Application of RcETC3 gene in regulating plant flower color

Abstract

The application provides application of RcETC3 gene in regulation of flower color of plants. The first aspect of the application provides application of RcETC3 gene in regulation of flower color of plants, and the RcETC3 gene is one of A1) to A3): A1) a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:1; A2) a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID NO:1 through substitution, deletion or addition of one or more nucleotides; and A3) a nucleotide sequence with at least 80% identity with SEQ ID NO:1. The application clones a transcription factor RcETC3 from Rosa chinensis 'Yueyue Fen', and experiments show that the color of petals of the Rosa chinensis is obviously deepened in a transient gene silencing strain of the Rosa chinensis, indicating that the RcETC3 gene plays an important role in regulation of flower color of the Rosa chinensis, and provides an important basis for breeding of the Rosa chinensis.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the RcETC3 gene in regulating plant flower color. Background Technology

[0002] The rose (Rosa chinensis Jacq.), belonging to the genus Rosa in the family Rosaceae, is a perennial woody plant. Its vibrant colors and the development of a rich variety of flower colors are a primary goal for flower breeders. The flower color of roses is mainly determined by anthocyanins. In anthocyanin synthesis, the expression of enzyme genes is mainly regulated by transcription factors. Currently, three classes of transcription factors have been identified as being involved in the regulation of anthocyanin synthesis: MYB genes, bHLH, and WD40. They typically form the MBW complex, promoting or inhibiting anthocyanin biosynthesis. MYB genes are widely distributed in higher plants and are one of the largest transcription factor families. Extensive research has shown that MYB factors can regulate anthocyanin synthesis. Among them, R2R3-MYB and R3-MYB transcription factors have been identified as inhibitors of anthocyanin synthesis, affecting flower color by reducing anthocyanin biosynthesis and accumulation. The regulatory functions of these transcription factors are manifested in different plant species. For example, in peony, PsMYB308 acts as a transcriptional repressor, inhibiting the expression of the PsDFR gene promoter by specifically binding to it, thereby reducing the area of ​​petal spots and the total anthocyanin content. In potato, the StMYBATV protein can bind to the bHLHs protein, preventing the biosynthesis of anthocyanins in potato cell culture. Discovering the transcription factors involved in flower color regulation in roses is one of the main research areas in rose breeding. Summary of the Invention

[0003] This invention provides the application of the RcETC3 gene in regulating plant flower color.

[0004] The first aspect of this invention provides the application of the RcETC3 gene in regulating plant flower color, wherein the RcETC3 gene is one of A1)-A3):

[0005] A1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:1;

[0006] A2) Nucleotide sequences derived from the nucleotide sequence shown in SEQ ID NO:1 by substitution, deletion, or addition of one or more nucleotides;

[0007] A3) is a nucleotide sequence that has at least 80% identity with SEQ ID NO:1.

[0008] Furthermore, downregulating the expression of the RcETC3 gene resulted in a darker color in the plant petals. Specifically, this was manifested in an increase in the anthocyanin content of the plant petals.

[0009] Furthermore, downregulating the expression of the RcETC3 gene includes silencing the expression of the RcETC3 gene in plants.

[0010] Furthermore, the expression of the RcETC3 gene in silent plants specifically includes:

[0011] The open reading frame of the RcETC3 gene is operatively linked to the pTRV2 vector to form a TRV2-RcETC3 recombinant vector containing the RcETC3 gene.

[0012] The TRV2-RcETC3 recombinant vector was introduced into Agrobacterium, and transgenic plants with silenced RcETC3 gene were obtained through Agrobacterium-mediated transformation.

[0013] Furthermore, the plant in question is a rose.

[0014] A second aspect of this invention provides a method for regulating flower color in plants, specifically by regulating the expression of the RcETC3 gene in plants; wherein the RcETC3 gene is one of A1-A3.

[0015] A1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:1;

[0016] A2) Nucleotide sequences derived from the nucleotide sequence shown in SEQ ID NO:1 by substitution, deletion, or addition of one or more nucleotides;

[0017] A3) is a nucleotide sequence that has at least 80% identity with SEQ ID NO:1.

[0018] Furthermore, regulating the expression of the RcETC3 gene in plants includes downregulating the expression of the RcETC3 gene.

