhibit the expression of the EIL 2. While EIL2 was able to bind directly to and repress the expression of the LCYB, IMP and MIOX promoters.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> transcription factor for regulating and controlling tomato fruit quality and application thereof
<130> 2020
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 614
<212> PRT
<213> Artificial sequence
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Asp Gln Ser Lys Val Leu Thr Ser Pro Phe Asn Ala Ser Ser Thr Asp
565 570 575
Asp Phe Asn Phe Met Phe Gly Ser Pro Phe Asn Met Gln Ser Thr Asp
580 585 590
Leu Ser Glu Cys Leu Ser Gly Ile Ser His Asp Asp Val Thr Lys Gln
595 600 605
Asp Ala Ser Val Trp Tyr
610
<210> 2
<211> 268
<212> PRT
<213> Artificial sequence
<400> 2
Met Ser Val Phe Ser Leu Ser Ala Ser Pro Ala Ser Gly Phe Ser Leu
1 5 10 15
Asn Pro Thr Lys Thr Ser Ser Tyr Leu Ser Phe Ser Ser Ser Ile Asn
20 25 30
Thr Ile Phe Ala Pro Leu Thr Ser Asn Thr Thr Lys Ser Phe Ser Gly
35 40 45
Leu Thr Tyr Lys Ala Ala Leu Pro Arg Asn Leu Ser Leu Thr Cys Arg
50 55 60
His Ser Asp Tyr Phe Glu Pro Gln Gln Gln Gln Gln Gln Leu Gln Gly
65 70 75 80
Ala Ser Thr Pro Lys Val Phe Val Gly Tyr Ser Ile Tyr Lys Gly Lys
85 90 95
Ala Ala Leu Thr Val Glu Pro Arg Ser Pro Glu Phe Ser Pro Leu Asp
100 105 110
Ser Gly Ala Phe Lys Leu Ser Lys Glu Gly Met Val Met Leu Gln Phe
115 120 125
Ala Pro Ala Ala Gly Val Arg Gln Tyr Asp Trp Ser Arg Lys Gln Val
130 135 140
Phe Ser Leu Ser Val Thr Glu Ile Gly Ser Ile Ile Ser Leu Gly Ala
145 150 155 160
Lys Asp Ser Cys Glu Phe Phe His Asp Pro Asn Lys Gly Arg Ser Asp
165 170 175
Glu Gly Arg Val Arg Lys Val Leu Lys Val Glu Pro Leu Pro Asp Gly
180 185 190
Ser Gly His Phe Phe Asn Leu Ser Val Gln Asn Lys Leu Ile Asn Leu
195 200 205
Asp Glu Asn Ile Tyr Ile Pro Val Thr Lys Ala Glu Phe Ala Val Leu
210 215 220
Val Ser Ala Phe Asn Phe Val Met Pro Tyr Leu Leu Gly Trp His Thr
225 230 235 240
Ala Val Asn Ser Phe Lys Pro Glu Asp Ala Ser Arg Ser Asn Asn Thr
245 250 255
Asn Pro Arg Ser Gly Ala Glu Leu Glu Trp Asn Arg
260 265
<210> 3
<211> 1845
<212> DNA
<213> Artificial sequence
<400> 3
atgatgatgt ttgaagacat tgggttttgt gctgatcttg atttcttccc tgctccgctg 60
aaggaggcgg aaacagtagc tgctgttcca ccaattgtgc cggagccgat gatggatgat 120
gatgatagtg atgaggagat cgatgtggat gagctggaga agaggatgtg gagggataag 180
atgaagctga aaaggctgaa agaaatgagc aagggcaagg aaggtgttga tgctgtcaaa 240
caacgccagt ctcaggagca agctaggagg aagaagatgt ccagggctca agatgggatc 300
ttgaagtaca tgttgaagat gatggaagtc tgtaaggctc agggttttgt ttatggaatt 360
atcccggaga aaggcaaacc ggtgactggg gcatcggata atctcaggga gtggtggaag 420
gataaagtga ggtttgatcg caatggacct gctgcgatag caaagtacca agctgacaat 480
gccatccctg gcaagaacga gggtgctaat ccgattggtc caacccctca taccttgcag 540
gagcttcaag ataccaccct tggttctcta ctgtcagctt taatgcaaca ttgtgatcct 600
cctcagaggc gatttccatt ggaaaaaggt gtatcacctc catggtggcc aaatggacag 660
gaggattggt ggcctcagtt gggactgcca aatgatcaag gtcctccacc ttacaagaag 720
cctcatgatc tgaagaaggc ttggaaggtt ggtgtcctca cagcggtgat caagcacatc 780
tcccctgata ttgctaagat acgcaagctg gtaaggcaat cgaagtgctt gcaggataag 840
atgacagcaa aggaaagtgc aacttggctt gccatcatca atcaggagga agttttggct 900
cgcgaacttt atcctgatcg ctgtccacct ttgtcctcag gtggtagtag tggaaccttc 960
actatgaacg acagcagtga gtatgatgtt gaaggtgcta ttgatgaccc tatctttgat 1020
gttcaagagc aaaaaccaaa ccatctcagt ttgctgaatg tcaatgttga gatgttcaag 1080
gagaagctgc ctctgctaca gcagtctcag ccaatgaagg gtgacatttt tgccaactta 1140
gatttcactc gcaagaggaa gccggctgat gacttgactt tcctgatgga tccgaagaca 1200
tatacttgcg agtgtcttca ttgccctcat agtgagcttc gcaatggttt tccagacaga 1260
tccagcagag acaatcatca gctaacttgc ctcttcagga atacttctca atttgtagtt 1320
ccaaactttc acatggagga ggtcaagcca gttgtcttcc ctcaacagta tgctgagcca 1380
aagcgggctt cgcttccggt caacccagct ccaccctcct ttgatacatc tggacttggg 1440
gttcctgcag atgggcagag ggtgatcaat gagcttatgt cattctatga aagtaatgtg 1500
