阅读说明：本技术 AcAMS1在调控植物类黄酮类物质合成中的应用 (Application of AcAMS1 in regulation and control of synthesis of plant flavonoids ) 是由 李晓杰 梁毅 焦棒棒 曹琳娇 于 2020-11-06 设计创作，主要内容包括：本发明公开了AcAMS1在调控植物类黄酮类物质合成,尤其使花青素合成中的应用,AcAMS1具体可为如下A1)编码链的编码序列是序列表中序列1的cDNA分子或DNA分子；A2)编码链的核苷酸是序列表中序列1的cDNA分子或DNA分子。本发明的实施例将AcAMS1导入在拟南芥中,拟南芥种子的种皮颜色变浅,类黄酮类物质,尤其是花青素的合成受到抑制,类黄酮类物质含量尤其是花青素含量显著降低。该基因有望被用于调控植物类黄酮类物质含量、花青素含量和种皮颜色。(The invention discloses application of AcAMS1 in regulation and control of synthesis of plant flavonoids, in particular synthesis of anthocyanin, wherein the AcAMS1 can be specifically A1) coding sequence of a coding chain is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table; A2) the nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table. According to the embodiment of the invention, when the AcAMS1 is introduced into the Arabidopsis, the seed coat color of the Arabidopsis seed becomes light, the synthesis of flavonoid substances, especially anthocyanin is inhibited, and the content of the flavonoid substances, especially the content of anthocyanin is obviously reduced. The gene is expected to be used for regulating and controlling the content of plant flavonoid substances, the content of anthocyanin and the seed coat color.)

1. Use of a protein characterized by: the protein is AcAMS1, and the application is at least one of the following X1-X5:

the application of X1 in regulating and controlling the synthesis of plant flavonoids;

the application of X2 in regulating and controlling the content of plant flavonoids;

the application of X3 in regulating and controlling the synthesis of plant anthocyanin;

the application of X4 in regulating and controlling the content of plant anthocyanin;

the application of X5 in regulating and controlling the color of plant seed coat;

the AcAMS1 protein is a protein of a1), a2) or A3) as follows:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein having 90% or more identity to the protein represented by A1) and having 6-phosphoglucose isomerase activity, which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the protein represented by A1);

A3) a fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

2. Use of at least one of Y1-Y5 as a substance regulating gene expression:

the application of Y1 in regulating and controlling the synthesis of plant flavonoids;

the application of Y2 in regulating and controlling the content of plant flavonoids;

the application of Y3 in regulating and controlling the synthesis of plant anthocyanin;

the application of Y4 in regulating and controlling the content of plant anthocyanin;

the application of Y5 in regulating and controlling the color of plant seed coat;

the gene encodes the AcAMS1 protein of claim 1.

3. Use according to claim 2, characterized in that: the substance for regulating gene expression is biological material related to the AcAMS1 protein; the biological material is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B1);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector.

4. Use according to claim 3, characterized in that: B1) the nucleic acid molecule is specifically b1) or b2) as follows:

b1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table;

b2) the nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table.

5. Plant reagent comprising the AcAMS1 protein of claim 1 or the biological material related to the AcAMS1 protein of any one of claims 3-4, the plant reagent functioning as one or more of W1-W3:

w1 reduces the content of flavonoid substances;

w2 reduces the anthocyanin content;

w3 lightened the color of the seed coat.

6. A method for producing a plant having a low flavonoid content, comprising introducing a gene encoding AcAMS1 protein of claim 1 into a plant of interest to obtain a plant having a low flavonoid content; the plant with low content of flavonoid substance has flavonoid substance content lower than that of the target plant.

7. A method for producing a plant having a low anthocyanin content, comprising introducing a gene encoding AcAMS1 protein of claim 1 into a plant of interest to produce a plant having a low anthocyanin content; the plant with low anthocyanin content has anthocyanin content lower than that of the target plant.

8. A method for producing a plant with light seed coat color, comprising introducing a gene encoding AcAMS1 protein of claim 1 into a plant of interest to obtain a plant with light seed coat color; the color of the seed coat of the plant with light seed coat color is lighter than that of the seed coat of the target plant.

