Application of QfAP1 gene and encoded protein in regulation and control of expression of target gene PAL

Abstract

The invention discloses an application of a QfAP1 gene and an encoded protein thereof in regulation and control of expression of a target gene PAL. The target gene PAL is a key enzyme gene in regulation and control of a synthetic route of condensed tannin in fruits. The QfAP1 gene sequence is as shown in (a) or (b): (a) a nucleotide sequence as shown in Seq ID No: 1; and (b) a nucleotide sequence encoding an amino acid sequence having at least 70% or more homology with the amino acid sequence as shown in SEQ ID NO: 2. According to the application, expression of a target gene PAL is reduced by inhibiting expression of the QfAP1 gene; or the expression of the target gene PAL is increased by overexpressing the QfAP1 gene. According to the application of the QfAP1 gene and the encoded protein thereof in regulation and control of expression of a target gene PAL, regulation and control of a key enzyme gene in a condensed tannin synthesis route can be realized, and a new route is provided for regulation and control of condensed tannin synthesis. Preferably, the expression of the target gene PAL can be reduced by inhibiting the expression of the QfAP1 gene, so that the content of condensed tannin in the white oak fruits is reduced, the astringency of the white oak fruits is reduced, and the white oak fruits can be directly eaten.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to an application of a QfAP1 gene and a coding protein in regulating the expression of a target gene PAL, wherein the target gene PAL is a key enzyme gene regulating the synthesis of condensed tannins in fruits. Background Art

[0002] Tannin is a natural polyphenol active substance, which is widely found in the skin, roots, leaves, flowers, fruits and other tissues of plants. According to its structure and properties, tannin can be divided into two categories: hydrolyzed tannin and condensed tannin.

[0003] Condensed tannins, also known as proanthocyanidins (PA), are polymerized from flavonoids and flavan-3-ols as structural units. Condensed tannins have a variety of biological activities. For example, condensed tannins can be combined with nucleic acids, proteins, enzymes, polysaccharides or other substances, and have good antioxidant, anti-inflammatory, antibacterial, antiparasitic and other biological activities. They are mainly used in chemical industry, materials and other fields. However, the presence of condensed tannins in fruits will also cause the fruits to have a distinct astringent taste. For example, the fruit of Quercus fabri Hance, commonly known as acorns, is rich in starch, protein, soluble sugar, fat, vitamins and other nutrients. It is an excellent woody food resource that can be developed and utilized. However, the condensed tannin content of Quercus fabri Hance fruit is relatively high, and its mass fraction can be as high as 12% of the fruit. Therefore, the fruit tastes distinctly astringent and cannot be eaten directly.

[0004] Therefore, it is necessary to study the biosynthesis pathway of condensed tannins. Previous researchers have conducted extensive research on the biosynthesis pathway of condensed tannins and cloned several key genes such as PAL, CHS, DFR, LAR, ANS and ANR. Phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) are considered to be key enzymes in the early stage of the biosynthesis pathway of condensed tannins in plants; colorless anthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) are considered to be key enzymes in the late stage of condensed tannin synthesis. However, the synthesis of condensed tannins is mainly regulated at the transcriptional level.

[0005] At present, the regulatory transcription factors involved in the synthesis of condensed tannins mainly include MYB, bHLH and WD40 proteins, which can act independently or form MBW (MYB-bHLH-WD40) complexes to regulate the biosynthesis of condensed tannins. In Arabidopsis, the MBW complex mainly regulates the expression levels of late structural genes in the condensed tannin synthesis pathway, while the expression levels of early structural genes in the condensed tannin synthesis pathway, such as CHS, CHI and F3H, are regulated by MYB11, MYB12 and MYB111.

[0006] At present, there is no research specifically targeting the regulation of the key enzyme gene PAL in the condensed tannin biosynthesis pathway in fruits. As a key enzyme gene in the early stage of the condensed tannin biosynthesis pathway, it plays a key role in the biosynthesis of condensed tannins.

