阅读说明：本技术 茄子SmBIC2基因及蛋白的用途 (Eggplant SmBIC2 gene and application of protein ) 是由 陈火英 何永军 刘杨 张信童 于 2020-07-24 设计创作，主要内容包括：本发明涉及生物技术领域,特别是涉及茄子SmBIC2基因及蛋白在抑制植物花青素合成或调控植物光形态建成中的用途。本发明中将SmBIC2基因在野生型拟南芥和茄子中过表达,可以促进拟南芥和茄子下胚轴的伸长,同时抑制了花青素合成。本发明针对目前茄子研究基础薄弱的现状,克隆光信号通路中的关键基因SmBIC2,为今后利用基因工程技术改良植物品质,获得具有高抗氧化性的药物或食物提供理论依据,具有很大的应用价值。(The invention relates to the technical field of biology, in particular to application of eggplant SmBIC2 gene and protein in inhibiting plant anthocyanin synthesis or regulating plant photomorphogenesis. According to the invention, the SmBIC2 gene is overexpressed in wild arabidopsis thaliana and eggplants, so that the elongation of hypocotyls of the arabidopsis thaliana and the eggplants can be promoted, and the synthesis of anthocyanin is inhibited. Aiming at the current situation that the research foundation of eggplants is weak at present, the key gene SmBIC2 in an optical signal path is cloned, so that a theoretical basis is provided for improving the plant quality by utilizing a genetic engineering technology and obtaining a medicine or food with high oxidation resistance in the future, and the method has great application value.)

1. The application of the eggplant SmBIC2 gene or protein in inhibiting plant anthocyanin synthesis or regulating plant photomorphogenesis.

2. Use according to claim 1, characterized in that the nucleotide sequence of the eggplant SmBIC2 gene is one of the following sequences:

1) a nucleotide sequence shown as SEQ ID NO.1, 1-579 bits;

2) a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in the 1st to 579 th positions shown in SEQ ID NO. 1;

3) a nucleotide sequence capable of hybridizing with the nucleotide sequences shown in the 1st to 579 th positions of SEQ ID NO. 1.

3. Use according to claim 1, characterized in that the amino acid sequence of the eggplant SmBIC2 protein is represented by SEQ ID No. 2.

4. The use of claim 1, wherein said modulating plant photomorphogenesis comprises promoting elongation of the hypocotyl of the plant.

5. Use according to claim 1, wherein the plants comprise Arabidopsis thaliana and eggplant.

6. The use of the eggplant SmBIC2 gene or protein as the raw material for preparing a product for inhibiting plant anthocyanin synthesis or regulating plant photomorphogenesis according to claim 1.

7. A method for inhibiting plant anthocyanin synthesis or regulating plant photomorphogenesis, which comprises the following steps:

1) obtaining eggplant SmBIC2 gene;

2) constructing an eggplant SmBIC2 transformation vector;

3) and infecting a target plant or tissues and organs thereof with the eggplant SmBIC2 transformation vector.

8. The method of claim 7, further comprising one or more of the following features:

1) after infection, antibiotic screening or continuous selfing is carried out;

2) the target plant is eggplant or arabidopsis thaliana.

9. The method as claimed in claim 7, wherein the nucleotide sequence of the eggplant SmBIC2 gene is one of the following sequences:

1) a nucleotide sequence shown as SEQ ID NO.1, 1-579 bits;

2) a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in the 1st to 579 th positions shown in SEQ ID NO. 1;

3) a nucleotide sequence capable of hybridizing with the nucleotide sequences shown in the 1st to 579 th positions of SEQ ID NO. 1.

10. The method as claimed in claim 7, wherein the eggplant SmBIC2 gene is obtained by PCR amplification, and the nucleotide sequences of a primer pair used for the PCR amplification of the eggplant SmBIC2 gene are shown as SEQ ID No.3 and SEQ ID No. 4.

Technical Field

The invention relates to the technical field of biology, in particular to an eggplant SmBIC2 gene and application of the gene and protein.

Background

BIC2 is an cryptochrome blue light inhibitor in plants, and can interact with blue light receptors CRY1 and CRY2 to regulate and control plant photomorphogenesis. It is found that under dark conditions, the CRYs exists as an inactive monomer, and light changes the CRYs into an active dimer, thereby triggering a series of photomorphogenesis, while the BIC2 protein can maintain the photosensitivity of cells by inhibiting the dimerization of the CRYs, thereby participating in the regulation of photomorphogenesis of plants.

