CN114014917B

CN114014917B - FvbHLH36 protein, and encoding gene and application thereof

Info

Publication number: CN114014917B
Application number: CN202111262122.4A
Authority: CN
Inventors: 连红莉; 许鹏博; 曹明浩; 肖坤; 李歆渝
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-10-28
Filing date: 2021-10-28
Publication date: 2023-09-08
Anticipated expiration: 2041-10-28
Also published as: CN114014917A

Abstract

The invention belongs to the technical field of molecular biology, and discloses FvbHLH36 protein, and a coding gene and application thereof. An FvbHLH36 protein which is a protein of (1) or (2) as follows: (1) A protein consisting of an amino acid sequence shown as SEQ ID NO. 2; (2) And (3) the protein which is derived from the (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown as SEQ ID NO. 2. The FvbHLH36 gene has the function of regulating and controlling plant anthocyanin synthesis and/or plant anthocyanin accumulation, and can promote plants to be colored faster and deepened. The FvbHLH36 gene provides a new gene resource for molecular breeding for promoting synthesis and/or accumulation of plant anthocyanin.

Description

FvbHLH36 protein, and encoding gene and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to FvbHLH36 protein, and a coding gene and application thereof.

Background

Strawberry belongs to the genus strawberry of the family Rosaceae, and is a perennial herb with higher economic value. At present, the strawberry planting area and yield in China are the first in the world. The strawberry fruit has juicy pulp and rich flavor, and the color is mainly red and dark red, so the color is mainly shown because the strawberry fruit is rich in a large amount of anthocyanin. In recent years, more and more evidence shows that anthocyanin in fruits has good health care function for human bodies. The anthocyanin not only has the functions of antioxidation and antibiosis, but also has the functions of resisting cancer, cardiovascular diseases, diminishing inflammation and the like. Due to the improvement of living standard, the demands of people for fruits with high nutritive value are gradually increased. The color of the fruit, as its external quality trait, is a direct factor in attracting consumers to purchase. Therefore, the method can provide a good theoretical basis for breeding new varieties by excavating genes for regulating and controlling anthocyanin content and controlling fruit color and analyzing regulating and controlling mechanisms, and has good application prospect.

The biosynthetic pathway and regulatory mechanism of anthocyanin have been studied intensively in petunia and in the model plant Arabidopsis thaliana. Among them, MYB and bHLH are two major families of transcription factors in plants, which not only play an extremely important role in secondary metabolism of plants, but also both have related genes that promote or inhibit anthocyanin synthesis, ultimately determining the coloration of plants. The MYB transcription factor FvMYB10/FaMYB10 currently identified in diploid forest strawberries and octaploid strawberries is a key gene that promotes anthocyanin accumulation in strawberry fruits. The research shows that the change of the amino acid sequence of FvMYB10 protein can cause that the FvMYB10 protein can not normally regulate and control the expression of the anthocyanin synthesis key enzyme genes, so that anthocyanin can not be accumulated in strawberry fruits. The overexpression of FvMYB10 gene in strawberry can cause the accumulation of anthocyanin in the leafstalk and fruit of strawberry, thereby verifying that FvMYB10 transcription factor has the function of regulating anthocyanin synthesis. The bHLH transcription factor is the second largest family of proteins following MYB. In plants, bHLH transcription factors are involved in the regulation of many cellular processes, such as development of floral organs, photomorphogenesis, development of trichomes, root hairs and stomata, hormonal responses, and the like. In addition, bHLH transcription factors in many species are also involved in the regulation of anthocyanin biosynthesis.

However, there are very limited reports of currently clear bHLH-like transcription factors involved in regulating anthocyanin synthesis in strawberry fruits. The inventor has found from earlier research that the strawberry FvbHLH36 can promote anthocyanin synthesis, so that the invention can perform functional research on the gene on the basis, thereby being beneficial to understanding plant anthocyanin synthesis in molecular aspect and being beneficial to molecular breeding work for screening varieties of plants with different colors.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide FvbHLH36 protein, and encoding gene and use thereof, thereby solving the problems of the prior art.

It is an object of the present invention to provide an FvbHLH36 protein which is a protein of the following (1) or (2):

(1) A protein consisting of an amino acid sequence shown as SEQ ID NO. 2;

(2) And (3) the protein which is derived from the (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown as SEQ ID NO. 2.

It is a second object of the present invention to provide a biological material related to the protein as described, comprising any one of the following:

a) A polynucleotide encoding a protein as described;

b) A recombinant expression vector comprising the polynucleotide of a);

c) A bioengineering bacterium containing the polynucleotide of a) or a bioengineering bacterium containing the recombinant expression vector of b);

d) A transgenic plant cell comprising the polynucleotide of a), or a transgenic plant comprising the recombinant expression vector of b).

According to the technical scheme of the invention, in the a), the sequence of the polynucleotide comprises a sequence shown as SEQ ID NO. 1.

It is a further object of the present invention to provide the use of said FvbHLH36 protein or said polynucleotide as a target in the preparation or screening of products regulating plant anthocyanin synthesis and/or anthocyanin accumulation.

According to the technical scheme of the invention, the product comprises an FvbHLH36 inhibitor, wherein the FvbHLH36 inhibitor refers to a compound with an inhibiting effect on FvbHLH36 or the FvbHLH36 inhibitor is the only active ingredient or one of the active ingredients of the product; or alternatively, the first and second heat exchangers may be,

the product comprises an FvbHLH36 accelerator, wherein the FvbHLH36 accelerator refers to a compound with an accelerating effect on FvbHLH36 or the FvbHLH36 accelerator is the only effective component or one of the effective components of the product.

The fourth object of the present invention is to provide the use of said protein or said biological material for modulating plant anthocyanin synthesis and/or anthocyanin accumulation.

According to the technical scheme of the invention, the application comprises one or more of the following:

1) Regulating and controlling synthesis of plant anthocyanin;

2) Regulating and controlling the anthocyanin content of plants;

3) Regulating and controlling plant anthocyanin accumulation;

4) Regulating and controlling the color change of plants;

5) Preparing the related products in 1) to 4);

6) Cultivating the relevant plants of 1) to 4).

It is a fifth object of the present invention to provide a method of modulating plant anthocyanin synthesis and/or anthocyanin accumulation, the method comprising one or more of the following steps:

d1 Introducing FvbHLH36 protein as described;

d2 Introducing a biological material as described;

d3 Introducing a FvbHLH36 promoter as described;

d4 Introducing an FvbHLH36 inhibitor as described;

d5 Knocking out or altering the gene encoding the FvbHLH36 protein as described, inactivating or reducing its function.

According to the technical scheme of the invention, one or more of the steps D1), D2) or D3) are used for improving the anthocyanin content and/or anthocyanin accumulation of plants;

or, one or both of steps D4) or D5) for reducing plant anthocyanin content and/or anthocyanin accumulation.

According to the technical scheme of the invention, the plant color comprises stem color, leaf color, flower color and fruit color.

According to the technical scheme of the invention, the plant is a dicotyledonous plant or a monocotyledonous plant, preferably strawberry or arabidopsis thaliana.