Technical Field

The invention relates to an application of AcAMS1 in the synthesis of plant flavonoids in the field of biotechnology.

Background

The flavonoid is a polyphenol secondary metabolite widely existing in plants, is a derivative of chromone or chromane, and is a natural product taking a C6-C3-C6 structure as a basic parent nucleus. Flavonoids are an important class of plant pigments, which affect the color of plant organs. Wanhuafang and Liangying research confirm that the seed coat pigment of Arabidopsis is determined by the content of flavonoid substances. The gazelle and the like summarize the biosynthesis pathway of flavonoid substances and can be divided into three stages: 1. synthesizing flavone and isoflavone; 2. synthesizing flavonol; 3. synthesis of anthocyanidin and proanthocyanidin.

The anthocyanin belongs to flavonoid substances, is a water-soluble pigment, and can change color along with the acid and alkali of cell sap. The cellular fluid is acidic and reddish, and the cellular fluid is alkaline and bluish. Anthocyanins (anthocyanins) are one of the main pigments that make up the color of petals and fruits, synthesized by the biosynthetic pathway of flavonoids.

Reference documents:

1. wanhua Fang, Liangying, research on the mechanism of formation of pigments in seed coat of Arabidopsis thaliana, the report of agricultural science in China, 5 th 2005.

2. Arborinus, Machulei, old, regulation of plant flavonoid biosynthetic pathway and important genes, natural product research and development, 2009, 21: 354-.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate the synthesis of plant flavonoid substances.

The first purpose of the invention is to provide the application of protein AcAMS1, wherein the application is at least one of the following X1-X5:

the application of X1 in regulating and controlling the synthesis of plant flavonoids;

the application of X2 in regulating and controlling the content of plant flavonoids;

the application of X3 in regulating and controlling the synthesis of plant anthocyanin;

the application of X4 in regulating and controlling the content of plant anthocyanin;

the application of X5 in regulating and controlling the color of plant seed coat;

the AcAMS1 protein is a protein of a1), a2) or A3) as follows:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein A1), has 90% or more of identity with the protein A1), and has 6-phosphoglucose isomerase activity;

A3) a fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

In the above application, the regulation of the synthesis of plant flavonoids can be the inhibition of the synthesis of plant flavonoids. The regulating and controlling of the content of the plant flavonoid substances can be the reduction of the content of the plant flavonoid substances. The regulating plant anthocyanin synthesis can be inhibiting plant anthocyanin synthesis. The regulating and controlling of the plant anthocyanin content can be reducing of the plant anthocyanin content. The color of the plant seed coat is regulated and controlled to be light.

In the above use, the plant may be a monocotyledon or a dicotyledon. The dicotyledonous plant may be a plant of the family cruciferae, such as arabidopsis thaliana.

In the above applications, the AcAMS1 protein may be synthesized artificially, or it may be obtained by synthesizing its encoding gene and then expressing it biologically.

In the above applications, the protein-tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag can be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc., for example, the AcAMS1 protein is expressed biologically by fusing cDNA encoding gene with GFP tag.

In the above applications, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Perresisure Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above uses, the 90% or greater identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The second object of the present invention is to provide the use of at least one of Y1-Y5 as a substance for regulating gene expression:

the application of Y1 in regulating and controlling the synthesis of plant flavonoids;

the application of Y2 in regulating and controlling the content of plant flavonoids;

the application of Y3 in regulating and controlling the synthesis of plant anthocyanin;

the application of Y4 in regulating and controlling the content of plant anthocyanin;

the application of Y5 in regulating and controlling the color of plant seed coat;

the gene encodes the AcAMS1 protein.

In the above application, the regulation of the synthesis of plant flavonoids can be the inhibition of the synthesis of plant flavonoids. The regulating and controlling of the content of the plant flavonoid substances can be the reduction of the content of the plant flavonoid substances. The regulating plant anthocyanin synthesis can be inhibiting plant anthocyanin synthesis. The regulating and controlling of the plant anthocyanin content can be reducing of the plant anthocyanin content. The color of the plant seed coat is regulated and controlled to be light.

