Sequence and application of gene PeANS3 for regulating and controlling petal color of phalaenopsis amabilis

Abstract

The invention discloses a sequence of a gene PeANS3 for regulating and controlling the petal color of phalaenopsis and application of the gene PeANS3. The gene sequence comprises one or more of the following nucleotide sequences: 1) a nucleotide sequence as shown in SEQ ID NO.1; 2) a nucleotide sequence which is derived by substitution, deletion or addition of one or more nucleotides of the nucleotide sequence as shown in SEQ ID NO.1; and 3) a nucleotide sequence having at least 80% homology with the SEQ ID NO.1. The gene sequence plays an important role in regulating and controlling flower color development of the phalaenopsis, the color of petals of the phalaenopsis is obviously lightened in a VIGS strain of the phalaenopsis, and an important basis is provided for phalaenopsis flower color breeding.

Description

Technical Field

[0001] The present invention relates to a gene sequence and application thereof, and in particular to a gene sequence of PeANS3 for regulating the color of Phalaenopsis petals and application thereof. Background Art

[0002] Phalaenopsis is a plant of the genus Phalaenopsis in the orchid family. Phalaenopsis has large and bright flowers with rich colors. It is often used for cut flowers, bouquets, potted plants, etc. and has important ornamental value, scientific research value and commercial value.

[0003] Flower color is one of the key ornamental traits of flower plants and has important research significance. Anthocyanin belongs to a class of flavonoids, which makes plants present a variety of flower colors and can protect against various biological and abiotic stresses. ANS (Anthocyanidin Synthase) belongs to 2-ketoglutarate-dependent dioxygenase. Its main function is to catalyze the conversion of colorless anthocyanins into colored anthocyanins. Its catalytic product is the first color-forming compound in the anthocyanin biosynthesis pathway, which plays an important role in the formation of plant organ color. By using RNA interference technology to inhibit ANS in blue butterfly grass, white butterfly grass with stable inheritance was cultivated; ANS mutation in gentian is an important reason for its flower color to become white. Inhibition or lack of its activity will cause the flower color to become lighter. However, there are few studies on the synthesis of colored anthocyanins in Phalaenopsis, and the molecular mechanism of its formation is not clear, which needs to be systematically studied. Summary of the invention

[0004] The purpose of the present invention is to provide a sequence of a gene PeANS3 for regulating the color of Phalaenopsis petals and its application. The gene sequence can affect the color of Phalaenopsis petals, making it have unique ornamental value.

[0005] To achieve the above object, the first aspect of the present invention provides a sequence of a gene PeANS3 that regulates the color of Phalaenopsis petals, wherein the sequence of the gene PeANS3 comprises one or more of the following nucleotide sequences:

[0006] 1) the nucleotide sequence shown in SEQ ID NO.1;

[0007] 2) A nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion, or addition of one or more nucleotides;

[0008] 3) A nucleotide sequence having at least 80% homology to SEQ ID NO.1.

[0009] The second aspect of the present invention provides an amino acid sequence, which can affect the gene of Phalaenopsis petal color, and the amino acid sequence is selected from one or more of the following sequences:

[0010] 1) the amino acid sequence shown in SEQ ID NO.2;

[0011] 2) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO.2 by substitution, deletion, or addition of one or more amino acids;

[0012] 3) An amino acid sequence having at least 80% homology to SEQ ID NO.2.

[0013] The third aspect of the present invention provides the use of the nucleotide sequence or the amino acid sequence described above in influencing the color of Phalaenopsis orchid petals.

[0014] Further, the Phalaenopsis orchids are primarily 'Cherry Tomato' Phalaenopsis orchids.

[0015] A fourth aspect of the present invention provides a method for regulating the color of Phalaenopsis orchid petals, comprising silencing the nucleotide sequence described above in a target plant.

[0016] Furthermore, the nucleotide sequence described above was connected to the CymMV virus vector through BP reaction, and then the recombinant plant expression vector was transferred into the target plant, and the PeANS3 silenced strain was obtained by cultivation.

