Lily anthocyanin synthesis regulation gene LvWRKY75 and application thereof

Abstract

The invention belongs to the technical field of plant molecular biology and genetic engineering, and particularly relates to a lily anthocyanin synthesis regulation gene LvWRKY75 and application thereof, and an overexpression vector and a silent expression vector of the gene are obtained. The overexpressed LvWRKY75 gene is transformed into wild arabidopsis thaliana to obtain a third-generation transgenic plant, and then the expression quantities of the LvWRKY75 gene and a downstream target gene in the transgenic plant are detected to prove that the LvWRKY75 gene has the effect of promoting anthocyanin synthesis. A yeast one-hybrid technology, a gel retardation experiment and a bimolecular fluorescent complementation experiment prove that the LvWRKY75 gene plays a positive regulation role in the process of regulating the flower color of the Scinnolia lilii, and the discovery provides important experimental and theoretical basis for further knowing a molecular mechanism and a genetic regulation network of plant flower color formation.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology and genetic engineering, and specifically relates to a lily anthocyanin synthesis regulatory gene LvWRKY75 and an application thereof, which is an important regulatory factor involved in the lily flower color synthesis process. Background Art

[0002] Ornamental plants are widely cultivated in parks, roadsides, and potted plants displayed indoors. On the one hand, they can beautify the environment, and on the other hand, they can purify the air and improve environmental quality. Therefore, the market demand is large, the cultivated area is expanding year by year, and the commercial value is quite high. Flowers are the main ornamental organs of ornamental plants. The color of flowers is the most important ornamental trait and one of the important factors that determine the ornamental value of flowers. Therefore, cultivating varieties with different flower colors is an important goal of ornamental plant breeding. At present, some transcription factors regulate the expression of structural genes related to anthocyanin biosynthesis, thereby affecting the activity of these encoding enzymes, thereby regulating the accumulation of anthocyanins. However, the specific regulatory mechanism still needs further in-depth study. Therefore, as one of the famous ornamental flowers, it is particularly important to explore the mechanism of flower color formation of lily. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a lily anthocyanin synthesis regulatory gene LvWRKY75 and application thereof.

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

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

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

[0007] Specifically, the preparation process of the above-mentioned recombinant vector is as follows: using cDNA of lily petals as a template, pRI101-LvWRKY75-F and pRI101-LvWRKY75-R as primers to amplify the full length of the LvWRKY75 gene coding region, and recovering the target small fragment; using SalⅠ and BamHI to digest the vector pRI101, and recovering the linear large fragment; using HD Cloning Plus to connect the LvWRKY75 target small fragment to the pRI101 linear large fragment, using the heat shock method to transfer the connection product into Escherichia coli DH5α, after colony PCR and sequencing identification, shaking the bacteria and extracting the plasmid to obtain the recombinant vector pRI101-LvWRKY75.

[0008] Provided is the application of the above-mentioned lily anthocyanin synthesis regulatory gene LvWRKY75 for regulating anthocyanin synthesis in plants.

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

[0010] The present invention discovered a lily anthocyanin synthesis regulatory gene LvWRKY75 in lily, and obtained an overexpression vector and a silent expression vector of the gene. After the overexpressed LvWRKY75 gene was transformed into wild-type Arabidopsis, the third-generation transgenic plants were obtained, and the expression levels of the LvWRKY75 gene and its downstream target genes in the transgenic strains were detected, proving that the LvWRKY75 gene has the effect of promoting anthocyanin synthesis. Yeast single hybridization technology, gel retardation experiment and bimolecular fluorescence complementation experiment were used to prove that the LvWRKY75 gene plays a positive regulatory role in the color regulation process of Sinored lily. These findings provide important experimental and theoretical basis for further understanding the molecular mechanism and genetic regulatory network of plant flower color formation. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0014] Figure 4Figure A shows the phenotype of the petals of 'Xinuohong' lily with transient overexpression of the LvWRKY75 gene (the three petals on the left in the figure are the petals infected with the empty vector, and the three petals on the right are the petals with transient overexpression of the LvWRKY75 gene). Figure B shows the comparison of anthocyanin content between the control group and the experimental group. Figure C shows the expression level of anthocyanin synthesis structural genes after transient overexpression of the LvWRKY75 gene;

[0015] Figure 5-1 This is the PCR electrophoresis diagram of LvWRKY75-OE transgenic Arabidopsis;

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

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

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to explain the present invention and are not used to limit the present invention.