BIC2 can regulate plant photomorphogenesis transcription factors, such as HY5, TT8, MYB1, LZF1, HFR1, CO, etc. BIC2 regulates elongation of seedling hypocotyl of Arabidopsis thaliana by inhibiting HY5, LZF1 and HFR1, and regulates photoperiod flowering of Arabidopsis thaliana by regulating protein stability of CO.

BIC2 regulates the photomorphogenesis of plants, how does light regulate BIC 2? Research shows that the transcription of BIC2 is not only regulated by blue light, red light and far-red light can promote the expression of BIC2, and red light, far-red light receptor and blue light receptor can regulate the expression of HY5 through COP, so the light is supposed to regulate the transcription of BIC2 through HY 5.

At present, the BIC2 gene is cloned only in Arabidopsis thaliana, and only the photomorphogenesis of the plant is functionally analyzed. However, the research on the BIC2 gene in the eggplant is relatively lagged, and no relevant literature report on the eggplant SmBIC2 gene and the protein coded by the same is reported at present.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide the use of eggplant SmBIC2 gene and protein for solving the problems of the prior art.

In order to achieve the above objects and other related objects, the invention provides the use of eggplant SmBIC2 gene and protein.

Preferably, the nucleotide sequence of the eggplant SmBIC2 gene is one of the following sequences:

1) a nucleotide sequence shown as SEQ ID NO.1, 1-579 bits;

2) a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in the 1st to 579 th positions shown in SEQ ID NO. 1;

3) a nucleotide sequence capable of hybridizing with the nucleotide sequences shown in the 1st to 579 th positions of SEQ ID NO. 1.

The amino acid sequence of the eggplant SmBIC2 protein is shown as SEQ ID No. 2.

As described above, the eggplant SmBIC2 gene and the use of the protein have the following beneficial effects:

1. the SmBIC2 gene is over-expressed in wild arabidopsis thaliana and eggplant, so that the elongation of the hypocotyl of arabidopsis thaliana and eggplant can be promoted, and the synthesis of anthocyanin is inhibited.

2. Aiming at the current situation that the research foundation of eggplants is weak at present, the key gene SmBIC2 in an optical signal path is cloned, so that a theoretical basis is provided for improving the plant quality by utilizing a genetic engineering technology and obtaining a medicine or food with high oxidation resistance in the future, and the method has great application value.

Drawings

FIG. 1 shows the results of amino acid sequence comparison (FASTA) of eggplant SmBIC2 protein of the present invention and Arabidopsis AtBIC2 protein, in which the same amino acid is indicated in dark color between the two sequences, and the CID domain is a part that interacts with the blue light receptor CRY;

FIG. 2 shows the expression of the eggplant SmBIC2 gene in different tissues;

FIG. 3 shows RT-PCR detection of SmBIC2 transgenic Arabidopsis plants;

FIG. 4 shows phenotypic changes of wild type Arabidopsis thaliana seedlings transformed with SmBIC2 gene, wherein the leftmost one is a WT enlarged diagram;

FIG. 5 shows the hypocotyl change of SmBIC2 transgenic wild type Arabidopsis seedlings;

FIG. 6 shows the change of anthocyanin synthesis in wild type Arabidopsis seedlings transformed with SmBIC2 gene;

FIG. 7 shows phenotypic changes of eggplant seedlings transformed with SmBIC2 gene (three plants in each group);

FIG. 8 shows the hypocotyl changes of eggplant seedlings transformed with SmBIC2 gene;

FIG. 9 shows the change of anthocyanin synthesis in eggplant seedlings transformed with SmBIC2 gene;

FIG. 10 shows the changes of the major structural genes for anthocyanin synthesis in eggplant seedlings transformed with SmBIC2 gene.

In the above figures, BIC2-OE1\ BIC2-OE2\ BIC2-OE3 or SmBIC2-OE1\ SmBIC2-OE2\ SmBIC2-OE3 are three biological repeats of BIC2 transgene; in the figure 116 represents the wild type.

Detailed Description

The invention provides application of eggplant SmBIC2 gene or protein in inhibiting plant anthocyanin synthesis or regulating plant photomorphogenesis.

The nucleotide sequence of the eggplant SmBIC2 gene is one of the following sequences:

1) a nucleotide sequence shown as SEQ ID NO.1, 1-579 bits;

2) a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in the 1st to 579 th positions shown in SEQ ID NO. 1;

3) a nucleotide sequence capable of hybridizing with the nucleotide sequences shown in the 1st to 579 th positions of SEQ ID NO. 1.