The invention has the following beneficial effects:

1. according to the invention, the FvbHLH36 gene is cloned from strawberry, a recombinant expression vector is successfully constructed, and the recombinant expression containing the FvbHLH36 gene is transformed into the model plant Arabidopsis thaliana by adopting an agrobacterium-mediated method, so that compared with the wild Arabidopsis thaliana, the FvbHLH36 gene is introduced, the whole plant of the Arabidopsis thaliana can be more quickly colored and darker in color, and the relative content of anthocyanin of the whole plant is improved by more than 8.5 times compared with that of the wild Arabidopsis thaliana, and the FvbHLH36 gene has the function of promoting anthocyanin synthesis and improving anthocyanin synthesis.

2. According to the invention, recombinant expression containing FvbHLH36 gene is transiently expressed in strawberry fruits by an agrobacterium-mediated method, the strawberry fruits are more rapid to color and darker in color, and compared with a control group, the relative content of anthocyanin in the strawberry fruits can be improved by more than 5.6 times, and the relative expression amount of FvbHLH36 in the fruits is improved by more than 4.3 times, so that the FvbHLH36 gene can promote anthocyanin synthesis in the strawberry fruits and improve anthocyanin content.

3. The recombinant expression vector containing the FvbHLH36 gene constructed by the invention not only can over-express the FvbHLH36 gene, but also can realize transient FvbHLH36 gene expression in strawberry fruits by adopting agrobacterium mediation to obtain a novel germplasm of the FvbHLH36 gene over-expression, thereby providing a novel gene resource for promoting molecular breeding of plant anthocyanin biosynthesis.

Drawings

FIG. 1 shows an electrophoretogram of PCR products of the FvbHLH36 gene coding region sequence in example 1 of the present invention.

FIG. 2A shows a phenotypic chart of the staining of T2-transgenic Arabidopsis and wild type Arabidopsis plants in example 3 of the present invention.

FIG. 2B is a graph showing the relative anthocyanin levels in T2-transgenic Arabidopsis and wild-type Arabidopsis plants in example 3 of the present invention.

FIG. 3A is a phenotypic chart showing the coloration of strawberry fruits after injection of Agrobacterium GV3101 containing the recombinant expression vector pHB-FvbHLH36-Flag in example 4 of the present invention.

FIG. 3B is a graph showing the relative anthocyanin content in strawberry fruits injected with Agrobacterium GV3101 containing the recombinant expression vector pHB-FvbHLH36-Flag in example 4 of the present invention.

FIG. 3C is a graph showing the relative expression level of FvbHLH36 gene in strawberry fruits injected with Agrobacterium GV3101 containing the recombinant expression vector pHB-FvbHLH36-Flag in example 4 of the present invention.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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 invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

The invention aims to overcome the defects in the prior art and provides an application of a transgenic FvbHLH36 gene in regulating and controlling plant anthocyanin synthesis and/or regulating and controlling plant anthocyanin accumulation. The gene cloning, vector construction, genetic transformation, molecular detection and anthocyanin content determination are used for the invention, and a method for stably improving plant anthocyanin accumulation and/or promoting plant anthocyanin synthesis is established.

In a first aspect of the present invention, there is provided a FvbHLH36 protein, which is a protein of (1) or (2) as follows: (1) A protein consisting of an amino acid sequence shown as SEQ ID NO. 2; (2) And (3) the protein which is derived from the (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown as SEQ ID NO. 2.

The sequence shown in SEQ ID NO.2 is specifically:

MDINQLKSEEQMEMMMMMQMDKISELCGAYNDVVSDLPSSEHFSGSATSMPHYSHDQNPHTVSSSPPSSFLNLHPSTISFTNSLEQSPEPQLASDKRASMAAMREMIFRIASMQPIHIDPESVKPPKRRNVKISKDPQSVAARHRRERISERIRILQRLVPGGTKMDTASMLDEAIHYVKFLKSQVQTLERAAVNNNNNRVQQTGIGFPVAMSSGSNYLSNSMAKAYQAHPPQHHQNVQHFGDA

in another aspect of the present invention, there is also provided a biological material associated with the protein described above, comprising any one of the following:

a) A polynucleotide encoding a protein as described above;

b) A recombinant expression vector comprising the polynucleotide of a);

c) A bioengineering bacterium containing a) the polynucleotide, or b) the recombinant expression vector;

d) A transgenic plant cell comprising a) said polynucleotide, or a transgenic plant comprising b) said recombinant expression vector.

In the present invention, a) the polynucleotide encoding the protein according to the first aspect may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA; the DNA may be single-stranded or double-stranded; the DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature protein may be identical to the coding region sequence in SEQ ID NO.1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein having the sequence shown in SEQ ID NO.2, but differs from the coding region sequence in SEQ ID NO. 1.

In one embodiment of the invention, the polynucleotide is extracted from the tissue of the forest strawberry "Rugen". The polynucleotide is shown as the sequence of SEQ ID NO. 1.

The sequence shown in SEQ ID NO.1 is specifically:

atggatatcaaccaactaaaatcagaggagcagatggaaatgatgatgatgatgcaaatggataaaatctccgagctctgtggcgcctacaacg

acgtcgtttccgacctcccttcatctgaacacttttctggtagtgcaacttccatgccacattacagccatgaccaaaaccctcacactgtttcttcttc

acctccatcctcatttcttaacctacacccttcaaccatatcattcaccaactcacttgaacaatcaccagaacctcaactggcatcggataagcgtg

cttcaatggcggctatgagggagatgatattcagaatcgcctcgatgcagccaatccacatagacccggagtcggtgaagccgccaaagagaa

ggaacgtgaagatttccaaggaccctcagagcgtggcggctcgtcacaggagggagaggattagcgagaggataagaatactccagcgact

agtccccgggggaactaaaatggacactgcatccatgctggacgaggctattcactacgtcaaattcttgaagtctcaagttcagacgctggaga

gagccgccgtgaataataacaataacagggtacaacaaaccgggattggttttccggtggcgatgtcaagtgggagtaattacctttctaattctat

ggccaaagcgtaccaagcccatcctccgcagcatcatcagaatgtgcagcatttcggcgacgcttga

in the present invention, the full-length polynucleotide sequence of FvbHLH36 gene or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the nucleotide sequences, particularly the open reading frame sequences, described in the examples of the present invention, and amplified using a commercially available cDNA library or cDNA prepared according to a conventional method known to those skilled in the art as a template to obtain the relevant sequences. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

In a specific embodiment of the invention, the polynucleotide shown in SEQ ID NO.1 is obtained by a method comprising the steps of: extracting total genome RNA of forest strawberries (Rugen), carrying out reverse transcription on the obtained total genome RNA by reverse transcriptase to obtain a first cDNA strand, wherein according to the polynucleotide shown as SEQ ID NO.1, an upstream primer is a sequence shown as SEQ ID NO.3, and a downstream primer is a sequence shown as SEQ ID NO. 4; and (3) taking the first strand of the cDNA as a template, taking the upstream primer and the downstream primer as primer pairs, and performing PCR amplification to obtain the cDNA.