In the above application, the plant may be a monocotyledon or a dicotyledon. The dicotyledonous plant may be a plant of the family cruciferae, such as arabidopsis thaliana.

In the above application, the substance for regulating gene expression is a biological material related to the AcAMS1 protein; the biological material is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B1);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, among others.

In the above application, in the biomaterial, the nucleic acid molecule B1) is a gene represented by B1) or B2) as follows:

b1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table;

b2) the nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table.

In the above applications, the expression cassette containing the nucleic acid molecule described in biomaterial B2) refers to a nucleic acid molecule capable of expressing AcAMS1 in a host cell. The nucleic acid molecule may include not only a promoter that initiates transcription of the AcAMS1 gene, but also a terminator that terminates transcription of AcAMS 1. Further, the expression cassette may also include an enhancer sequence.

The recombinant expression vector containing the AcAMS1 gene expression cassette can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When a plant expression vector is constructed using the gene of the present invention, an enhancer may also be used. In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding a marker gene for antibiotics (e.g., a gene conferring resistance to the antibiotic hygromycin).

In the above application, the recombinant microorganism in the biological material may be yeast, bacteria, algae and fungi.

In order to solve the above technical problems, the present invention also provides a plant agent which functions as one or more of W1-W3:

w1 reduces the content of flavonoid substances;

w2 reduces the anthocyanin content;

w3 lightened the color of the seed coat.

The plant reagent provided by the invention contains the AcAMS1 protein or/and biological materials related to the AcAMS1 protein.

The active ingredient of the plant agent can be AcAMS1 protein or/and biological materials related to the AcAMS1 protein, the active ingredient of the plant agent can also contain other biological ingredients or/and non-biological ingredients, and the other active ingredients of the plant agent can be used by a person skilled in the art according to the reduction effect of the content of plant flavonoids and/or the content of anthocyanin or the lightening effect of seed coats.

In order to solve the above technical problems, the present invention also provides a method for producing a plant having a low content of flavonoids. The method for producing the plant with low content of the flavonoid substances comprises the steps of introducing a gene coding the AcAMS1 protein into a target plant to obtain the plant with low content of the flavonoid substances; the plant with low content of flavonoids has lower content of flavonoids than the target plant.

The invention also provides a method of producing a plant with low anthocyanin content. The method for producing the plant with low anthocyanin content comprises the steps of introducing a gene coding the AcAMS1 protein into a target plant to obtain the plant with low anthocyanin content; the plant with low anthocyanin content has anthocyanin content lower than that of the target plant.

In order to solve the technical problem, the invention also provides a method for producing the plant with light seed coat color. The method for producing the plant with light seed coat color provided by the invention comprises the steps of introducing a gene coding the AcAMS1 protein into a target plant to obtain the plant with light seed coat color; the color of the seed coat of the plant with light seed coat color is lighter than that of the seed coat of the target plant.

The plant of interest may be a monocot or a dicot that does not contain the AcAMS1 gene. The dicotyledonous plant may be a crucifer, such as Arabidopsis thaliana.

The AcAMS1 gene can be introduced into plant cells by conventional biotechnological methods such as agrobacterium transformation.

The plant having a low flavonoid content and/or the plant having a low anthocyanin content and/or the plant having a light seed coat color may be a transgenic plant or a plant obtained by a conventional breeding technique such as crossing.

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In the embodiment of the invention, the AcaMS1 is introduced into the Arabidopsis, the seed coat color of the Arabidopsis seed is lightened, the content of flavonoid substances is proved to be reduced, the influence of the AcAMS1 on the content of plant anthocyanin is further examined, and the result shows that the content of the plant anthocyanin is obviously reduced after the AcAMS1 is expressed. The gene is expected to be used for regulating and controlling synthesis and content of flavonoids, especially anthocyanin, and further regulating and controlling plant organ colors such as seed coat color and the like.

Drawings

FIG. 1 is a bar graph of the expression level of onion AcAMS1 in white and red skin onions measured by real-time experiments in example 1, wherein Xiu Qiu is red skin onion variety and Ring master is white skin onion variety. Data are presented as mean ± standard deviation.

