Lilium cv anthocyanin synthesis regulation gene Lvwrky75 and application thereof

Abstract

The present application belongs to the field of plant molecular biology and genetic engineering technology, and particularly relates to a lily anthocyanin synthesis regulation gene LvWRKY75 and application thereof, and the present application has obtained an overexpression vector and a silencing expression vector of the gene. The overexpression LvWRKY75 gene is transformed into wild-type Arabidopsis, and after obtaining the third generation of transgenic plants, the expression amount of the LvWRKY75 gene and downstream target genes in the transgenic lines is detected, and it is proved that the LvWRKY75 gene has the effect of promoting anthocyanin synthesis. The yeast one-hybrid technology, gel retardation experiment and double-molecule fluorescence complementation experiment prove that the LvWRKY75 gene plays a positive regulation role in the regulation of Westin red lily color, and these findings provide important experimental and theoretical basis for further understanding the molecular mechanism and genetic regulation network of plant flower color formation.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and genetic engineering technology, specifically relating to a lily anthocyanin synthesis regulatory gene LvWRKY75 and its application, which is an important regulatory factor involved in the lily flower color synthesis process. Background Technology

[0002] Ornamental plants are widely cultivated in parks, along roadsides, and as potted plants displayed indoors. They beautify the environment and purify the air, improving environmental quality. Consequently, market demand is high, cultivation area is expanding year by year, and commercial value is considerable. Flowers are the main ornamental organs of ornamental plants, and flower color is the most important ornamental trait and one of the key factors determining a flower's ornamental value. Therefore, cultivating varieties with different flower colors is an important goal of ornamental plant breeding. Currently, some transcription factors regulate the expression of structural genes related to anthocyanin biosynthesis, thereby affecting the activity of these encoded enzymes and thus regulating anthocyanin accumulation. However, the specific regulatory mechanisms still require further in-depth research. Therefore, as one of the most famous ornamental flowers, exploring the mechanism of flower color formation in lilies is particularly important. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a gene LvWRKY75 that regulates anthocyanin synthesis in lilies and its applications.

[0004] The present invention is achieved by providing a lily anthocyanin synthesis regulatory gene LvWRKY75, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0005] A protein encoded by the lily anthocyanin synthesis regulatory gene LvWRKY75 is provided, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] Primers for amplifying the above-mentioned lily anthocyanin synthesis regulatory gene LvWRKY75 are provided, and a recombinant vector containing the above-mentioned lily anthocyanin synthesis regulatory gene LvWRKY75 is provided.

[0007] Specifically, the preparation process of the above recombinant vector is as follows: using cDNA from lily petals as a template, pRI101-LvWRKY75-F and pRI101-LvWRKY75-R are used as primers to amplify the full-length coding region of the LvWRKY75 gene, and the target small fragment is recovered; the vector pRI101 is digested with SalⅠ and BamHI, and the linear large fragment is recovered; the target small fragment of LvWRKY75 is ligated to the linear large fragment of pRI101 using HD Cloning Plus, and the ligation product is transformed into Escherichia coli DH5α using the heat shock method. After colony PCR and sequencing identification, the recombinant vector pRI101-LvWRKY75 is obtained by shaking and plasmid extraction.

[0008] The application of the aforementioned lily anthocyanin synthesis regulatory gene LvWRKY75 is provided for regulating anthocyanin synthesis in plants.

[0009] Compared with the prior art, the advantages of the present invention are as follows:

[0010] This invention discovered a gene, LvWRKY75, that regulates anthocyanin synthesis in lilies, and obtained overexpression and silencing vectors for this gene. After transforming wild-type Arabidopsis thaliana with overexpressed LvWRKY75, and obtaining third-generation transgenic plants, the expression levels of LvWRKY75 and its downstream target genes in these transgenic lines were detected, demonstrating that LvWRKY75 promotes anthocyanin synthesis. Using yeast one-hybrid assays, gel retardation experiments, and bimolecular fluorescence complementation experiments, it was confirmed that LvWRKY75 plays a positive regulatory role in the flower color regulation of *Lilium sinense*. These findings provide an important experimental and theoretical foundation for further understanding the molecular mechanisms and genetic regulatory networks of plant flower color formation. Attached Figure Description

[0011] Figure 1 Different stages of petal growth and development of the 'Xinuohong' lily;

[0012] Figure 2 The changing trend of LvWRKY75 gene expression at different stages of petal growth and development in 'Xinuohong' lily;

[0013] Figure 3 Image A shows the phenotype of the LvWRKY75 gene transiently silenced in the petals of the 'Xinuohong' lily (the three petals on the left are those infected with the empty vector, and the three petals on the right are those with the LvWRKY75 gene transiently silenced by VIGS). Image B shows the comparison of anthocyanin content between the control group and the experimental group. Image C shows the expression level of the anthocyanin synthesis structure gene after transient silencing of the LvWRKY75 gene.