In the examples of the present invention, the term "polynucleotide" may be used to refer to a polynucleotide which has been isolated from a sequence which is naturally located on either side of the polynucleotide, or it may be used to refer to a polynucleotide which has been isolated from a component which naturally accompanies a nucleic acid and which has been isolated from a protein which accompanies a cell.

In the present invention, b) a recombinant expression vector comprising the polynucleotide of a). In the present invention, the polynucleotide sequence encoding the protein may be inserted into an expression vector to form a recombinant expression vector. The term "expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements. The expression vector in the present invention is not limited to the PHB vector mentioned in the following examples. Methods well known to those skilled in the art can be used to construct expression vectors containing nucleotide sequences encoding FvbHLH36 protein and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In a specific embodiment of the application, the PCR amplification product of the FvbHLH36 gene obtained in the first aspect is ligated to a cloning vector to obtain an intermediate vector; then, taking the intermediate vector as a template, respectively introducing PstI restriction enzyme cutting sites and BamH restriction enzyme cutting sites before and after FvbHLH36 genes to obtain full-length fragments of target genes, then using PstI enzyme and BamH restriction enzyme cutting expression vectors, recovering FvbHLH36 gene fragments and expression vector fragments, connecting and transforming, selecting monoclonal, extracting plasmids for PCR detection and restriction enzyme cutting verification, and obtaining recombinant expression vectors.

Preferably, the cloning vector is pLB vector, and intermediate vector pLB-FvbHLH36 is obtained; the expression vector is pHB, and a recombinant expression vector pHB-FvbHLH36-Flag is obtained.

Specifically, the construction method of the recombinant expression vector pHB-FvbHLH36-Flag comprises the following steps: the intermediate vector pLB-FvbHLH36 is taken as a template, pstI restriction enzyme sites and BamHI restriction enzyme sites are introduced, a forward primer and a reverse primer are taken as primer pairs, wherein the forward primer comprises a sequence shown as SEQ ID NO.5, the reverse primer comprises a sequence shown as SEQ ID NO.6, fvbHLH36 gene products containing the restriction enzyme sites are obtained, and the FvbHLH36 gene products are connected to a pHB vector after restriction enzyme through ligase T4 DNA ligase after PCR amplification, so that the recombinant expression vector pHB-FvbHLH36-Flag is obtained.

In the present application, the bioengineering bacterium containing the polynucleotide of a) or the bioengineering bacterium containing the recombinant expression vector of b) is contained in c). The bioengineering bacteria contain the recombinant expression vector or the polynucleotide as described above integrated in the genome. The bioengineering bacteria are cultured to enable expression of the protein. The bioengineered bacteria may be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: coli, streptomyces, agrobacterium; fungal cells such as yeast; plant cells, and the like. Not limited to the Agrobacterium GV3101 used in the present application.

In the present application, d) a transgenic plant cell containing the polynucleotide of a), or a transgenic plant containing the recombinant expression vector of b). In the present application, the plant suitable for use in the present application is not particularly limited as long as it is suitable for performing a gene transformation operation.

In another aspect of the application there is also provided the use of a FvbHLH36 protein or a polynucleotide as described above as a target in the preparation or screening of a product that modulates plant anthocyanin synthesis and/or anthocyanin accumulation.

In the invention, the FvbHLH36 gene serving as a target is used for preparing or screening a product for regulating and controlling plant anthocyanin synthesis and/or anthocyanin accumulation, and the product specifically comprises the following components: the FvbHLH36 gene or FvbHLH36 protein is taken as an acting object, and products are screened to find products which can promote or inhibit the expression level of the FvbHLH36 gene or promote or inhibit the expression or activity of the FvbHLH36 protein as alternative products for regulating the synthesis and/or accumulation of anthocyanin in plants.

In the invention, the product for regulating and controlling the synthesis and/or the accumulation of the plant anthocyanin comprises molecules which can specifically inhibit the transcription or translation of the FvbHLH36 gene or can specifically inhibit the expression or activity of the FvbHLH36 protein, thereby reducing the expression level of the FvbHLH36 gene in the plant and achieving the purpose of inhibiting the synthesis of the plant anthocyanin or reducing the content of the plant anthocyanin.

In the invention, the product for regulating and controlling the synthesis of the plant anthocyanin or the anthocyanin content comprises molecules which can specifically promote the transcription or translation of the FvbHLH36 gene or can specifically promote the expression or activity of the FvbHLH36 protein, thereby improving the expression level of the FvbHLH36 gene in the plant and achieving the purposes of promoting the synthesis of the plant anthocyanin or improving the anthocyanin content of the plant.

In the invention, the product comprises an FvbHLH36 inhibitor, wherein the FvbHLH36 inhibitor refers to a compound with an inhibiting effect on FvbHLH36 or the FvbHLH36 inhibitor is the only active ingredient or one of the active ingredients of the product; or alternatively, the first and second heat exchangers may be,

In the present invention, the product necessarily includes an FvbHLH36 inhibitor or FvbHLH36 accelerator, and the FvbHLH36 inhibitor or FvbHLH36 accelerator is used as an active ingredient for the aforementioned efficacy.

In the invention, the effective component in the product can be only FvbHLH36 inhibitor or FvbHLH36 promoter, and can also contain other molecules with inhibiting or promoting effects.

In the present invention, the product may be a single component substance or a multi-component substance.

In the present invention, the form of the product is not particularly limited, and may be various forms of substances such as solid, liquid, gel, semifluid, aerosol, and the like.

In the invention, the object to which the product is mainly directed is a plant. The plant includes a dicotyledonous plant or a monocotyledonous plant. The dicotyledonous plant is preferably strawberry, arabidopsis thaliana or tomato.

In the present invention, the products include, but are not limited to, pesticides, fertilizers, and the like.

In the present invention, the FvbHLH36 inhibitor or the FvbHLH36 promoter includes, but is not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses.

In the invention, the FvbHLH36 inhibitor or the FvbHLH36 promoter is adopted to regulate and control the synthesis and/or the anthocyanin accumulation of the plant, and the aim of regulating and controlling the synthesis and/or the anthocyanin content of the plant is mainly achieved by promoting the expression level of FvbHLH36 gene in the plant or inhibiting the expression level of FvbHLH36 gene in the plant.

In another aspect of the invention there is provided the use of a protein as hereinbefore described or a biological material as hereinbefore described for modulating plant anthocyanin synthesis and/or anthocyanin accumulation.

In the present invention, the use includes one or more of the following:

1) Regulating and controlling synthesis of plant anthocyanin;

2) Regulating and controlling the anthocyanin content of plants;

3) Regulating and controlling plant anthocyanin accumulation;

4) Regulating and controlling the color change of plants;

5) Preparing the related products in 1) to 4);

6) Cultivating the relevant plants of 1) to 4).

In another aspect of the invention, there is also provided a method of modulating plant anthocyanin synthesis and/or anthocyanin accumulation, the method comprising one or more of the following steps:

D1 Introducing FvbHLH36 protein as described;

d2 Introducing a biological material as described;

d3 Introducing a FvbHLH36 promoter as described;

d4 Introducing an FvbHLH36 inhibitor as described;

In the present invention, one or more of steps D1) or D2) or D3) are used to increase plant anthocyanin content and/or anthocyanin accumulation; or, one or both of steps D4) or D5) for reducing plant anthocyanin content and/or anthocyanin accumulation.