[0017] The fragments shown by 1-294bp, 429-729bp, and 753-1049bp of the PeANS3 gene cloned from Phalaenopsis provided by the present invention are respectively connected to the CymMV virus vector, and then mixed and injected into the leaves of Phalaenopsis, and the PeANS3 silenced strain is obtained by 30-40 days of cultivation, indicating that the gene can affect the color of Phalaenopsis petals.

[0018] The advantages of the present invention are: the PeANS3 of the present invention plays an important role in regulating the flower color development of Phalaenopsis, and in the VIGS strain, the color of the petals becomes significantly lighter, which provides an important basis for regulating the flower color of Phalaenopsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The sampling and observation are done at different stages of flower bud development of wild-type Phalaenopsis.

[0020] Figure 2 is the expression level of PeANS3 at different developmental stages of flower buds.

[0021] Figure 3 These are pictures of the phenotype of the silenced strain 30 days after injection of CymMV-PeANS3.

[0022] Figure 4The PeANS3 expression levels in the wild-type petal S4 period and in the silenced strain injected with CymMV-PeANS3 30 days later. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The reagents used are commercially available or publicly available unless otherwise specified.

[0025] Example 1 Obtaining Phalaenopsis anthocyanin synthase gene PeANS3

[0026] Total RNA was extracted from wild-type Phalaenopsis petals using an RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich) (commercially available), and total RNA was reverse transcribed into cDNA using a reverse transcription kit (commercially available). Primers were designed based on the sequencing results of the Phalaenopsis genome, and a 1083 bp band was amplified from the Phalaenopsis cDNA. The PCR product was recovered to obtain the PeANS3 gene, whose nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2, which consists of 360 amino acid residues and has a molecular weight of 39.9 kilodaltons.

[0027] Example 2 Verification of expression profiles of Phalaenopsis orchids at different developmental stages

[0028] (1) Extraction Figure 1 As shown, the RNA of Phalaenopsis at different developmental stages was extracted using the RNAprep PurePlant Plus Kit (Polysaccharides & Polyphenolics-rich) (commercially available), and the total RNA was reverse transcribed into cDNA using a reverse transcription kit (commercially available).

[0029] (2) Real-time fluorescence quantitative primers were designed based on the nucleotide sequence data. The primer sequences are shown in SEQ ID NO. 3 (5'-GGGAAGGGGTTCTTCGAATTGC-3') and SEQ ID NO. 4 (5'-CTTGTTTTGGTCGAGGCCAAGAC-3').

[0030] (3) Using the cDNA of Phalaenopsis orchids at different developmental stages obtained by reverse transcription as template, the expression profile of PeANS at different developmental stages was verified. Figure 2 As shown, the expression of PeANS3 was significantly induced at different developmental stages (S1-S4), indicating that PeANS3 may be involved in the formation of petal color in Phalaenopsis.

[0031] Example 3

[0032] CymMV virus induces silencing of PeANS3 gene in cherry tomato and Phalaenopsis

[0033] The open reading frame 200-300 bp of the PeANS3 gene was operably linked to the CymMV viral vector to form a CymMV-PeANS3 vector containing the gene fragment, which was then transferred into Agrobacterium (EHA105) and injected into the leaves of cherry tomatoes and Phalaenopsis through the Agrobacterium-mediated method. The mixture was cultured for 30-40 days and the phenotype was observed. Figure 3 As shown, the petal color of the silenced strain became significantly lighter.

[0034] (1) The open reading frame 200-300 bp of PeANS3 is operably linked to the CymMV viral vector to form a CymMV-PeANS3 vector containing the gene fragment.

[0035] (2) The vector in step (1) was transformed into Agrobacterium (EHA105).

[0036] (3) Take the Agrobacterium (EHA105) obtained in step (2) and culture it in 5 mL of LB medium containing 100 μM acetosyringone and 50 μg / mL kanamycin at 28° C. and 200 rpm for 16 h.

[0037] (4) The Agrobacterium obtained in step (3) was subcultured in 50 mL LB medium containing 100 μM acetosyringone and 50 μg / mL kanamycin, and incubated at 28° C., 200 rpm for 13-16 h until the OD600 reached 0.8-1.0.

[0038] (5) Take the Agrobacterium culture solution in step (4), transfer it to a 50 ml centrifuge tube, and centrifuge it at 4°C, 3000g for 10 minutes.

