阅读说明：本技术 调控番茄果实品质的转录因子及其应用 (Transcription factor for regulating and controlling tomato fruit quality and application thereof ) 是由 庄焜扬 孟庆伟 吕巍 马娜娜 于 2020-05-18 设计创作，主要内容包括：本发明涉及涉及植物生物技术领域,具体涉及调控番茄果实品质的转录因子及其应用。本发明研究发现,EIL2转录因子能够直接结合到LCYB的启动子上抑制其表达,减少番茄红素的转化；WHY1转录因子又能够结合到EIL2的启动子上抑制其表达,从而促进了LCYB的转录,推进番茄红素向β-胡萝卜素的转化；EIL2还能够通过直接结合启动子元件的方式抑制肌醇单磷酸酶(IMP,肌醇合成限速酶)及肌醇加氧酶(MIOX,肌醇途径合成抗坏血酸的限速酶)基因的表达,减少抗坏血酸的合成。WHY1则能够通过抑制EIL2促进抗坏血酸的合成。本发明的WHY1-EIL2介导的调控机制对番茄果实番茄红素及抗坏血酸的积累有重要的意义。(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.)

1. The use of the transcription factor described in the following 1) or 2) in the regulation of 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.

2. The use according to claim 1, characterized in that the transcription factor WHY1 regulates the quality of tomato fruits by inhibiting EIL2 expression by binding directly to the promoter of EIL 2.

3. Use according to claim 1 or 2, wherein said modulating tomato fruit quality comprises: regulating and controlling the content of lycopene and ascorbic acid in tomato fruits.

4. Use of a gene encoding the transcription factor of claim 1 for modulating tomato fruit quality; wherein the gene comprises:

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.

5. Use according to claim 4, wherein said modulating tomato fruit quality comprises: regulating and controlling the content of lycopene and ascorbic acid in tomato fruits.

6. A method for regulating and controlling the content of lycopene in tomato fruits is characterized by comprising the following steps:

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.

7. A method for regulating and controlling the content of ascorbic acid in tomato fruits, which is characterized by comprising the following steps:

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.

8. The method of claim 7, wherein the expression of myo-inositol monophosphatase and myo-inositol oxygenase is promoted by inhibiting the expression of EIL2, thereby activating the myo-inositol pathway of ascorbic acid synthesis and increasing the content of ascorbic acid.

9. Use of the transcription factor of claim 1 or the gene encoding the transcription factor of claim 4 for breeding transgenic tomato.

10. Use according to claim 9, wherein the cultivated transgenic tomato has altered color and quality of tomato fruit compared to the tomato starting plant.

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 effecting lycopene accumulation in the lycopene synthesis and metabolic pathways is 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; Lorenceet 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, VitaminC) 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.