In the present invention, a method for growing plants having increased anthocyanin synthesis or accumulation or content, comprising increasing the content of FvbHLH36 protein as described in the plants or increasing the activity of FvbHLH36 protein as described in the plants or increasing FvbHLH36 promoter as described in the plants, to obtain plants having increased anthocyanin content.

In the present invention, a method for growing a plant with reduced anthocyanin synthesis or accumulation or content comprising reducing the content of FvbHLH36 protein as described in the plant or reducing the activity of FvbHLH36 protein as described in the plant or increasing the FvbHLH36 promoter as described in the plant, to obtain a plant with reduced anthocyanin content.

In the present invention, a method for growing a plant color-changing plant comprising changing the amount of FvbHLH36 protein as described in the plant or changing the activity of FvbHLH36 protein as described in the plant or increasing FvbHLH36 promoter or FvbHLH36 inhibitor as described in the plant, to obtain a plant color-changing plant.

In the present invention, the plant colors include stem color, leaf color, flower color, and fruit color.

The plant is a dicotyledonous or monocotyledonous plant, preferably strawberry or Arabidopsis thaliana.

In some embodiments of the invention, methods of growing plants with increased anthocyanin synthesis or accumulation or content can be accomplished by introducing into the plant a gene encoding a FvbHLH36 protein as described above and allowing expression of the gene, or by introducing into the plant a gene encoding a FvbHLH36 protein as described above or a recombinant expression vector as described above. Wherein, the method for introducing the polynucleotide is electroporation method, microinjection and gene gun; the method for introducing the recombinant expression vector is an agrobacterium-mediated method.

In one embodiment of the present invention, a recombinant expression vector containing the FvbHLH36 gene is introduced into a model plant arabidopsis thaliana by agrobacterium mediation, comprising the steps of: transforming a recombinant expression vector pHB-FvbHLH36-Flag into agrobacterium; agrobacterium containing the recombinant expression vector pHB-FvbHLH36-Flag was co-cultured with the model plant. Wherein the co-cultivation comprises the steps of: soaking the mode plant, transferring the mode plant into a co-culture medium, adding activated 1/2MS suspension of agrobacterium GV3101 containing pHB-FvbHLH36-Flag recombinant expression vector, fully contacting the mode plant with bacterial liquid, and performing dark culture at 28 ℃ for 24 hours to obtain transgenic arabidopsis; and meanwhile, the wild type arabidopsis thaliana is taken as a control group.

The transgenic arabidopsis plant with increased anthocyanin content is obtained through phenotype observation of coloring of the transgenic arabidopsis plant and anthocyanin content measurement. The relative anthocyanin content in transgenic arabidopsis is 8.5 times that of the control group, which shows that FvbHLH36 gene can promote the mode plants to accumulate more anthocyanin.

In one embodiment of the invention, the instant transformation of the FvbHLH36 gene in tsaoko fruits by agrobacterium mediation comprises the steps of: injecting the activated YEP liquid culture medium heavy suspension of agrobacterium tumefaciens GV3101 containing pHB-FvbHLH36-Flag recombinant expression vector into strawberry fruits, and culturing for 5-7 days to obtain transiently expressed strawberry fruits; strawberry fruits injected with a suspension of YEP liquid medium of Agrobacterium GV3101 containing no target gene but pHB-Flag were used as a control group.

And obtaining the transgenic strawberry with improved anthocyanin content through phenotype observation of coloring of the transiently expressed strawberry fruits, and measurement of the relative anthocyanin content and the relative FvbHLH36 expression. After transient expression, the relative content of anthocyanin in the strawberry fruits is more than 5.6 times that of a control group, and the relative expression amount of FvbHLH36 gene in the strawberry fruits is 4.3 times that of the control group, so that the FvbHLH36 gene can promote anthocyanin synthesis in the strawberry fruits and improve anthocyanin content, and plant varieties with different colors, such as strawberries or arabidopsis thaliana, can be screened by controlling the FvbHLH36 gene.

FvbHLH36 can regulate and control plant anthocyanin synthesis or plant anthocyanin content, especially can promote plant anthocyanin synthesis or improve plant anthocyanin content, and anthocyanin not only has important effect on plants, but also is a natural antioxidant for people, and can remove oxygen free radicals in the human body, so that collective aging is delayed. Therefore, the FvbHLH36 gene is subjected to functional research, so that the synthesis mechanism of plant anthocyanin is well known in molecular aspects, and molecular breeding work of screening plant varieties with high anthocyanin is well facilitated, a gene intelligent manufacturer of anthocyanin is provided for human health, and candidate genes are provided for improvement of plant anthocyanin content traits in the future.

A more specific embodiment of the invention is:

(1) Acquisition of full-length FvbHLH36 coding nucleic acid sequence

a. Taking leaves of forest strawberries Rugen as a material, extracting total RNA of the leaves, and then obtaining a cDNA first chain through reverse transcription;

b. designing a pair of specific primers FvbHLH36-F1 and FvbHLH36-R1, and obtaining the full-length nucleotide sequence of FvbHLH36 gene by PCR amplification by taking the first strand of cDNA as a template;

Wherein, the specific primer is:

FvbHLH36-F1：5'-ATGGATATCAACCAACTAAAATCAGAG-3'(SEQ ID NO.3)

FvbHLH36-R1：5'-TCAAGCGTCGCCGAAATGCTG-3'(SEQ ID NO.4)

(2) Construction of the overexpression vector pHB-Flag-FvbHLH36

a. Designing a pair of specific primers FvbHLH36-F2 and FvbHLH36-R2 containing enzyme cutting sites, taking the FvbHLH36 gene product obtained in the step (1) as a template, and obtaining the full length of the FvbHLH36 gene containing enzyme cutting sites through PCR amplification;

wherein the PstI cleavage site is introduced into the upstream primer FvbHLH36-F2, and the BamHI cleavage site is introduced into the downstream primer FvbHLH 36-R2. The specific sequences of the primers FvbHLH36-F2 and FvbHLH36-R2 are:

FvbHLH36-F2：AAACTGCAG ATGGATATCAACCAACTAAAATCAGAG(SEQ ID NO.5)

FvbHLH36-R2：GCCGGATCCAGCGTCGCCGAAATGCTG(SEQ ID NO.6)

note that: underlined bases in FvbHLH36-F2 and FvbHLH36-R2 primer sequences are protective bases and cleavage sites, are artificially introduced for constructing interference vectors, and do not belong to the sequence of FvbHLH36 gene. The FvbHLH36-R2 primer removes the stop codon due to the requirement of constructing the Flag fusion expression vector.

b. Double-enzyme cutting is carried out on the full length of the FvbHLH36 gene with the enzyme cutting site obtained in the step a by using restriction enzymes PstI and BamHI, and the enzyme cutting product is purified and recovered to obtain the full length product of the FvbHLH36 gene with the sticky end;

c. double-enzyme digestion is carried out on the pHB-Flag vector by using restriction enzymes PstI and BamHI, agarose gel electrophoresis is carried out on enzyme digestion products, and gel digestion recovery is carried out, so that the linearized pHB-Flag vector with sticky ends is obtained;

d. And c, connecting the FvbHLH36 gene with the sticky end obtained in the steps b and c with the pHB-Flag vector through T4 DNA ligase, then converting the connection product into escherichia coli competent JM107, identifying positive clones through colony PCR, shaking the positive clones, extracting plasmids and carrying out sequencing verification, thus obtaining the overexpression vector pHB-FvbHLH36-Flag.