[0019] Furthermore, downregulating the expression of the RcETC3 gene includes silencing the expression of the RcETC3 gene in plants.

[0020] Furthermore, the expression of the RcETC3 gene in silent plants specifically includes:

[0021] The open reading frame of the RcETC3 gene is operatively linked to the pTRV2 vector to form a TRV2-RcETC3 recombinant vector containing the RcETC3 gene.

[0022] The TRV2-RcETC3 recombinant vector was introduced into Agrobacterium, and transgenic plants with silenced RcETC3 gene were obtained through Agrobacterium-mediated transformation.

[0023] Furthermore, the plant in question is a rose.

[0024] This invention cloned the transcription factor RcETC3 from the rose variety 'Yueyuefen'. Experiments showed that in rose lines with transient gene silencing, the color of rose petals became significantly darker, indicating that the RcETC3 gene plays an important role in regulating rose flower color and providing an important foundation for rose breeding. Attached Figure Description

[0025] Figure 1 Photos of flower buds or flowers at different developmental stages of the 'Monthly Pink' rose variety;

[0026] Figure 2 Anthocyanin content in flower buds or flowers of the rose 'Yueyuefen' at different developmental stages; different letters indicate significant differences between groups, t-test, p<0.05;

[0027] Figure 3 The expression levels of the RcETC3 gene in flower buds or flowers of the rose 'Yueyuefen' at different developmental stages; different letters indicate significant differences between groups, t-test, p<0.05;

[0028] Figure 4 Phenotypes of petal discs from the control and experimental groups of the 'Monthly Pink' rose variety after 5 days of vacuum inoculation with TRV2-RcETC3.

[0029] Figure 5 The anthocyanin content in the small round petals of the 'Monthly Pink' rose variety was determined by vacuum inoculation with TRV2-RcETC3 and compared with that in the control and experimental groups after 5 days. Different letters indicate the significance of differences between groups. The t-test showed that p < 0.05.

[0030] Figure 6 This study investigated the expression levels of genes related to anthocyanin formation in the small round petals of the 'Monthly Pink' rose variety after 5 days of vacuum infection with TRV2-RcETC3. In the figures, A represents the relative expression level of the RcETC3 gene, B represents the relative expression level of RcANS, and C represents the relative expression level of RcDFR. Different letters indicate significant differences between groups. A t-test was used, with p < 0.05 and ** indicating p < 0.01. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0032] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all reagents used were commercially available or publicly available.

[0033] In this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids.

[0034] The primers and their nucleotide sequences involved in the following examples are shown in Table 1.

[0035] Table 1. Primers and their nucleotide sequences involved in the embodiments of the present invention.

[0036]

[0037] Example 1: Cloning of the RcETC3 gene in roses

[0038] Total RNA was extracted from the petals of the ancient rose 'Yueyuefen' using a commercially available RNAplant extraction kit. The total RNA was then reverse transcribed into cDNA using a commercially available reverse transcription kit. Primers were designed based on the transcriptome sequencing results, and their sequences are shown in SEQ ID NO:3 and SEQ ID NO:4. An RT-PCR method was used to amplify a 339 bp band from the rose cDNA. The PCR product was recovered to obtain the RcETC3 gene, whose nucleotide sequence is shown in SEQ ID NO:1. The amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO:2, consisting of 112 amino acids with a molecular weight of 26.58 kilodaltons (kDa).

[0039] Example 2: Determination of anthocyanin content in roses at different developmental stages

[0040] Select as Figure 1 The flower buds or flowers of 'Monthly Powder' at different developmental stages were weighed and placed in 1.5 mL centrifuge tubes. 1 mL of a 1:99 mixture of hydrochloric acid and methanol was added, and the mixture was stored in the dark at 4°C overnight. The extract was collected, and the absorbance (A) was measured at 530 nm and 657 nm. Based on the absorbance (A530) at 530 nm and (A657) at 657 nm, the anthocyanin content of the flower buds or flowers at different developmental stages was calculated using the formula: anthocyanin content = (A530 - 0.25 × A657) / fresh weight.

[0041] The measurement results are as follows Figure 2 As shown, the anthocyanin content is higher in the S5-S6 period.