[0014] Figure 4Image A shows the phenotype of the 'Xinuohong' lily petals transiently overexpressing the LvWRKY75 gene (the three petals on the left are those infected with an empty vector, and the three petals on the right are those transiently overexpressing the LvWRKY75 gene). Image B shows the comparison of anthocyanin content between the control and experimental groups. Image C shows the expression level of the anthocyanin synthesis structure gene after transient overexpression of the LvWRKY75 gene.

[0015] Figure 5-1 This is a PCR electrophoresis image of LvWRKY75-OE transgenic Arabidopsis thaliana.

[0016] Figure 5-2 A shows the gene expression level of LvWRKY75-OE in three randomly selected transgenic Arabidopsis thaliana plants. B shows a comparison of the color of Arabidopsis thaliana seed coat and the color of the base of the stem. C shows the anthocyanin extraction results of the homozygous transgenic Arabidopsis thaliana lines. D shows the expression level of the anthocyanin synthesis structure gene in transgenic Arabidopsis thaliana.

[0017] Figure 6 A shows the analysis of WRKYs binding sites in the LvMYB5 promoter. B shows the yeast one-hybrid assay of LvWRKY75 and proLvMYB5 promoters. C shows the gel migration assay of the LvWRKY75 and proLvMYB5 promoters. D shows the luciferase complementation assay of the interaction between LvWRKY75 and LvMYB5. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the invention.

[0019] Example 1: Analysis of the expression pattern of the LvWRKY75 gene in lilies at different developmental stages under natural conditions:

[0020] refer to Figure 1 Transcriptome analysis was performed on petals from three different coloring stages of the 'Xinuohong' lily variety: S1 (bud stage), S2 (coloring stage), and S3 (full bloom stage). RNA was extracted from the petals using the RNAprep pure plant kit (DP432, Tiangen). After electrophoresis to check the RNA extraction efficiency, the RNA was reverse transcribed into cDNA using the QuantScript RT kit (KR103-04, Tiangen). Real-time quantitative PCR was performed using the obtained cDNA as a template, qF and qR as primers, and qActin-F and qActin-R as internal control primers, using the QuantScript RT kit (KR103-04, Tiangen) and a Roche real-time PCR instrument. Data analysis was performed using the 2-ΔΔCt method. The results are shown in [Figure number missing]. Figure 2The results showed that the trend of this expression level change was consistent with the trend of petal color change, suggesting that this gene may be involved in the regulation of lily flower color. Therefore, this gene was selected as a candidate gene for further investigation. The vectors and primers used in the process are shown in Tables 1, 2, and 3.

[0021] Table 1. Vector Names and Resistance

[0022]

[0023] Table 2. LvWRKY75-specific primers used during vector construction.

[0024]

[0025]

[0026] Table 3 Primers for Real-Time Quantitative PCR

[0027]

[0028] Example 2: Construction of recombinant vectors pRI101-LvWRKY75 and pTRV2-LvWRKY75

[0029] Using cDNA from petals of *Lilium sinosum* at the S3 stage as a template, the full-length coding region of the LvWRKY75 gene was amplified using primers pRI101-LvWRKY75-F and pRI101-LvWRKY75-R, and the target small fragment was recovered. The vector pRI101 was digested with SalⅠ and BamHI in the following reaction system: 7 μL pRI101 plasmid, 1 μL 10×T buffer, 1 μL SalⅠ, 1 μL BamHI, incubated at 37℃ for 3 h, and the linear large fragment was recovered. The LvWRKY75 target small fragment was ligated to the pRI101 linear large fragment using HD Cloning Plus (TaRaKa, 638910). The reaction system consisted of 6 μL linear large fragment, 2 μL target small fragment, and 2 μL 5×In-Fusion HD Enzyme Premix, incubated at 50℃ for 15 min. The ligation product was transformed into *E. coli* DH5α using a heat shock method. After colony PCR and sequencing identification, the recombinant plasmid pRI101-LvWRKY75 was obtained by shaking and plasmid extraction. For the construction of pTRV2-LvWRKY75, cDNA from petals of *Lilium sinense* at the S3 stage was used as a template. The full-length coding region of the LvWRKY75 gene was amplified using primers pTRV2-LvWRKY75-F and pTRV2-LvWRKY75-R, and the target fragment was recovered. The vector pTRV2 was digested with EcoRI and BamHI, with the reaction time and specific steps as described above.