(3) Overexpression of FvbHLH36 gene to promote accumulation of anthocyanin in Arabidopsis

Transferring pHB-FvbHLH36-Flag over-expression vector into agrobacterium through freeze thawing process, infecting Arabidopsis wild type through soaking process to obtain transgenic plant over-expressing FvbHLH36, photographing and measuring anthocyanin content.

(4) Instant injection of strawberry fruits and FvbHLH36 gene function verification

Transferring the pHB-FvbHLH36-Flag over-expression vector into agrobacterium through a freeze thawing method, injecting the agrobacterium into strawberry fruits in a gingko stage by using a disposable injector, observing the color of the strawberry fruits after 5-7 days, and determining the anthocyanin content and the expression condition of FvbHLH36 genes. The strawberries used when the strawberries were injected were octaploid cultivated strawberries "red color".

The Agrobacterium GV3101 strain according to the present invention can be obtained by commercial means.

The test materials used in the invention are as follows:

preparation of LB medium (1L):

preparation of YEP Medium (1L):

preparation of MS medium (1L):

EXAMPLE 1 cloning of strawberry FvbHLH36 Gene

In this example, cloning of strawberry FvbHLH36 gene was performed, including the following:

1.1 obtaining plant Material

Tender leaves of forest strawberry "Rugen" 0.1g were taken, placed in a clean 1.5mL centrifuge tube, placed in liquid nitrogen for rapid cooling, and then ground into powder at low temperature for use in extracting RNA.

1.2 extraction of RNA

Taking the powder obtained in the step 1.1 as a target, extracting total RNA according to the operation instruction of a Tiangen RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (purchased from Tiangen biochemistry, cat: DP 441).

1.3 Synthesis of first strand of cDNA

The total RNA obtained according to step 1.2) was used as a template, and was synthesized according to the full-scale gold cDNA synthesis kit (Cat: AE 311-02) gave a first strand of cDNA.

1.4 cloning of the gene FvbHLH36

Based on the nucleotide sequence of strawberry FvbHLH36 gene (SEQ ID NO: 1), an upstream primer and a downstream primer for amplifying the coding region are designed:

FvbHLH36-F1 primer: 5'-ATGGATATCAACCAACTAAAATCAGAG-3' (SEQ ID NO. 3)

FvbHLH36-R1 primer: 5'-TCAAGCGTCGCCGAAATGCTG-3' (SEQ ID NO. 4)

And (3) carrying out PCR amplification reaction by taking the first strand of the cDNA obtained in the step 1.3) as a template according to the following reaction system and reaction procedure to obtain a PCR product.

The PCR reaction system is as follows: 12.5 mu L PrimeSTAR Max Premix, primers FvbHLH36-F1 and FvbHLH36-R1 each 0.4. Mu.L, cDNA 1. Mu.L, and finally sterile distilled water was added to make up to 25. Mu.L.

PCR reaction procedure: pre-denaturation at 98℃for 1min; denaturation at 98℃for 10s, annealing at 60℃for 20s, extension at 72℃for 20s, and 30 cycles from denaturation to extension; extending at 72deg.C for 10min, and storing at 25deg.C.

The PCR product obtained was subjected to agarose gel electrophoresis.

FIG. 1 is an electrophoretogram of the PCR amplification product obtained in this example.

As can be seen from FIG. 1, the fragment size of the PCR amplified product was 735bp, which was identical to the FvbHLH36 gene size, indicating that the target fragment was obtained by PCR amplification.

A band of the target gene FvbHLH36 was excised from the agarose gel under ultraviolet light, and the target band was recovered according to the protocol of the gel recovery kit (Bio-organism, cat: B518131).

The above recovered target fragment was seamlessly ligated to pLB vector (Tiangen Biochemical, cat: VT 205) to obtain recombinant vector pLB-FvbHLH36, and then intermediate vector pLB-FvbHLH36 was transformed into E.coli competent cell JM107 by chemical transformation, spread on LB plate (containing 100mg/L ampicillin) and cultured overnight at 37 ℃.

Performing PCR (polymerase chain reaction) identification on the overnight bacterial colony, extracting plasmids from the positive clone bacteria, and sequencing to obtain the full-length gene sequence of FvbHLH36, wherein the full-length gene sequence is shown as SEQ ID NO. 1; the amino acid sequence is shown as SEQ ID NO. 2.

EXAMPLE 2 construction of recombinant expression vector pHB-FvbHLH36-Flag

In this example, a recombinant expression vector pHB-FvbHLH36-Flag was constructed, comprising the following:

2.1 introduction of cleavage sites in the FvbHLH36 Gene

Using the intermediate vector pLB-FvbHLH36 obtained in step 1.4 of example 1 as a template, a forward primer for introducing the Pst I cleavage site and a reverse primer for introducing the BamH I cleavage site were designed as follows:

the Pst I cleavage site is introduced into the forward primer FvbHLH 36-F2:

5'-AAACTGCAGATGGATATCAACCAACTAAAATCAGAG-3'(SEQ ID NO.5)

the BamH I cleavage site is introduced into the reverse primer FvbHLH 36-R2:

5'-GCCGGATCCAGCGTCGCCGAAATGCTG-3'(SEQ ID NO.6)

Then, PCR amplification was performed according to the following PCR reaction system to obtain FvbHLH36 gene product containing the cleavage site.

The PCR reaction system is as follows: 12.5 mu L PrimeSTAR Max Premix, primers FvbHLH36-F2 and FvbHLH36-R2 each 0.4. Mu.L, intermediate vector pLB-FvbHLH 36.1. Mu.L, and finally adding sterile distilled water to make up to 25. Mu.L.

And (3) purifying PCR amplification products by the products, and recovering the full-length fragment of the FvbHLH36 gene containing the enzyme cutting site.

2.2 cleavage reaction

The full-length fragment of FvbHLH36 gene containing the cleavage site obtained in step 2.1 was double-digested with restriction enzymes Pst I and BamH I, and the vector pHB-Flag was double-digested with restriction enzymes Pst I and BamH I.

The double enzyme digestion reaction system is as follows: 10 XDiget Buffer 5. Mu.L, full-length FvbHLH36 gene fragment containing enzyme cutting site 15. Mu.L, endonuclease PstI and BamH I1. Mu.L each, finally adding sterile distilled water to make up to 50. Mu.L, instantaneous centrifugation, mixing well, and placing in 37 ℃ for enzyme cutting for 2h.

The digested products were purified separately to obtain FvbHLH36 gene fragment with cohesive ends and pHB-Flag linearized vector with cohesive ends.