[0007] The MADS-box transcription factor AP1 (APETALA1) gene is a floral meristem characteristic gene and a floral organ morphology characteristic gene of plants. It is both a floral meristem characteristic gene and a floral organ morphology characteristic gene, and plays an important role in controlling the floral meristem characteristics and floral organ formation of plants. There has been no report on the study of the regulation of PAL by the MADS-box transcription factor AP1 gene. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide an application of a QfAP1 gene and its encoded protein in regulating the expression of a target gene PAL, in order to provide a new regulatory pathway for the synthesis of condensed tannins in fruits.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] On the one hand, the present invention provides an application of a QfAP1 gene in regulating the expression of a target gene PAL, wherein the target gene PAL is a key enzyme gene in regulating the synthesis pathway of condensed tannins in fruits;

[0011] The QfAP1 gene sequence is shown in (a) or (b):

[0012] (a) the nucleotide sequence shown in Seq ID No: 1;

[0013] (b) A nucleotide sequence encoding an amino acid sequence that is at least 70% homologous to the amino acid sequence shown in SEQ ID NO:2.

[0014] As a further improvement of the present invention, the promoter sequence of the target gene PAL is shown in Seq ID No:3.

[0015] As a further improvement of the present invention, the expression of the target gene PAL is reduced by inhibiting the expression of the QfAP1 gene.

[0016] As a further improvement of the present invention, the expression of the target gene PAL is increased by overexpressing the QfAP1 gene.

[0017] As a further improvement of the present invention, the expression level of the QfAP1 gene is inhibited by knocking out the QfAP1 homologous gene in the plant, thereby reducing the expression of the target gene PAL.

[0018] As a further improvement of the present invention, the fruit is Quercus alba fruit.

[0019] In a second aspect, the present invention provides an application of a protein encoded by a QfAP1 gene in regulating the expression of a target gene PAL, wherein the target gene PAL is a key enzyme gene in regulating the synthesis pathway of condensed tannins in fruits;

[0020] The protein sequence is shown in (a) or (b):

[0021] (a) the amino acid sequence shown in SEQ ID NO: 2;

[0022] (b) an amino acid sequence that is at least 70% homologous to the amino acid sequence shown in SEQ ID NO: 2.

[0023] As a further improvement of the present invention, the expression of the target gene PAL is reduced by inhibiting the expression of the protein encoded by the QfAP1 gene.

[0024] As a further improvement of the present invention, the expression of the target gene PAL is increased by overexpressing the protein encoded by the QfAP1 gene.

[0025] The present invention screens out a MADS-box transcription factor from the transcriptome data of white oak. The MADS-box transcription factor AP1 (APETALA1) gene (named QfAP1) belongs to the characteristic gene of plant floral meristem and floral organ morphology. Based on the present invention, the present invention conducts research on it and unexpectedly finds that it can regulate the expression of the key enzyme gene PAL in the condensed tannin synthesis pathway of the fruit. Therefore, the condensed tannin synthesis pathway in the fruit can be regulated by regulating the expression of PAL, that is, the content of condensed tannins in the fruit can be regulated, such as reducing the content of condensed tannins in the fruit of white oak, thereby reducing its astringency and making it directly edible. Based on the high conservatism of the QfAP1 gene, it can also be applied to the expression regulation of the key enzyme gene PAL in the condensed tannin synthesis pathway in the fruit of other plant varieties. According to actual needs, the expression of PAL can be increased by overexpression, thereby increasing the content of condensed tannins in the fruit, or the expression of PAL can be reduced by inhibiting expression, thereby reducing the content of condensed tannins in the fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0027] Figure 1 This is a protein sequence alignment diagram of QfAP1 and homologous proteins, where black represents 100% similarity, pink represents 75% similarity, and light blue represents 50% similarity.

[0028] Figure 2The figure is a graph of the expression level and condensed tannin content of AP1 in overexpression and knockout Micro-Tom plant lines.

[0029] (A) Expression levels of QfAP1 in overexpressing Micro-Tom line 5 (QfAP1-OE5) and line 8 (QfAP1-OE8).

[0030] (B) Condensed tannin content in overexpressed Micro-Tom line 5 (QfAP1-OE5) and line 8 (QfAP1-OE8).