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

[0020] refer to Figure 1 Petals of 'Xinuohong' lily at three different coloring stages, S1 (lily bud stage), S2 (lily coloring stage) and S3 (lily blooming stage), were selected for transcriptome analysis. RNA from petals was extracted using RNAprep pure plant kit (DP432, Tiangen). After electrophoresis to detect the RNA extraction effect, RNA was reverse transcribed into cDNA using Quant Script RT kit (KR103-04, Tiangen). Real-time quantitative PCR was performed using the obtained cDNA as template, qF and qR as primers, qActin-F and qActin-R as internal reference primers, using QuantScript RT kit (KR103 04, Tiangen) and Roche fluorescent quantitative PCR instrument. Data analysis was performed using the 2-△△Ct method, and the results are shown in Figure 2The results showed that the expression trend was consistent with the petal color trend, suggesting that the gene may be involved in the regulation of lily flower color. Therefore, this gene was further explored as a candidate gene. The vectors and primers used in the process are shown in Tables 1, 2, and 3.

[0021] Table 1 Vector name and resistance

[0022]

[0023] Table 2 LvWRKY75 specific primers for vector construction

[0024]

[0025]

[0026] Table 3 Real-time fluorescence quantitative PCR related primers

[0027]

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

[0029] The cDNA of petals of Sinored Lily in S3 period was used as template, pRI101-LvWRKY75-F and pRI101-LvWRKY75-R were used as primers to amplify the full length of LvWRKY75 gene coding region, and the target small fragment was recovered. The vector pRI101 was digested with SalⅠ and BamHI. The reaction system was: 7μL pRI101 plasmid, 1μL 10×T buffer, 1μL SalⅠ, 1μL BamHI, and reacted at 37℃ for 3h to recover the linear large fragment. HD Cloning Plus (TaRaKa, 638910) was used to connect the LvWRKY75 target small fragment to the pRI101 linear large fragment. The reaction system was: 6μL linear large fragment, 2μL target small fragment, 2μL 5×In-Fusion HD Enzyme Premix, and reacted at 50℃ for 15min. The ligation product was transferred into Escherichia coli DH5α by heat shock method, and after colony PCR and sequencing identification, the recombinant plasmid pRI101-LvWRKY75 was obtained by shaking the bacteria and extracting the plasmid. The construction of pTRV2-LvWRKY75 used the cDNA of the petals of the S3 period of Sinored Lily as a template, pTRV2-LvWRKY75-F and pTRV2-LvWRKY75-R as primers to amplify the full length of the LvWRKY75 gene coding region, and the target small fragment was recovered. The vector pTRV2 was digested with EcoR I and BamH I, and the reaction time and specific steps were the same as above.

[0030] refer to Figure 3 and Figure 4 , indicating that in the petals of 'Xinuohong' lily, VIGS transiently silenced the LvWRKY75 gene, resulting in a decrease in the expression of structural genes related to anthocyanin synthesis, which in turn led to a decrease in the synthesis of anthocyanins in the petals, causing the petals to become lighter in color. Therefore, it is preliminarily speculated that the LvWRKY75 gene plays an important role in positive regulation in the regulation of petal color of 'Xinuohong' lily.

[0031] Example 3, stable genetic analysis of overexpressed LvWRKY75:

[0032] Since the stable transformation system of lily in the group is not yet mature, we chose to carry out stable genetic transformation in Arabidopsis to further verify its function. The successfully constructed LvWRKY75 gene overexpression vector was transformed into Agrobacterium GV3101 and transformed into Arabidopsis wild-type (Col-0) plants using the traditional flower-dipping method. The transformed plants were screened with hygromycin and tested by PCR to obtain T3 generation LvWRKY75-OE transgenic plants under Col-0 background conditions. In order to accurately verify the correctness of LvWRKY75-OE transgenic Arabidopsis, DNA of T3 generation LvWRKY75-OE transgenic Arabidopsis plants was extracted, and PCR detection was performed using primers LvWRKY75-F and 1300-R. Figure 5-1 The electrophoresis results showed that there was no band in the PCR results using Col-0 Arabidopsis plant DNA as a template, proving that there was no LvWRKY75 gene in Col-0 Arabidopsis, which could be used as a negative control. Nine Arabidopsis plants were randomly selected from the T3 generation LvWRKY75-OE transgenic plants, and the PCR results using their Arabidopsis plant DNA as a template obtained a gene fragment of about 475bp, which was consistent with the target band size. It can be determined 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, the gene was able to affect the synthesis process of anthocyanins in Arabidopsis by regulating the structural genes of anthocyanin biosynthesis. This regulatory effect led to significant changes in the accumulation of anthocyanins in different parts of Arabidopsis plants, such as the seed coat, stem base and hypocotyl. This discovery reveals the important role of the LvWRKY75 gene in regulating anthocyanin metabolism in Arabidopsis, and further proves the function of this gene in flower color regulation. The synthesis and accumulation of anthocyanins are then regulated by regulating the key enzyme genes in the anthocyanin biosynthesis pathway.

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

[0035] First, a yeast single hybrid experiment is performed, that is, specific primers are designed for PCR amplification of the target fragment, the PCR product is gel-recovered, and the target fragment is extracted. The vector is digested with EcoR I and BamH I enzymes, and the digestion product is purified. The target fragment is connected to the vector and transformed into an appropriate host cell. Sequencing verification. After confirmation, the recombinant plasmid is obtained for subsequent experiments or applications. Then the recombinant plasmid of the pGADT7 transcription factor CDS is constructed. The recombinant plasmid of the pAbAi promoter (or transient element) is constructed. Prepare yeast competence and transform yeast. Then a subsequent gel retardation experiment is performed, first inducing the protein, that is, detecting whether the protein is an inclusion body protein and determining the optimal induction concentration, and finally running electrophoresis detection. Finally, pGreenII-0800-LUC, pGreenII-62-SK, proLvMYB5-LUC, and LvWRKY75-62-SK Agrobacterium are used. A dual luciferase reporter gene experiment is performed. Through the analysis of promoter sequences, yeast single hybridization, EMSA, LUC and other experiments within the research group, it was proved that LvWRKY75 positively regulates the synthesis and accumulation of anthocyanin biosynthesis during the development of the petals of the 'Xinuohong' lily by binding to the promoter of the anthocyanin biosynthesis gene LvMYB5. Figure 6 shown).

Claims

1. A lily anthocyanin synthesis regulatory gene LvWRKY75, characterized in that: The nucleotide sequence of the lily anthocyanin synthesis regulatory gene LvWRKY75 is shown in SEQ ID NO.

1.

2. A protein encoded by the lily anthocyanin synthesis regulatory gene LvWRKY75, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Primers for amplifying the lily anthocyanin synthesis regulatory gene LvWRKY75 according to claim 1, characterized in that: The invention comprises the lily anthocyanin synthesis regulating gene LvWRKY75 as claimed in claim 1.

4. A recombinant vector, characterized in that: The invention comprises the lily anthocyanin synthesis regulating gene LvWRKY75 as claimed in claim 1.

5. The recombinant vector according to claim 4, characterized in that The cDNA of lily petals was used as a template and pRI101-LvWRKY75-F and pRI101-LvWRKY75-R were used as primers to amplify the full-length coding region of LvWRKY75 gene, and the target small fragment was recovered; the vector pRI101 was digested with SalⅠ and BamHI to recover the linear large fragment; the target small fragment of LvWRKY75 was ligated to the linear large fragment of pRI101 using HD Cloning Plus, and the ligation product was transferred into Escherichia coli DH5α using the heat shock method. After colony PCR and sequencing identification, the recombinant vector pRI101-LvWRKY75 was obtained by shaking the bacteria and extracting the plasmid.

6. The use of the lily anthocyanin synthesis regulatory gene LvWRKY75 according to claim 1, characterized in that: Used to regulate anthocyanin synthesis in plants.

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