[0042] Example 3: Validation of RcETC3 expression profiles in roses at different developmental stages

[0043] Select as Figure 1 RNA was extracted from flower buds or flowers at different developmental stages of *Echeveria elegans* using an RNAplant kit (commercially available). The total RNA was then reverse transcribed into cDNA using a reverse transcription kit (commercially available). Using the cDNA obtained from the reverse transcription of *Echeveria elegans* at different developmental stages as templates, PCR amplification was performed using primer pairs with nucleotide sequences SEQ ID NO:5 and SEQ ID NO:6, respectively, to detect the relative expression level of the RcETC3 gene. Using *Echeveria elegans* RcActin as an internal reference gene, the primer pairs for amplifying the internal reference gene had nucleotide sequences SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0044] Test results as follows Figure 3 As shown, the expression level of RcETC3 varies at different developmental stages. Higher expression is observed in petal S2, suggesting that RcETC3 may be involved in changes in rose petal color.

[0045] Example 4: Silencing the RcETC3 gene in the 'Monthly Pink' rose variety

[0046] The 339bp open reading frame of the RcETC3 gene obtained in Example 1 was operatively ligated into the pTRV2 vector to form the TRV2-RcETC3 vector containing the gene fragment. This vector and the blank control TRV2-Ev were transformed into Agrobacterium GV3101. The small round petals of rose were vacuum-infected using the Agrobacterium-mediated method (the second and third layers of rose petals were punched with a punch to obtain small round petals). The phenotype of the 'Yueyuefen' rose was observed after 4-7 days of culture.

[0047] Photo of small round rose petals as shown Figure 4 As shown, the petal discs of the 'Monthly Pink' gene-silenced individuals become noticeably darker in color.

[0048] Example 5: Determination of anthocyanin content in the small round petals of the 'Yueyuefen' rose with gene silencing

[0049] Will Figure 4 The petal discs from the experimental and control groups were weighed and placed in 1.5 mL centrifuge tubes. The anthocyanin content in the petal discs was tested using the same method as in Example 2.

[0050] Test results are as follows Figure 5 As shown, the anthocyanin content is significantly increased in the small round petals of gene-silenced petals.

[0051] Example 6: Verification of the expression of the RcETC3 gene and anthocyanin synthesis-related genes in the gene-silenced petal discs of the 'Yueyuefen' rose.

[0052] Using RNAplant (commercially available) Figure 4 Total RNA was extracted from the petal discs of the experimental and control groups, and the total RNA was reverse transcribed into cDNA using a commercially available reverse transcription kit. Using the cDNA obtained from the reverse transcription of the control and gene-silenced petal discs as templates, the relative expression levels of RcETC3 and the anthocyanin synthesis-related genes RcANS and RcDFR were measured using primer pairs from SEQ ID NO: 5-10, with RcActin as an internal reference gene. The test results are shown below. Figure 6 As shown.

[0053] according to Figure 6 It can be seen that the expression of RcETC3 was significantly reduced in the silent petal discs of 'Yueyuefen' rose, and its relative expression levels with those of the anthocyanin synthesis genes RcANS and RcDFR were significantly increased, indicating that RcETC3 can regulate the color of 'Yueyuefen' rose petals.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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. Silence RcETC3 The application of genes in deepening the color of the monthly pink rose is characterized by, The RcETC3 Genes are nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, Silent Monthly Pink Rose RcETC3 Gene expression specifically includes: The RcETC3 The open reading frame of the gene is operatively linked to the pTRV2 vector to form a vector containing the gene described above. RcETC3 TRV2-RcETC3 recombinant vector of the gene; The TRV2-RcETC3 recombinant vector was introduced into Agrobacterium, and Agrobacterium-mediated transformation was used to obtain... RcETC3 Genetically modified monthly rose with silenced genes.

3. A method for deepening the color of the monthly pink rose, characterized in that, Silent Monthly Pink Rose RcETC3 Gene expression; the aforementioned RcETC3 Genes are nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that, Silent Monthly Pink Rose RcETC3 Gene expression specifically includes: The RcETC3 The open reading frame of the gene is operatively linked to the pTRV2 vector to form a vector containing the gene described above. RcETC3 TRV2-RcETC3 recombinant vector for the gene; The TRV2-RcETC3 recombinant vector was introduced into Agrobacterium, and Agrobacterium-mediated transformation was used to obtain... RcETC3 Genetically modified monthly rose with silenced genes.

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