FIG. 2 is a graph showing the results of measuring the expression level of AcAMS1 in transgenic Arabidopsis thaliana of example 1, in which OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9 are all AcAMS1 transgenic lines, and Col is a control Arabidopsis thaliana wild type Col.

FIG. 3 is a photograph of a seed of transgenic Arabidopsis thaliana of example 1. In the figure, OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9 are all AcAMS1 transgenic lines, tt2-5 and tt8-1 are all anthocyanin synthesis regulatory gene mutants of Arabidopsis thaliana, and Col is a control Arabidopsis thaliana wild type Col.

FIG. 4 is a histogram of anthocyanin content in transgenic Arabidopsis thaliana of example 1. In the figure, OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9 are all AcAMS1 transgenic lines, Col is a control arabidopsis wild type Col, and tt2-5 and tt8-1 are all anthocyanin synthesis regulatory gene mutants of arabidopsis. Data are presented as mean ± sd with a repetition number of 3.

FIG. 5 is a histogram of the flavonoid content of transgenic Arabidopsis thaliana in example 1. In the figure, OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9 are all AcAMS1 transgenic lines, Col is a control arabidopsis wild type Col, and tt2-5 and tt8-1 are all anthocyanin synthesis regulatory gene mutants of arabidopsis. Data are presented as mean ± sd with a repetition number of 3.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

In the following examples, the vector pCAMBIA1300 is described in the non-patent literature "DEXH Box RNA Helicase-media specific Production in Arabidopsis thaliana proteins across proteins, [ J ] Plant Cell,2012,24(5): 1815-" 1833 ". The public can obtain from agriculture and forestry academy of sciences of Beijing city to repeat the experiment of the application, and can not be used as other purposes.

In the examples described below, Agrobacterium GV3101(BC304-01) is a product of Beijing Bomaide Gene technology, Inc.

In the examples described below, Arabidopsis thaliana mutant tt2-5(SALK _005260, 2105593) is the product of European Arabidopsis Stork Center.

In the examples described below, Arabidopsis thaliana mutant tt8-1(SALK _030966, 530966) is the European Arabidopsis Stork Center product.

In the following examples, red skin Onion "Xiu Qiu" and white skin Onion "Ring master" are described in the non-patent document "Transcriptome Sequencing and Metabolism Analysis developments of the role of Cyanidin Metabolism in Dark-red On (Allium cepa L.) Bulb, Scientific Reports,2018, 8(1): 14109". The application experiment is repeated by the public, which is available from agriculture and forestry academy of sciences in Beijing, and the application can not be used as other applications.

In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

Example 1

The ABORTED MICROPORES (AMS) gene in Arabidopsis thaliana is a transcription regulatory factor that regulates the development of pollen walls and the synthesis of sporopollenin, and this gene causes pollen abortion after mutation. The expression level of the homologous gene of the AtAMS in the onion, namely the AcAMS1 gene, in the white-skin onion is far higher than that of the red-skin onion. The coding sequence of the coding chain of the AcAMS1 gene is a sequence 1 in a sequence table, and the coding amino acid sequence is a protein AcAMS1 of a sequence 2 in the sequence table.

The inventor carries out real-time experiments on the expression level of the onion acaams 1 in white-skin onions and red-skin onions, and the specific results are shown in fig. 1, which indicates that the expression level of the onion acaams 1 in white-skin onions is far higher than that of the red-skin onions.

The results of the gene expression differences suggested that AcAMS1 may be an inhibitor of anthocyanin synthesis. The cDNA sequence of AcAMS1 (SEQ ID NO: 1 in the sequence Listing) was ligated into the engineered overexpression vector SUP1300(SUP1300 was an overexpression vector engineered according to the method described on page 1829 of "DEXH Box RNA Helicase-Mediated Mitochondrial Reactive Oxygen specifications Production in Arabidopsis thaliana Mediates Crosstalk. J.plant Cell,2012,24(5): 1815-1833.") using pCAMBIA1300 as the starting vector.