[0030] refer to Figure 3 and Figure 4 The results indicate that transient silencing of the LvWRKY75 gene by VIGS in the petals of the 'Xinuohong' lily leads to a decrease in the expression of genes related to anthocyanin synthesis, which in turn reduces anthocyanin synthesis in the petals, resulting in a lighter petal color. Therefore, it is preliminarily speculated that the LvWRKY75 gene plays an important positive regulatory role in the regulation of petal color in the 'Xinuohong' lily.

[0031] Example 3: Stable genetic analysis of LvWRKY75 overexpression:

[0032] Since the stable transformation system for lilies within the group is not yet mature, stable genetic transformation was performed in Arabidopsis thaliana to further verify its function. The successfully constructed LvWRKY75 gene overexpression vector was transformed into Agrobacterium GV3101, and then transformed into wild-type (Col-0) Arabidopsis plants using the traditional pollination method. The transformed plants were screened using hygromycin and PCR to obtain T3 generation LvWRKY75-OE transgenic plants under Col-0 background conditions. To accurately verify the correctness of the LvWRKY75-OE transgenic Arabidopsis, DNA was extracted from the T3 generation LvWRKY75-OE transgenic Arabidopsis plants, and PCR detection was performed using primers LvWRKY75-F and 1300-R. Figure 5-1 Electrophoresis results showed that PCR using Col-0 Arabidopsis plant DNA as a template yielded no bands, proving that the LvWRKY75 gene is absent in Col-0 Arabidopsis and can serve as a negative control. Nine Arabidopsis plants were randomly selected from the T3 generation LvWRKY75-OE transgenic plants, and PCR using their Arabidopsis plant DNA as a template yielded a gene fragment of approximately 475 bp, consistent with the size of the target band. This confirms that the LvWRKY75 gene was successfully and stably inherited in these nine T3 generation Arabidopsis plants.

[0033] Referring to Figure 5, the results show that after the LvWRKY75 gene was transferred into Arabidopsis thaliana, it was able to influence the anthocyanin synthesis process by regulating structural genes involved in anthocyanin biosynthesis. This regulatory effect led to significant changes in anthocyanin accumulation in different parts of the Arabidopsis plant, such as the seed coat, stem base, and hypocotyl. This finding reveals the important role of the LvWRKY75 gene in regulating anthocyanin metabolism in Arabidopsis thaliana and further demonstrates its function in flower color regulation. Furthermore, it regulates anthocyanin synthesis and accumulation by modulating key enzyme genes in the anthocyanin biosynthesis pathway.

[0034] Example 4: Functional identification of the interaction between LvWRKY75 and the anthocyanin synthesis pathway gene LvMYB5:

[0035] First, a yeast one-hybrid assay was performed, involving the design of specific primers for PCR amplification of the target fragment, followed by gel recovery of the PCR product to extract the target fragment. The vector was digested with EcoRI and BamHI enzymes, and the digestion products were purified. The target fragment was ligated into the vector and transformed into appropriate host cells. Sequencing verification was performed. After confirmation, a recombinant plasmid was obtained for subsequent experiments or applications. Next, a recombinant plasmid for the pGADT7 transcription factor CDS was constructed. A recombinant plasmid for the pAbAi promoter (or transient element) was also constructed. Yeast competent cells were prepared and transformed into yeast. Subsequent gel retardation experiments were then performed, first inducing the protein (detecting whether the protein is an inclusion body protein and determining the optimal induction concentration), and finally detecting it by electrophoresis. Finally, Agrobacterium tumefaciens pGreenII-0800-LUC, pGreenII-62-SK, proLvMYB5-LUC, and LvWRKY75-62-SK were used for a dual-luciferase reporter gene assay. Through promoter sequence analysis, yeast one-hybrid assays, EMSA, and LUC experiments within the research group, it was demonstrated that LvWRKY75 positively regulates the synthesis and accumulation of anthocyanins during the development of 'Xinuohong' lily petals by binding to the promoter of the anthocyanin synthesis gene LvMYB5. Figure 6 (As shown).

Claims

1. A gene regulating anthocyanin synthesis in lilies LvWRKY75 The application, characterized in that, The lily anthocyanin synthesis regulatory gene is used to regulate anthocyanin synthesis in lilies. LvWRKY75 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The lily anthocyanin synthesis regulatory gene according to claim 1 LvWRKY75 The application, characterized in that, The gene regulating anthocyanin synthesis in lilies LvWRKY75 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

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