[0039] (6) After centrifugation, remove the supernatant. Add 300 μL of MS liquid culture medium containing 100 μM acetosyringone to resuspend the cell pellet. Let stand at room temperature for 0.5 h.

[0040] (7) Use a 1 mL syringe with a needle to absorb the Agrobacterium transformation solution from step (6) and inject it into the leaves of Phalaenopsis orchids and cherry tomato Phalaenopsis orchids.

[0041] (8) After injection, observe the orchid petals for 30-40 days. Figure 3 As shown, the petal color of the silent mutant strains of Phalaenopsis was significantly lighter.

[0042] Example 4

[0043] Verification of the expression of PeANS3 gene and anthocyanin synthesis-related genes in the mutant lines of cherry tomato and Phalaenopsis induced by CymMV virus

[0044] (1) Select Figure 1 and Figure 3 Total RNA was extracted from petals of the control and silent mutant lines using RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich) (commercially available), and the total RNA was reverse transcribed into cDNA using a reverse transcription kit (commercially available).

[0045] (2) Design real-time fluorescence quantitative primers based on the nucleotide sequence data. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0046] (3) Using the cDNA of the petals of the control and silent mutant lines obtained by reverse transcription as a template, the gene silencing efficiency of the PeANS gene was verified, such as Figure 4 As shown, PeANS3 expression was significantly reduced in the petals of the silenced strain, proving that PeANS3 has a high silencing efficiency, and further illustrating that PeANS3 is involved in the formation of Phalaenopsis petal color.

[0047] In summary, when the PeANS3 gene of Phalaenopsis is silenced, the color of the petals becomes significantly lighter. ANS3 can regulate the flower color, which provides a theoretical basis and new ideas for the targeted improvement of the flower color of Phalaenopsis in the future.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sequence of a gene PeANS3 that regulates the color of Phalaenopsis petals, characterized in that: The sequence of the gene PeANS3 comprises one or more of the following nucleotide sequences: 1) the nucleotide sequence shown in SEQ ID NO.1; 2) A nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO.1 by substitution, deletion, or addition of one or more nucleotides; 3) A nucleotide sequence having at least 80% homology to SEQ ID NO.

1.

2. An amino acid sequence for regulating the Phalaenopsis petal color gene PeANS3, characterized in that: The amino acid sequence is encoded by the nucleotide sequence of claim 1.

3. The amino acid sequence according to claim 2, wherein The amino acid sequence comprises one or more of the following sequences: 1) the amino acid sequence shown in SEQ ID NO.2; 2) An amino acid sequence derived from the amino acid sequence shown in SEQ ID NO.2 by substitution, deletion, or addition of one or more amino acids; 3) An amino acid sequence having at least 80% homology to SEQ ID NO.

2.

4. Use of the gene PeANS3 sequence according to claim 1 or the amino acid sequence according to any one of claims 2 to 3, characterized in that: Used to control the color of Phalaenopsis orchid petals.

5. A method for regulating the color of Phalaenopsis orchid petals, characterized in that: Regulate the expression of the sequence of the gene PeANS3 as claimed in claim 1 in Phalaenopsis.

6. The method for regulating the color of Phalaenopsis petals according to claim 5, wherein: The Phalaenopsis orchid is a 'cherry tomato' Phalaenopsis orchid.

7. A method for lightening the color of Phalaenopsis orchid petals, characterized in that: Inhibit or silence the expression of the sequence of the gene PeANS3 as claimed in claim 1 in Phalaenopsis.

8. The method for lightening the color of Phalaenopsis orchid petals according to claim 7, wherein: The nucleotide sequence is connected to the CymMV virus plasmid, and then the recombinant vector is introduced into Agrobacterium, and then the target plant is infected by Agrobacterium, and Phalaenopsis with lighter petal color is obtained through cultivation.

9. The method for lightening the color of Phalaenopsis orchid petals according to claim 7 or 8, wherein: The Phalaenopsis orchid is a control and silenced strain.

10. The method for lightening the color of Phalaenopsis orchid petals according to claim 7 or 8, wherein: The Phalaenopsis orchid is a 'cherry tomato' Phalaenopsis orchid.

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