The specific connection steps are as follows: the primers AMS1-SalI-F and AMS1-KpnI-TAA-R were used to add SalI enzyme recognition site sequence at the 5 'end and KpnI enzyme recognition site sequence at the 3' end of the sequence 1 fragment by PCR. AMS 1-SalI-F5' -CCGGTCGACATGATAGAAGCACTGAGGCCAC-3' (the underlined sequence is the SalI enzyme recognition site sequence);

AMS1-KpnI-TAA-R:(the sequence indicated by the double underline is the KpnI enzyme recognition site sequence).

The sequence 1 fragment is obtained by PCR from a cDNA library of red skin onion 'Xiu Qiu', the sequence 1 and the SUP1300 vector are cut by two enzymes of SalI and KpnI, then are connected by T4 ligase, and are transformed into escherichia coli competence, and positive clones are selected for sequencing identification.

Through sequencing identification, the fragment (including a small fragment including SalI enzyme recognition site and KpnI enzyme recognition site) between the SalI and KpnI recognition sites of the restriction endonuclease of the SUP1300 vector replaced by the sequence 1 in the sequence table is kept unchanged by other sequences of the SUP1300 vector, and the recombinant expression vector of the AcAMS1 is obtained and is named as pSUP1300-AcAMS 1.

Agrobacterium GV3101 was transformed with a positive clone of pSUP1300-AcAMS1, and Columbia ecotype Arabidopsis Col (T) was transfected by pollen tube infection₀Generation). HarvestingT₁The generation seeds are screened by hygromycin of 30mg/L to obtain 12 positive plants, wherein the positive plants comprise plants with the plant numbers of OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9.

Taking Columbia ecotype arabidopsis thaliana Col as a wild type arabidopsis thaliana control (a control for short), carrying out AcAMS1 gene expression detection on T3 generation positive plants, respectively extracting total RNA of arabidopsis thaliana seedlings, carrying out reverse transcription, respectively taking cDNA obtained by the reverse transcription as a template, and carrying out Real Time-PCR identification by using AcAMS1 gene specific primers of AcAMS1-F and AcAMS 1-R.

AcAMS1-F：5’-ATTCTAGGAGATGCGATTGAGTACG-3’；

AcAMS1-R：5’-TTGTTGATGGTGTTGGTAATGAA-3’。

The Arabidopsis AtActin2 gene is used as an internal standard, and the used internal standard primers are Primer-TF and Primer-TR.

Primer-TF：5’-AGCACTTGCACCAAGCAGCATG-3’；

Primer-TR：5’-ACGATTCCTGGACCTGCCTCATC-3’。

The results are shown in FIG. 2, which shows that there is expression of the AcAMS1 gene in OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9, whereas there is no expression of the AcAMS1 gene in the wild type Arabidopsis control, and cDNA accumulation of AcAMS1 can be detected in the AcAMS1 overexpression lines OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9, indicating that these three lines are overexpression positive lines of AcAMS 1.

The color of the seed coat was observed under a microscope, and the results are shown in FIG. 3, which are similar to those of Arabidopsis wild type Col (in comparison, the seed coat colors of the AcAMS1 overexpression lines OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9 and Arabidopsis mutant tt2-5(SALK _005260) and Arabidopsis mutant tt8-1(SALK _030966) of the anthocyanin synthesis regulatory gene of Arabidopsis), all yellow and lighter in color, indicating that the synthesis of flavonoids is inhibited and the content of flavonoids in the seed coat is reduced.

The anthocyanidin content of the seed coats of the T3 generation Acams1 overexpression lines OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9 was further determined, and Columbia ecotype Arabidopsis Col, Arabidopsis mutant tt2-5(SALK _005260) and Arabidopsis mutant tt8-1(SALK _030966) were used as controls.

Experimental methods for determining the Anthocyanin content reference is made to the Radish Basic Helix-Loop-Helix transformation Factor, RsTT8 Acts a Positive Regulator for Anthocy biosynthesis, front Plant Sci 8: 1917. The experiment was set up in 3 replicates and the results of the assay are shown in figure 4, showing that the content of anthocyanins in seeds of acaams 1 overexpressing lines oeacaams-5, oeacaams-7, oeacaams-9 was significantly reduced compared to arabidopsis wild type Col, consistent with the phenotype observed in the microscope. The experimental results show that the overexpression of the AcAMS1 is really an inhibitor of anthocyanin synthesis.