2.3 ligation reaction

And (3) performing a ligation reaction on the FvbHLH36 with the sticky end obtained in the step (2.2) and pHB-Flag by using T4 DNA ligase, wherein the ligation reaction is performed according to the operation instruction of a DNA ligation kit (Cat.6022) of a precious organism, and constructing a recombinant expression vector pHB-FvbHLH36-Flag.

2.4 screening of Positive clone strains, sequencing

The recombinant expression vector pHB-FvbHLH36-Flag obtained in step 2.3 was transformed into E.coli competent cells JM107, plated on LB plates (containing 50mg/L of kanamycin), and cultured overnight at 37 ℃.

And (3) carrying out PCR (polymerase chain reaction) on the colonies after overnight, carrying out shaking on the positive clones, extracting plasmids, and verifying the correctness of the FvbHLH36 gene sequence through sequencing.

Example 3 Agrobacterium-mediated FvbHLH36 Gene conversion Pattern plants

In this example, the recombinant expression vector pHB-FvbHLH36-Flag sequenced correctly in example 2 was transformed into Agrobacterium GV3101 and further transformed into Arabidopsis thaliana, a model plant, comprising the following:

3.1 recombinant expression vector pHB-FvbHLH36-Flag transformation of Agrobacterium

One competent cell of Agrobacterium GV3101 is placed on ice, after the competent cell is dissolved, 5 mu L of recombinant expression vector pHB-FvbHLH36-Flag which is obtained by the step 2.4 in the example 2 is added, and after the competent cell is dissolved, the competent cell is gently sucked and stirred by a pipettor, and then placed on ice for standing for 30min, then placed on liquid nitrogen for cooling for 1min, and then placed on 37 ℃ for 3min for thawing. Subsequently, 1mL of YEP liquid medium containing no antibiotic was added, and the culture was carried out in a shaking table at 30℃for 3 hours at 220rpm, and then centrifuged at 13000rpm at room temperature for 1min, and after 900. Mu.L of the supernatant was removed, the resuspended cell pellet was aspirated. Finally, 100 mu L of bacterial liquid is coated on a YEP solid culture medium containing 50mg/L Rif (rifampin), 50mg/L Gm (gentamicin) and 50mg/L Kan (kanamicin), the culture is inverted at 30 ℃ for 3d, monoclonal is selected for PCR detection, positive clone shaking bacteria are selected, and agrobacterium GV3101 containing a recombinant expression vector pHB-FvbHLH36-Flag is obtained.

3.2 transformation mode plants

3.2.1 Preagrobacteria: the agrobacterium GV3101 containing recombinant expression vector pHB-FvbHLH36-Flag obtained in step 3.1 is inoculated into YEP liquid culture medium containing Kan (kanamycin) 50mg/L and Rif (rifampicin) 25mg/L, and cultured at 28 ℃ under shaking at 200rpm for 24 hours to obtain agrobacterium liquid.

3.2.2. 2. Agrobacterium expansion: 5mL of the pre-shaken agrobacterium liquid obtained in step 3.2.1 is taken, 200mL of liquid YEP liquid culture medium is added, the mixture is placed at 28 ℃ and is subjected to shaking culture at 200rpm for 20 hours, and after 5000rpm, the thallus is collected by centrifugation at 5min, and thus the agrobacterium thallus is obtained.

3.2.3 transformed plants: the "Columbia" wild type Arabidopsis thaliana was grown in containers, and nutrient soil for the Arabidopsis thaliana was watered out the day before transformation at the beginning of bud formation.

The agrobacteria collected in step 3.2.2 were resuspended with 100mL of MS resuspension (1/2ms+5% suspension, ph=5.7); then, 40. Mu.L of Silwet L-77 and 2. Mu.L of 6-BA (mother liquor: 1 mg/mL) were added and stirred uniformly to form a bacterial liquid for transformation.

Then, the wild type arabidopsis thaliana with the bud is soaked in the bacterial liquid for transformation for 10s, after transformation, a container for cultivating the arabidopsis thaliana is placed in a tray, covered with a black box and cultivated for 24h in a dark place. And then watering and normally culturing, so as to ensure that the arabidopsis has sufficient water.

3.2.4 screening of transgenic positive lines: and collecting seeds after the transformed plants are fully mature to obtain transgenic T0 generation seeds, sowing the transgenic T0 generation seeds in a plug tray, screening seedling resistance by using 0.05% (v/v) glyphosate to obtain T1 generation transgenic positive plants, and continuously screening until T2 generation homozygous transgenic plants are obtained for later observation of phenotypes.

Phenotype identification of 3.2.5 transgenic positive plants: seeds of T2 generation homozygote transgenic arabidopsis and wild arabidopsis are paved on an MS culture plate and are cultured for 7d under the condition of (16 h illumination/8 h darkness), anthocyanin accumulation conditions are observed, photographing is carried out, and the relative anthocyanin content is measured.

3.3 analysis of anthocyanin accumulation in transgenic Arabidopsis

FIG. 2A is a photograph of T2-transformed Arabidopsis thaliana and wild type Arabidopsis thaliana in the present example. Wherein OE-1 and OE-2 represent two different T2 generation homozygotes transgenic Arabidopsis thaliana.

As can be seen from FIG. 2A, the plants of transgenic Arabidopsis thaliana are more colored and darker than the wild type Arabidopsis thaliana.

3.4 anthocyanin content analysis of transgenic Arabidopsis thaliana

1) 20 seedlings of Arabidopsis thaliana were taken, immersed in 600. Mu.L of 1% HCL in methanol, and incubated overnight at 4 ℃;

2) 400. Mu.L ddH was added ₂ After O, centrifuging at 3800rpm for 10min to obtain supernatant;

3) Collecting supernatant, adding 1mL of chloroform, mixing, and centrifuging at 13000rpm for 5min;

4) After centrifugation, the upper aqueous phase was collected, absorbance values were measured at 530nm and 657nm using a spectrophotometer, and the relative anthocyanin content was calculated using the following formula:

(A530-A657)/Fw

wherein A530 is the absorbance measured at 530nm with a spectrophotometer, A657 is the absorbance measured at 657nm with a spectrophotometer, fw represents the fresh weight per gram.

FIG. 2B is a graph showing the relative amounts of anthocyanin in T2-transgenic Arabidopsis and wild-type Arabidopsis in this example.

From fig. 2B, it can be seen that the relative content of anthocyanin in transgenic arabidopsis was higher compared to wild type arabidopsis. The relative content of anthocyanin in the transgenic arabidopsis thaliana is 9.67+/-1.02 and 6.82+/-0.88 respectively; the relative content of anthocyanin in wild type arabidopsis is only 0.80+/-0.25, and the relative content of anthocyanin in transgenic arabidopsis is more than 8.5 times of that of wild type.

From fig. 2A and 2B, it can be seen that the FvbHLH36 gene can promote the accumulation of more anthocyanin in arabidopsis thaliana, which is a model plant, and increase the anthocyanin content in arabidopsis thaliana.