[0031] (C) Expression levels of AP1 in knockout Micro-Tom line 1 (AP1-KO1) and line 2 (AP1-KO2).

[0032] (D) Condensed tannin content in knockout Micro-Tom strain 1 (AP1-KO1) and strain 2 (AP1-KO2).

[0033] The data in (AD) above are mean values. Error bars represent the standard deviation of three biological replicates. Different letters indicate significant differences (P<0.05).

[0034] (E) Phenotypic images of Micro-Tom fruit at different developmental stages. Scale bar = 1 cm.

[0035] Figure 3 This is a graph of the expression levels of important genes involved in the condensed tannin synthesis pathway in Micro-Tom overexpression and knockout strains. Among them:

[0036] (A) Expression levels of PAL genes involved in the condensed tannin biosynthesis pathway in overexpression and knockout strains.

[0037] (B) Expression levels of CHS genes involved in the condensed tannin biosynthesis pathway in overexpression and knockout strains.

[0038] (C) Expression levels of ANR genes involved in the condensed tannin biosynthesis pathway in overexpression and knockout strains.

[0039] (D) Expression levels of 4CL genes involved in the condensed tannin biosynthesis pathway in overexpression and knockout strains.

[0040] The data in (AD) above are mean values. Error bars represent the standard deviation of three biological replicates. Different letters indicate significant differences (P<0.05).

[0041] Figure 4 This is a diagram of the promoter elements of the PAL gene that the QfAP1 protein binds to and activates.

[0042] (A) Schematic representation of the PAL promoter fragment (start codon-2000 bp) and the interaction between QfAP1 and this fragment in yeast cells.

[0043] (B) QfAP1 induces PAL expression in a dual-luciferase assay. Data are mean values, and error bars represent standard deviations of three biological replicates (P < 0.05).

[0044] (C) Schematic diagram of the PAL promoter fragment used for EMSA experiment and the EMSA experimental results showed that QfAP1 protein can bind to the PAL gene promoter fragment. DETAILED DESCRIPTION

[0045] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the biochemical reagents, carriers, consumables, etc. used in the examples are all commercially available products.

[0046] The regulation of the condensed tannin content in fruits is of great significance. Taking the fruit of white oak as an example, a high content of condensed tannins will make the fruit taste very astringent and cannot be eaten directly. Therefore, it is necessary to reduce the condensed tannin content in the fruit.

[0047] The target gene PAL is a key enzyme gene in the early stage of the condensed tannin synthesis pathway in fruits, and plays a key role in the biosynthesis of condensed tannins. The regulation of condensed tannin synthesis in fruits can be achieved by regulating it.

[0048] The present invention screens out a MADS-box transcription factor from the transcriptome data of white oak. The MADS-box transcription factor AP1 (APETALA1) gene belongs to the floral meristem characteristic gene and floral organ morphology characteristic gene of the plant. It is both a floral meristem characteristic gene and a floral organ morphology characteristic gene, and plays an important role in controlling the floral meristem characteristics of the plant and the formation process of floral organs. The present invention conducts research based on it and unexpectedly finds that it can regulate the key enzyme gene PAL in the condensed tannin synthesis pathway, and then can regulate the fruit condensed tannin synthesis pathway, and it plays an important role in the synthesis of condensed tannins.

[0049] The present invention is described in detail below in conjunction with specific embodiments.

[0050] Example 1 QfAP1 gene cloning and protein sequence alignment

[0051] In this example, a transcript annotated as MADS-box family protein APETALA1 (AP1) was screened out from Quercus alba transcriptome data (accession number in the National Center for Biotechnology Information (NCBI) database: PRJNA913393).

[0052] The transcript sequence was predicted and aligned in ORFfinder, and the sequence had a complete open reading frame (ORF). Primers for cloning the gene were designed with reference to the ORF sequence, and the gene was cloned by RT-PCR. The MADS-box gene was named QfAP1, with a length of 753 bp, and its encoding nucleotide sequence was as shown in Seq ID No: 1; it encoded 250 amino acids, as shown in Seq ID No: 2.