caaggaaaca aaagttcaat ggcggggaac tctgtgatgt ccaaagagca gcctcttcaa 1560
caacctagca ttcaacagaa caattacctt caaagccaag ggaatgtgtt ggagggaagc 1620
atctttgggg acaccaacat ttctgctaac aactccatgt ttgtgcaggg tgatcggttt 1680
gatcagagca aggttttaac ttcaccattc aatgcaagct ctactgatga tttcaatttc 1740
atgtttggat ctccattcaa catgcaatcc actgatctct ctgaatgtct ttctgggatt 1800
tcacatgatg acgtgacgaa gcaagatgcc tcggtttggt actag 1845
<210> 4
<211> 807
<212> DNA
<213> Artificial sequence
<400> 4
atgtctgtct tctctctttc tgcttcccct gcttcaggtt ttagtctaaa ccctactaaa 60
acctcttctt atctctcttt ttcctcttcc attaatacca tttttgctcc tttaacttcc 120
aacacaacaa aaagcttttc tggtttgacc tataaagcag ctttgcctag aaatctttct 180
ttaacatgtc gccattctga ttattttgaa ccccaacagc aacagcagca gctacagggg 240
gcatctacgc ctaaggtttt tgttggatac tcaatataca aagggaaggc agctctcact 300
gtggagcctc ggtcaccaga gttctcacct ttagattcag gggccttcaa gctgtcaaaa 360
gagggtatgg tgatgcttca atttgcaccc gctgctggtg ttcgtcaata tgattggagt 420
agaaagcagg tcttctcgct gtcagtgact gaaattggat ctatcatcag ccttggggca 480
aaagattcat gtgagttttt ccatgatcca aacaaaggaa gaagtgatga aggtagagtc 540
aggaaagtgt tgaaggttga gccacttcca gatggctccg gtcacttctt taatctcagt 600
gttcagaaca agcttattaa tttggacgag aacatttaca tcccagttac aaaggcagag 660
ttcgcagttc ttgtctccgc attcaatttt gttatgccat accttttagg ttggcacact 720
gctgtaaatt ccttcaagcc tgaagacgcc agtcgctcaa acaatactaa tccaagatca 780
ggtgctgaac ttgaatggaa tcgatag 807
Claims (7)
1. The use of the transcription factor of the following 1) or 2) in regulating the content of lycopene and ascorbic acid in tomato fruits:
1) the amino acid sequence of the transcription factor EIL2 is shown in SEQ ID NO. 1;
2) the amino acid sequence of the transcription factor WHY1 is shown in SEQ ID NO. 2.
2. The use as claimed in claim 1, characterized in that the transcription factor WHY1 is produced by direct binding toEIL2Inhibiting the expression of EIL2 on the promoter to regulate the quality of tomato fruits.
3. Use of a gene encoding a transcription factor as claimed in claim 1 for modulating the lycopene and ascorbic acid content in tomato fruits; wherein the gene comprises:
encoding transcription factorsEIL2The nucleotide sequence of the gene of (1) is shown in SEQ ID NO. 3; or
Encoding transcription factorsWHY1The nucleotide sequence of the gene of (1) is shown in SEQ ID NO. 4.
4. A method for regulating and controlling the content of lycopene in tomato fruits is characterized by comprising the following steps:
up regulationEIL2Expression or down regulation ofWHY1To increase the lycopene content in the tomato fruit;
or, down-regulation ofEIL2Expression or upregulation ofWHY1To reduce the lycopene content in the tomato fruit;
EIL2the nucleotide sequence of (A) is shown as SEQ ID NO. 3;WHY1the nucleotide sequence of (A) is shown in SEQ ID NO. 4.
5. A method for regulating and controlling the content of ascorbic acid in tomato fruits, which is characterized by comprising the following steps:
down-regulation ofEIL2Expression or upregulation ofWHY1To increase the ascorbic acid content in tomato fruits;
or, adjust upwardEIL2Expression or down regulation ofWHY1To reduce the level of ascorbic acid in tomato fruits;
EIL2the nucleotide sequence of (A) is shown as SEQ ID NO. 3;WHY1the nucleotide sequence of (A) is shown in SEQ ID NO. 4.
6. The method of claim 5, wherein the inhibition is byEIL2To promote the expression of myoinositol monophosphatase and myoinositol oxygenase, thereby activating the myoinositol pathway of ascorbic acid synthesis and increasing the content of ascorbic acid.
7. Use of the transcription factor as set forth in claim 1 or the gene encoding the transcription factor as set forth in claim 3 for breeding transgenic tomato;
compared with the original tomato plant, the content of lycopene and ascorbic acid in the tomato fruit is changed.
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