Example 4 strawberry transient FvbHLH36 Gene expression and analysis

In this example, agrobacterium GV3101 containing recombinant expression vector pHB-FvbHLH36-Flag obtained in example 3 was injected into strawberry fruits for transient expression, and the relative anthocyanin content and the FvbHLH36 gene expression level in strawberry fruits were measured. Comprises the following steps:

4.1 construction of transient FvbHLH36 Gene expression System

Experimental group: agrobacterium GV3101 containing the recombinant expression vector pHB-FvbHLH36-Flag obtained in example 3 was inoculated into YEP liquid medium containing 50mg/L Kan (kanamicin) and cultured overnight in a shaker at 30℃and 220rpm to obtain an Agrobacterium solution containing the recombinant expression vector pHB-FvbHLH 36-Flag. Control group: agrobacterium GV3101, a vector pHB-Flag, was inoculated into YEP liquid medium containing 50mg/L Kan (kanamicin) and cultured overnight in a shaker at 30℃and 220rpm to give an Agrobacterium solution containing the vector pHB-Flag.

Subsequently, 200. Mu.L of the agrobacteria solution of the experimental and control groups were transferred to 5mL of YEP liquid medium containing 50mg/L Kan (kanamicin), and Acetosyringone (AS) was added thereto at a final concentration of 200nM, and the culture was continued in a shaker at 30℃and 220rpm for 20-24 hours. Subsequently, the cells were collected by centrifugation at 4000rpm for 10 min.

Subsequently, the mixture was treated with a solution containing 10mM MgCl ₂ And 10mM MES (pH 5.6)The YEP liquid medium was resuspended until the OD600 was 0.6, and MES (pH 5.6) and 200. Mu.M AS were added at a final concentration of 10mM, and the mixture was left at room temperature for 3 hours to give a suspension. Injecting the suspension into the red strawberry fruits selected in advance and in the gingko stage by using a disposable injector, and continuously culturing.

Picking up strawberry fruits 5-7 days after injection, observing anthocyanin accumulation, photographing and observing, and determining the relative content of anthocyanin and the expression quantity of FvbHLH36 gene in the strawberry fruits.

4.2 analysis of anthocyanin accumulation in strawberry fruit after transient expression

FIG. 3A is a photograph showing the actual injection of Agrobacterium GV3101 containing the recombinant expression vector pHB-FvbHLH36-Flag into strawberry fruits in this example.

As can be seen from FIG. 3A, the strawberry fruits after the injection of Agrobacterium GV3101 containing the recombinant expression vector pHB-FvbHLH36-Flag were colored faster and darker than the control group.

4.3 analysis of the relative content of anthocyanin in strawberry fruit after transient expression

1) Strawberry fruits were ground into powder, 0.1g of the powder was immersed in 600. Mu.L of 1% HCL diluted with methanol, and incubated overnight at 4 ℃.

The other steps are the same as 3.4 in example 3. FIG. 3B is a graph showing the relative anthocyanin content of strawberry fruits in this example after injection of Agrobacterium GV3101 containing the recombinant expression vector pHB-FvbHLH 36-Flag.

As can be seen from FIG. 3B, the relative anthocyanin content of strawberry fruits after injection of Agrobacterium GV3101 containing recombinant expression vector pHB-FvbHLH36-Flag was higher than that of the control group. The relative anthocyanin content of strawberry fruits after being injected with agrobacterium GV3101 containing recombinant expression vector pHB-FvbHLH36-Flag is 5.68+/-0.93; the relative content of anthocyanin in the control group is only 1.01+/-0.45, and the average value of the relative content of anthocyanin in the strawberry fruits after transient expression is more than 5.6 times of that of the control group.

From fig. 3A and 3B, it can be seen that after the FvbHLH36 is transiently expressed in the strawberry fruit, the accumulation of anthocyanin in the strawberry fruit can be promoted, and the anthocyanin content in the strawberry fruit can be improved.

4.4 analysis of relative expression level of FvbHLH36 Gene in strawberry fruit after transient expression

4.4.1 acquisition of materials: picking up strawberry fruits 5-7 days after instant injection, photographing, respectively wrapping with aluminum platinum paper, and immediately putting into liquid nitrogen for standby.

Extraction of 4.4.2RNA: and then extracting total RNA of the fruits by using a total RNA extraction kit of the Tiangen RNAprep Pure polysaccharide polyphenol plants.

4.4.3 obtaining of cDNA: the total RNA of 500ng obtained in step 4.4.2 was used as a template, and the first strand of cDNA was obtained by reverse transcription according to the instructions of the full-scale gold cDNA synthesis kit.

4.4.4 fluorescent quantitative PCR analysis: specific primers were designed to analyze the amount of expression of FvbHLH36 gene in strawberry fruits.

According to the nucleotide sequence of the target gene FvbHLH36, a Primer premier v6.0 Primer design software is utilized to design a specific Primer for FvbHLH36 gene quantitative analysis in Real-time PCR.

The primers for the FvbHLH36 gene were as follows:

forward primer FvbHLH36-F3:5'-ACCAACTCACTTGAACAATC-3' (SEQ ID NO. 7)

Reverse primer FvbHLH36-R3:5'-TCTTATCCTCTCGCTAATCC-3' (SEQ ID NO. 8)

The internal reference gene is Fv26S gene, and the primers are as follows:

Fv26S-F：5′-TAACCGCATCAGGTCTCCAA-3′(SEQ ID NO.9)

Fv26S-R：5′-CTCGAGCAGTTCTCCGACAG-3′(SEQ ID NO.10)

real-time fluorescence quantitative analysis of target genes in strawberry fruits to be detected: the first strand of cDNA synthesized in the step 4.4.3 is used as a template, and the specific primer amplification of the gene FvbHLH36 and the reference gene Fv26S is used for carrying out fluorescence quantitative PCR analysis, and the Real-time PCR reaction is carried out on a BIO-RAD Chromo 4 Real-time quantitative instrument. The reaction system was 20. Mu.L, and the reaction was denatured at 94℃for 20s using a three-step method followed by 36 cycles: 94 ℃ for 15s;58 ℃ for 15s; 25s at 72 ℃.

After the reaction was completed, the data were exported to Excel table using 2 ^-△△Ct Calculation of genes by formulasRelative expression level.

FIG. 3C is a graph showing the relative expression levels of FvbHLH36 gene in the control group and strawberry fruits injected with Agrobacterium GV3101 containing the recombinant expression vector pHB-FvbHLH36-Flag in this example.

As can be seen from FIG. 3C, the expression level of FvbHLH36 gene in strawberry fruits injected with Agrobacterium GV3101 containing recombinant expression vector pHB-FvbHLH36-Flag was significantly increased as compared with the control group, 4.3 times as much as that of the control group.