[0053] Comparison with homologous proteins from other species (tomato, Arabidopsis, California white oak, grape, and Brazilian rubber tree, etc.) showed that almost 80% (at least more than 70%) of the amino acids in QfAP1 are conserved ( Figure 1 ). Such a large number of conserved amino acid sequences indicate that AP1 has very similar functions in different plants.

[0054] Example 2 The expression level of QfAP1 gene can positively affect the yield of condensed tannins in fruits

[0055] There are several major plant secondary metabolites in tomatoes, such as alkaloids, terpenes, flavonoids, and carotenoids, which change with their growth and development. Therefore, tomatoes have been used as a model plant for studying plant secondary metabolic pathways. This example studies the regulation of condensed tannins by QfAP1 in transgenic small tomatoes Micro-Tom (model plant).

[0056] By transferring the exogenous QfAP1 overexpression plasmid into wild-type (WT) Micro-Tom plants and positively screening the obtained transgenic plants, 12 positive overexpression (QfAP1-OE) lines were obtained. Real-time quantitative PCR detection was performed on fruits in the middle stage of fruit development (Stage 3) of these 12 positive overexpression lines, and it was found that the expression level of QfAP1 was the highest in QfAP1-OE8, followed by QfAP1-OE5 ( Figure 2 A, E). Subsequently, the content of condensed tannins in the fruits of these two strains was measured and compared with that of the wild type. The results showed that as the expression level of QfAP1 increased, the content of condensed tannins in the plants increased significantly ( Figure 2 B).

[0057] By inhibiting gene expression, this example knocked out the AP1 homologous gene in the wild-type Micro-Tom plant, and the obtained gene-edited plants were positively screened to obtain 6 positive gene knockout (AP1-KO) strains. The fruits in the middle stage of fruit development (Stage 3) of these 6 positive gene knockout strains were selected for real-time quantitative PCR detection, and it was found that the expression level of AP1 was the lowest in AP1-KO2, followed by AP1-KO1 ( Figure 2 C). Subsequently, the content of condensed tannins in the fruits of these two strains was also measured and compared with the wild type. The results showed that as the expression level of AP1 decreased, the content of condensed tannins in the plants decreased significantly ( Figure 2 D).

[0058] The results of these two experiments showed that the expression level of the AP1 gene can effectively have a positive effect on the yield of condensed tannins in the fruit.

[0059] Example 3 Overexpression or knockout of the QfAP1 gene changes the expression levels of key enzyme genes involved in the condensed tannin synthesis pathway

[0060] In this example, the key enzyme genes PAL, early CHS and late ANR in the conserved condensed tannin synthesis pathway in white oak and Micro-Tom were selected as research objects. Their expression levels in the fruits of overexpressed and knocked-out plants were analyzed using quantitative real-time PCR. The results showed that their expression trends were consistent with the expression trends of AP1 and were also consistent with the final yield of condensed tannins ( Figure 3 AC). We also selected the 4CL enzyme gene, which is immediately downstream of the PAL gene in the condensed tannin synthesis pathway, as the research object and analyzed the trend of its expression level change. The results are also consistent with the expression level change trend of the previous genes ( Figure 3 D) Therefore, it is speculated that the expression level of one or more of these genes is regulated by the AP1 transcription factor.

[0061] Example 4 QfAP1 specifically binds to and activates the promoter of PAL

[0062] In order to study whether QfAP1 can regulate the expression levels of the above genes, this example first analyzed the presence of MADS-box transcription factor binding elements in the promoter regions of PAL, CHS, ANR and 4CL genes. By cloning and sequencing the promoter fragments of these four genes, this example identified several CArG-box elements within the 2Kbp promoter range of the PAL gene. The promoter sequence of PAL is the nucleotide sequence shown in Seq ID No: 3. Subsequently, this example performed a yeast single hybrid assay and found that QfAP1 was able to bind to the PAL promoter ( Figure 4 A).