In particular, the at least 70% homology may be at least 80% homology, at least 90% homology, at least 95% homology, at least 98% homology or at least 99% homology.

The amino acid sequence of the eggplant SmBIC2 protein is shown as SEQ ID No. 2. The eggplant SmBIC2 protein has 192 amino acid residues in total, the molecular weight is 22.25KDa, and the theoretical isoelectric point (pI) is 9.853.

In one embodiment, mutations may also be introduced into the protein sequences of the present invention by chemical synthesis.

The plant photomorphogenesis is the process of controlling plant development by controlling the differentiation, structure and function of cells by relying on light, and finally integrating the plant photomorphogenesis into tissues and organs, namely, the process of controlling the plant development by light, and is called photomorphogenesis.

The regulating plant photomorphogenesis comprises promoting elongation of the hypocotyl of the plant.

In one embodiment, the plants are arabidopsis thaliana and eggplant. Eggplants are widely planted vegetable crops, the peel of the purple eggplants contains rich anthocyanin, and the antioxidation health-care effect of the purple eggplants is the best in common vegetable crops.

In one embodiment, the use further comprises the use of the eggplant SmBIC2 gene or protein in the preparation of a product for inhibiting plant anthocyanin synthesis or a product for regulating plant photomorphogenesis.

The effective substances in the product are eggplant SmBIC2 gene and/or protein.

The product is selected from a pharmaceutical product or an agent.

On the basis of nucleic acid homology or expressed protein homology, the eggplant SmBIC2 nucleotide sequence and the amino acid sequence provided by the invention can also be used for screening eggplant SmBIC2 related homologous genes or homologous proteins.

The eggplant SmBIC2 gene can also be used for obtaining a dot matrix of the gene related to the eggplant SmBIC 2. Can use³²P screening eggplant cDNA library by using DNA probe obtained by radioactive labeling all or part related to eggplant SmBIC 2. A suitable cDNA library for screening is a library from eggplant. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the field of molecular biology. In addition, many such cDNA libraries are also commercially available, for example, from Clontech, Stratagene, Palo Alto, Calif. This screening method allows to identify the nucleotide sequences of the gene family associated with eggplant SmBIC 2.

The invention provides a method for inhibiting plant anthocyanin synthesis or regulating plant photomorphogenesis, which comprises the following steps:

the method comprises the steps of obtaining an eggplant SmBIC2 gene, constructing an eggplant SmBIC2 transformation vector, and infecting a target plant or tissues and organs thereof with the eggplant SmBIC2 transformation vector.

Specifically, the method further comprises antibiotic screening or continuous selfing after infection.

The transformation vector is selected from overexpression vectors driven by 35S promoters. In one embodiment, the transformation vector is selected from PHB.

In one embodiment, the target plant is eggplant or arabidopsis thaliana.

The nucleotide sequence of the eggplant SmBIC2 gene is one of the following sequences:

1) a nucleotide sequence shown as SEQ ID NO.1, 1-579 bits;

2) a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in the 1st to 579 th positions shown in SEQ ID NO. 1;

3) a nucleotide sequence capable of hybridizing with the nucleotide sequences shown in the 1st to 579 th positions of SEQ ID NO. 1.

In particular, the at least 70% homology may be at least 80% homology, at least 90% homology, at least 95% homology, at least 98% homology or at least 99% homology.

The full-length eggplant SmBIC2 nucleotide sequence or a fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For the PCR amplification method, primers can be designed based on the nucleotide sequences disclosed herein, and amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. The sequence of interest is then obtained in large quantities by recombinant methods, usually by cloning it into a vector, transferring it into cells, and isolating the sequence of interest from the propagated host cells by conventional methods.

In one embodiment, the eggplant SmBIC2 gene is obtained by PCR amplification. The nucleotide sequences of the primer pair for amplifying the eggplant SmBIC2 gene by PCR are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The expression pattern of the eggplant SmBIC2 gene product can be analyzed by a real-time fluorescent quantitative PCR method, namely, the existence and the quantity of mRNA transcripts of the eggplant SmBIC2 gene in cells are analyzed.

In one embodiment, the nucleotide sequences of the primer pair for the eggplant SmBIC2 gene product used for the fluorescent quantitative PCR analysis are shown as SEQ ID No.5 and SEQ ID No. 6.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.