According to the invention, the FvbHLH36 gene is cloned from strawberry, a recombinant expression vector is successfully constructed, and the recombinant expression containing the FvbHLH36 gene is transformed into model plant Arabidopsis thaliana by adopting an agrobacterium-mediated method, so that compared with wild Arabidopsis thaliana, the FvbHLH36 gene is introduced, the whole plant of Arabidopsis thaliana can be more quickly colored and has darker color, and the relative content of anthocyanin of the whole plant is improved by more than 8.5 times compared with that of the wild Arabidopsis thaliana; the recombinant expression containing the FvbHLH36 gene is transiently expressed in strawberry fruits by an agrobacterium-mediated method, so that the relative content of anthocyanin in the strawberry fruits can be increased by more than 5.6 times, the relative expression amount of FvbHLH36 in the fruits can be increased by more than 4.3 times, the FvbHLH36 gene can promote the synthesis and/or the accumulation of anthocyanin in plants, the invention provides a basis for regulating and controlling the synthesis and/or the content of anthocyanin in plants by changing the expression of the FvbHLH36 gene, and provides candidate genes for changing the synthesis and/or the content of anthocyanin in plants in molecular breeding, in particular to plants such as strawberries or arabidopsis thaliana.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, many modifications and variations of the methods and compositions of the invention set forth herein will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Sequence listing

<110> Shanghai university of transportation

<120> FvbHLH36 protein, and encoding gene and use thereof

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atggatatca accaactaaa atcagaggag cagatggaaa tgatgatgat gatgcaaatg 60

gataaaatct ccgagctctg tggcgcctac aacgacgtcg tttccgacct cccttcatct 120

gaacactttt ctggtagtgc aacttccatg ccacattaca gccatgacca aaaccctcac 180

actgtttctt cttcacctcc atcctcattt cttaacctac acccttcaac catatcattc 240

accaactcac ttgaacaatc accagaacct caactggcat cggataagcg tgcttcaatg 300

gcggctatga gggagatgat attcagaatc gcctcgatgc agccaatcca catagacccg 360

gagtcggtga agccgccaaa gagaaggaac gtgaagattt ccaaggaccc tcagagcgtg 420

gcggctcgtc acaggaggga gaggattagc gagaggataa gaatactcca gcgactagtc 480

cccgggggaa ctaaaatgga cactgcatcc atgctggacg aggctattca ctacgtcaaa 540

ttcttgaagt ctcaagttca gacgctggag agagccgccg tgaataataa caataacagg 600

gtacaacaaa ccgggattgg ttttccggtg gcgatgtcaa gtgggagtaa ttacctttct 660

aattctatgg ccaaagcgta ccaagcccat cctccgcagc atcatcagaa tgtgcagcat 720

ttcggcgacg cttga 735

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Met Asp Ile Asn Gln Leu Lys Ser Glu Glu Gln Met Glu Met Met Met

1 5 10 15

Met Met Gln Met Asp Lys Ile Ser Glu Leu Cys Gly Ala Tyr Asn Asp

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Val Val Ser Asp Leu Pro Ser Ser Glu His Phe Ser Gly Ser Ala Thr

35 40 45

Ser Met Pro His Tyr Ser His Asp Gln Asn Pro His Thr Val Ser Ser

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Ser Pro Pro Ser Ser Phe Leu Asn Leu His Pro Ser Thr Ile Ser Phe

65 70 75 80

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

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Arg Ala Ser Met Ala Ala Met Arg Glu Met Ile Phe Arg Ile Ala Ser

100 105 110

Met Gln Pro Ile His Ile Asp Pro Glu Ser Val Lys Pro Pro Lys Arg

115 120 125

Arg Asn Val Lys Ile Ser Lys Asp Pro Gln Ser Val Ala Ala Arg His

130 135 140

Arg Arg Glu Arg Ile Ser Glu Arg Ile Arg Ile Leu Gln Arg Leu Val

145 150 155 160

Pro Gly Gly Thr Lys Met Asp Thr Ala Ser Met Leu Asp Glu Ala Ile

165 170 175

His Tyr Val Lys Phe Leu Lys Ser Gln Val Gln Thr Leu Glu Arg Ala

180 185 190

Ala Val Asn Asn Asn Asn Asn Arg Val Gln Gln Thr Gly Ile Gly Phe

195 200 205

Pro Val Ala Met Ser Ser Gly Ser Asn Tyr Leu Ser Asn Ser Met Ala

210 215 220

Lys Ala Tyr Gln Ala His Pro Pro Gln His His Gln Asn Val Gln His

225 230 235 240

Phe Gly Asp Ala

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<213> Artificial Sequence

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atggatatca accaactaaa atcagag 27

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<213> Artificial Sequence

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tcaagcgtcg ccgaaatgct g 21

<210> 5

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<213> Artificial Sequence

<400> 5

aaactgcaga tggatatcaa ccaactaaaa tcagag 36

<210> 6

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<213> Artificial Sequence

<400> 6

gccggatcca gcgtcgccga aatgctg 27

<210> 7

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<213> Artificial Sequence

<400> 7

accaactcac ttgaacaatc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

tcttatcctc tcgctaatcc 20

<210> 9

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<213> Artificial Sequence

<400> 9

taaccgcatc aggtctccaa 20

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<213> Artificial Sequence

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ctcgagcagt tctccgacag 20

Claims

Use of FvbHLH36 protein or a biological material related to FvbHLH36 protein in at least one of:

1) Promoting synthesis of plant anthocyanin;

2) Improving the anthocyanin content of plants;

3) Improving the accumulation of plant anthocyanin;

4) Promoting plant coloration;

the FvbHLH36 protein is a protein consisting of an amino acid sequence shown as SEQ ID NO. 2;

the biological material related to FvbHLH36 protein is any one of the following:

a) A polynucleotide encoding a FvbHLH36 protein;

b) A recombinant expression vector comprising the polynucleotide of a);

c) A bioengineering bacterium containing the polynucleotide of a) or a bioengineering bacterium containing the recombinant expression vector of b);

the promotion of plant anthocyanin synthesis, plant anthocyanin content increase, plant anthocyanin accumulation increase and plant coloring promotion are realized through over-expression of FvbHLH 36;

the plant is dicotyledon.
2. The use according to claim 1, wherein the sequence of the polynucleotide encoding FvbHLH36 protein is as shown in SEQ ID No. 1.
3. A method for promoting plant anthocyanin synthesis, comprising the steps of:

d1 Introducing the FvbHLH36 protein of claim 1;

d2 Introducing a biological material related to FvbHLH36 protein as set forth in claim 1;

the promotion of plant anthocyanin synthesis is realized by over-expression of FvbHLH36, and the plant is dicotyledonous plant.
4. A method for increasing the anthocyanin content of a plant, comprising the steps of:

d1 Introducing the FvbHLH36 protein of claim 1;

D2 Introducing a biological material related to FvbHLH36 protein as set forth in claim 1;

the improvement of the anthocyanin content of the plant is realized by over-expressing FvbHLH36, and the plant is dicotyledon.
5. A method for increasing plant anthocyanin accumulation, comprising the steps of:

d1 Introducing the FvbHLH36 protein of claim 1;

d2 Introducing a biological material related to FvbHLH36 protein as set forth in claim 1;

the improvement of anthocyanin accumulation in plants is realized by over-expression of FvbHLH36, and the plants are dicotyledonous plants.
6. A method for promoting plant coloration, said method comprising the steps of:

d1 Introducing the FvbHLH36 protein of claim 1;

d2 Introducing a biological material related to FvbHLH36 protein as set forth in claim 1;

the promotion of plant coloration is achieved by over-expression of FvbHLH36, and the plant is a dicotyledon.