[0063] In order to further analyze how QfAP1 regulates the expression of PAL, that is, whether it has a positive or negative effect on the activation of the promoter fragment after effectively binding to the PAL promoter element, this example uses the Dual-LUC test for detection. The experimental results show that in the presence of QfAP1 transcription factor, the activity of the PAL promoter is activated, thereby completing the function of positively regulating the expression level of PAL ( Figure 4 B).

[0064] Subsequently, this example constructed a QfAP1-His fusion vector to induce the labeled target protein in prokaryotic cells. After purifying the target protein, this example performed an EMSA experiment to verify whether QfAP1 can bind to the labeled probe of the CArG-box element and cause the migration of the band to be blocked. At the same time, this example also tested whether increasing the concentration of the unlabeled probe in the system would lead to a gradient decrease in the amount of QfAP1 binding to the labeled probe. These experimental results once again indicate that QfAP1 can directly bind to the CArG-box element in the PAL gene promoter to regulate gene transcription activity ( Figure 4 C).

[0065] Based on the above research results, the present invention unexpectedly discovered that the QfAP1 gene can regulate the expression of the key enzyme gene PAL in the condensed tannin synthesis pathway of the fruit, providing a new way for the regulation of condensed tannin synthesis. The condensed tannin synthesis pathway of the fruit can be regulated by regulating the expression of PAL, that is, the content of condensed tannins in the fruit can be regulated, such as reducing the content of condensed tannins in the fruit of white oak, thereby reducing its astringency and improving its practicality. Of course, based on the high conservatism of the QfAP1 gene, it can also be applied to the expression regulation of the key enzyme gene PAL in the condensed tannin synthesis pathway in the fruits of other plant varieties. Overexpression can be performed according to actual needs to increase the expression of PAL, thereby increasing the content of condensed tannins in the fruit, or the expression of PAL can be reduced by inhibiting expression, thereby reducing the content of condensed tannins in the fruit.

[0066] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.

Claims

1. An application of QfAP1 gene in regulating the expression of target gene PAL, characterized in that: The target gene PAL is a key enzyme gene that regulates the synthesis of condensed tannins in fruits; The QfAP1 gene sequence is shown in (a) or (b): (a) the nucleotide sequence shown in Seq ID No: 1; (b) A nucleotide sequence encoding an amino acid sequence that is at least 70% homologous to the amino acid sequence shown in SEQ ID NO:

2.

2. The use of the QfAP1 gene in regulating the expression of the target gene PAL according to claim 1, characterized in that: The promoter sequence of the target gene PAL is shown in Seq ID No:

3.

3. The use of the QfAP1 gene in regulating the expression of the target gene PAL according to claim 1, characterized in that: The expression of the target gene PAL was reduced by inhibiting the expression of QfAP1 gene.

4. The use of the QfAP1 gene in regulating the expression of the target gene PAL according to claim 1, characterized in that: The expression of the target gene PAL was increased by overexpressing the QfAP1 gene.

5. The use of the QfAP1 gene in regulating the expression of the target gene PAL according to claim 3, characterized in that: By knocking out the QfAP1 homologous gene in plants, the expression level of the QfAP1 gene is inhibited, thereby reducing the expression of the target gene PAL.

6. The use of the QfAP1 gene in regulating the expression of the target gene PAL according to claim 3 or 5, characterized in that: The fruit is a Quercus alba fruit.

7. Use of a protein encoded by the QfAP1 gene in regulating the expression of a target gene PAL, characterized in that: The target gene PAL is a key enzyme gene that regulates the synthesis of condensed tannins in fruits; The protein sequence is shown in (a) or (b): (a) the amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence that is at least 70% homologous to the amino acid sequence shown in SEQ ID NO:

2.

8. The use of the protein encoded by the QfAP1 gene in regulating the expression of the target gene PAL according to claim 7, characterized in that: The expression of the target gene PAL is reduced by inhibiting the expression of the protein encoded by the QfAP1 gene.

9. The use of the protein encoded by the QfAP1 gene in regulating the expression of the target gene PAL according to claim 7, characterized in that: The expression of the target gene PAL was increased by overexpressing the protein encoded by the QfAP1 gene.

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