To further determine the change in flavonoid content in transgenic plant seeds, we measured the flavonoid content in the seed coat using a plant flavonoid content detection kit (Solarbio, BC1330) using spectrophotometry, with controls of columbia ecotype arabidopsis thaliana Col, arabidopsis thaliana mutant tt2-5(SALK _005260), and arabidopsis thaliana mutant tt8-1(SALK _ 030966). As a result, as shown in FIG. 5, compared with wild-type Columbia ecotype Arabidopsis Col, the seed coat flavonoid contents of the ACAMS1 overexpression strains OEAcAMS-5, OEAcAMS-7 and OEAcAMS-9 were all decreased, indicating that the overexpression of AcAMS1 indeed inhibited the synthesis of flavonoids in the seed coat of Arabidopsis thaliana.

The results show that the overexpression of the AcAMS1 inhibits the synthesis of flavonoid substances, particularly the synthesis of anthocyanin, so that the content of the flavonoid substances, particularly the content of the anthocyanin in the Arabidopsis is reduced, and the color of the seed coat of the Arabidopsis is lightened.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> agriculture and forestry academy of sciences of Beijing City

Application of <120> AcAMS1 in regulation and control of synthesis of plant flavonoids

<130> GNCSY202366

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1458

<212> DNA

<213> onion (Allium cepa)

<400> 1

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aaattatctc ctgatcaaag attcttattg tggaatggat gctactgttc tggtgctgat 120

catgcaaaag tcaaaaacgg agtgatgtta ccggctacaa cactttgcag agatacaaaa 180

ttcgagcatc catggacaaa ttcatgcagc gatctttcag agtttccttc ttctgttcca 240

ctggactctt ctcttccgat ttatgcacaa gtactgatgt ctaaccaacc aatctggcaa 300

catttagacc atcctggttc aaccagctca caagaaacaa taggagcaaa aacaagggtt 360

cttgttccgg ttacgggtgg gatagttgag ctctttgtgt cgaaacaagt gacagaagat 420

caacaaatca cagacttcat catgtctcag tgcaacgcgg actctgactt tcttgaaaac 480

aatggtcatg agataaatca ctggatctcg ggagaacata acaactttca aaacggctca 540

gaccagtttg ataactctgc atttaaactg tttgattcta cacttactgc agtgtacaac 600

aataatcagc agcagcagtc tgtacatgat gctgattcag tgaagcagga ggcgccaacc 660

gtacgtggag acaattctgg aagcgaagga agtgaggatg atgacgaagg tggttctaga 720

acagttggaa gaaacgggaa gagacatcac tcaaagaatc taatggcaga gaggaagagg 780

aggaagaagc ttaatgatag gctatatgca ctaagagcat tggttcctaa gatcactaag 840

atggatagag catcaattct aggagatgcg attgagtacg tgatggagtt gcagaagcaa 900

gtaaaggagc ttgaggagga gttggaaaat gaaacgaatc atgatgacga ggcaaagcaa 960

tatgaaagca acttcgatat ggtaacgacg aatttgaatg ggttgttgaa tgatcaggcc 1020

atggagcttg atgaatctcc aaaactgagc aagctggagc attcaagttc ggaagaaaag 1080

gttaatcaga tggagccaca agttgaagtg aagcaattag atgggaatga gttttacctc 1140

aagattttgt gtgaacacaa gtttggagga ttttcaaggt tgatggaggc aataagttcg 1200

ctcggacttg aagttacaaa tgcgaccgtg accacgcaac aaagtttagt acttaacgta 1260

ttcacagttc agagaaggga taatgaaaca atgcaagtgg accaagtgag ggactcattg 1320

ctggagctga ctcgagggcc agttggaaac tggatggagt ctggaattgt tgcagcggtg 1380

aataatggtg attatcagca ggagcattgt cataatcaac accaccatca tcttcattac 1440

caacaccatc aacaatga 1458

<210> 2

<211> 485

<212> PRT

<213> onion (Allium cepa)

<400> 2

Met Ile Glu Ala Leu Arg Pro Leu Leu Cys Ser Asn Gly Trp Asp Tyr

1 5 10 15

Cys Ile Leu Trp Lys Leu Ser Pro Asp Gln Arg Phe Leu Leu Trp Asn

20 25 30

Gly Cys Tyr Cys Ser Gly Ala Asp His Ala Lys Val Lys Asn Gly Val

35 40 45

Met Leu Pro Ala Thr Thr Leu Cys Arg Asp Thr Lys Phe Glu His Pro

50 55 60

Trp Thr Asn Ser Cys Ser Asp Leu Ser Glu Phe Pro Ser Ser Val Pro

65 70 75 80

Leu Asp Ser Ser Leu Pro Ile Tyr Ala Gln Val Leu Met Ser Asn Gln

85 90 95

Pro Ile Trp Gln His Leu Asp His Pro Gly Ser Thr Ser Ser Gln Glu

100 105 110

Thr Ile Gly Ala Lys Thr Arg Val Leu Val Pro Val Thr Gly Gly Ile

115 120 125

Val Glu Leu Phe Val Ser Lys Gln Val Thr Glu Asp Gln Gln Ile Thr

130 135 140

Asp Phe Ile Met Ser Gln Cys Asn Ala Asp Ser Asp Phe Leu Glu Asn

145 150 155 160

Asn Gly His Glu Ile Asn His Trp Ile Ser Gly Glu His Asn Asn Phe

165 170 175

Gln Asn Gly Ser Asp Gln Phe Asp Asn Ser Ala Phe Lys Leu Phe Asp

180 185 190

Ser Thr Leu Thr Ala Val Tyr Asn Asn Asn Gln Gln Gln Gln Ser Val

195 200 205

His Asp Ala Asp Ser Val Lys Gln Glu Ala Pro Thr Val Arg Gly Asp

210 215 220

Asn Ser Gly Ser Glu Gly Ser Glu Asp Asp Asp Glu Gly Gly Ser Arg

225 230 235 240

Thr Val Gly Arg Asn Gly Lys Arg His His Ser Lys Asn Leu Met Ala

245 250 255

Glu Arg Lys Arg Arg Lys Lys Leu Asn Asp Arg Leu Tyr Ala Leu Arg

260 265 270

Ala Leu Val Pro Lys Ile Thr Lys Met Asp Arg Ala Ser Ile Leu Gly

275 280 285

Asp Ala Ile Glu Tyr Val Met Glu Leu Gln Lys Gln Val Lys Glu Leu

290 295 300

Glu Glu Glu Leu Glu Asn Glu Thr Asn His Asp Asp Glu Ala Lys Gln

305 310 315 320

Tyr Glu Ser Asn Phe Asp Met Val Thr Thr Asn Leu Asn Gly Leu Leu

325 330 335

Asn Asp Gln Ala Met Glu Leu Asp Glu Ser Pro Lys Leu Ser Lys Leu

340 345 350

Glu His Ser Ser Ser Glu Glu Lys Val Asn Gln Met Glu Pro Gln Val

355 360 365

Glu Val Lys Gln Leu Asp Gly Asn Glu Phe Tyr Leu Lys Ile Leu Cys

370 375 380

Glu His Lys Phe Gly Gly Phe Ser Arg Leu Met Glu Ala Ile Ser Ser

385 390 395 400

Leu Gly Leu Glu Val Thr Asn Ala Thr Val Thr Thr Gln Gln Ser Leu

405 410 415

Val Leu Asn Val Phe Thr Val Gln Arg Arg Asp Asn Glu Thr Met Gln

420 425 430

Val Asp Gln Val Arg Asp Ser Leu Leu Glu Leu Thr Arg Gly Pro Val

435 440 445

Gly Asn Trp Met Glu Ser Gly Ile Val Ala Ala Val Asn Asn Gly Asp

450 455 460

Tyr Gln Gln Glu His Cys His Asn Gln His His His His Leu His Tyr

465 470 475 480

Gln His His Gln Gln

485