Transcription factor for regulating and controlling syringa oblata anthocyanin synthesis and application thereof

Abstract

The invention discloses a transcription factor for regulating syringa oblata anthocyanin synthesis and application thereof, the amino acid sequence of the transcription factor is a sequence 6 or a sequence 7 in a sequence table, and the nucleotide sequence of the transcription factor is a sequence 3 or a sequence 4 in the sequence table. The transcription factor can be used for cultivating syringa oblata germplasm with higher anthocyanin content compared with wild type syringa oblata germplasm.

Description

Technical Field

[0001] The invention relates to a transcription factor for regulating the synthesis of lilac anthocyanin and application thereof. Background Art

[0002] Flower color is an important indicator of flower quality and is of great significance for improving the ornamental and economic value of flowers. There are many types of anthocyanins in nature. Anthocyanins, as the most important flavonoids, are the main pigments that determine pink, red, blue, purple and other colors. Their biosynthesis is one of the most well-studied plant secondary metabolic pathways. Anthocyanin 3-O-glucosyltransferase (UFGT) is located at the end of the anthocyanin biosynthesis pathway and directly participates in (catalyzes) the synthesis of anthocyanins. It plays a vital role in plant anthocyanin metabolism. Syringa oblata, as an important ornamental shrub in gardens, changes color of its petals during the opening process of its flowers, but its internal molecular mechanism is rarely reported. Transcriptome sequencing of Syringa oblata at different flower development stages was performed, and combined with existing genomic data, it was found that the SoUFGT1 gene had obvious expression differences, with the highest expression in the bud stage and lower expression in the initial and full flowering stages. It is speculated that the SoUFGT1 gene may be involved in regulating the color changes of Syringa oblata during the opening process of its flowers. Summary of the invention

[0003] Therefore, one object of the present invention is to provide a transcription factor that regulates the synthesis of anthocyanins in lilac.

[0004] The transcription factors regulating the synthesis of anthocyanins in lilac provided by the present invention are named SoMYB44 and SobHLH130 respectively;

[0005] The sequence of the SoMYB44 gene is shown as Sequence 3 in the sequence listing, and the sequence of the SobHLH130 gene is shown as Sequence 4 in the sequence listing.

[0006] The SoMYB44 gene encodes the transcription factor protein described in sequence 6 (composed of 279 amino acids) and the SobHLH130 gene encodes the transcription factor protein described in sequence 7 (composed of 340 amino acids) in the sequence table.

[0007] Another object of the present invention is to provide a recombinant expression vector, which contains the transcription factor.

[0008] Another object of the present invention is to provide a recombinant host bacterium, wherein the recombinant host bacterium contains the recombinant expression vector.

[0009] The above-mentioned transcription factors can regulate the expression of the Syringa syringa SoUFGT1 gene. The nucleotide sequence of the Syringa syringa SoUFGT1 gene is shown in Sequence 1 in the sequence table. The SoUFGT1 gene encodes the protein (composed of 381 amino acids) described in Sequence 5 in the sequence table.

[0010] The transcription factor can activate the SoUFGT1 gene promoter, and the nucleotide sequence of the SoUFGT1 gene promoter is shown in Sequence 2 in the sequence list.

[0011] The transcription factor of the present invention can be used to cultivate lilac germplasm.

[0012] In certain embodiments, the Syringa syringa germplasm contains higher anthocyanins than wild type.

[0013] Another object of the present invention is to provide a method for cultivating lilac germplasm, comprising overexpressing the transcription factor in lilac by transgenic technology to obtain lilac plants containing higher anthocyanins than the wild type.

[0014] The transcription factor for regulating the synthesis of lilac anthocyanins of the present invention provides a reliable basis for breeding excellent lilac germplasm, and is of great significance for promoting the high-quality development of the flower industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the relative expression level of SoUFGT1 and its upstream transcription factors in different flower development stages and different organs;

[0016] Figure 2 This is the result of functional validation of SoUFGT1 in Syringa syringae;

[0017] Figure 3 The results of functional validation of SoMYB44 and SobHLH130 in Syringa syringae;

[0018] Figure 4 The results are as follows: the regulatory effects of SoMYB44 and SobHLH130 on SoUFGT1 in lilac (yeast one-hybrid and dual-luciferase assays);

[0019] Figure 5 This is the interaction verification result between SoMYB44 and SobHLH130 of Syringa japonica (yeast two-hybrid and bimolecular fluorescence complementation assay). DETAILED DESCRIPTION

[0020] In order to better illustrate the purpose, complete technical route and advantages of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments and drawings. However, the embodiments described below are only part of the embodiments of the present invention and do not include all the embodiments. Based on the embodiments of the present invention, other embodiments that can be obtained by those skilled in the art without paying creative work all belong to the protection scope of the present invention.

[0021] In the embodiments described below, unless otherwise specified, the conventional methods used are technical means generally recognized or commonly used by those skilled in the art, and the instruments, reagents, consumables, etc. used in the embodiments of the present invention can be obtained through regular commercial channels.

[0022] Example 1. Acquisition and application of lilac transcription factors SoMYB44 and SobHLH130

[0023] 1. Test materials

[0024] The experimental material was Syringa chinensis planted in the Beijing Agricultural Science and Technology Park in Tingzizhuang, Changping District, Beijing. The petals of Syringa chinensis at different flower development stages (bud stage, early flowering stage, full flowering stage and late flowering stage) and samples of different organs (roots, stems, leaves, flowers) at full flowering stage were collected, frozen with liquid nitrogen and stored in a -80℃ refrigerator. Nicotiana benthamiana seedlings were sown in 5×10 plug trays and cultured in the culture room of the Garden Building of Beijing Agricultural College (16h light / 8h dark, 24℃) for subcellular localization, dual luciferase and bimolecular fluorescence complementation experiments. Sterile seedlings of Syringa chinensis were cultivated, callus tissue was induced using leaves, and placed in the culture room of the Garden Building of Beijing Agricultural College (22℃, 16h light / 8h dark).

[0025] 2. Test methods and results

[0026] 2.1 Gene cloning and expression analysis

[0027] Under liquid nitrogen precooling conditions, petal samples and different organ samples of Syringa arguta at different stages were ground into powders, then placed in 1.5 ml centrifuge tubes, RNA was extracted and reverse transcribed into cDNA according to the instructions, and the samples were stored in a -20°C refrigerator after dilution.

[0028] Table 1. Primer sequences

[0029]

[0030]

[0031] The primers in Table 1 were used to perform PCR amplification with the synthesized cDNA as a template, and the gene fragments SoUFGT1, SoMYB44 and SobHLH130 were obtained after correct sequencing.

[0032] Total RNA was extracted and reverse transcribed into single-stranded cDNA, and qRT-PCR was performed to analyze the expression changes of genes SoUFGT1, SoMYB44, and SobHLH130 in different flower development stages and different organs, with the internal reference gene SoACTIN. Real-time quantitative PCR instrument was used for detection, and the reaction system is shown in Table 2:

[0033] Table 2. qRT-PCR amplification system

[0034] Reaction components Component volume / μL cDNA 1 YG-F 0.4 YG-R 0.4 SYBR Green Pro Taq Mix 10 <![CDATA[ddH2O]]> Up to 20

[0035] The sequence lengths of the obtained lilac genes SoUFGT1 gene, SoMYB44 gene, and SobHLH130 gene were 1146bp, 840bp, and 1023bp, respectively. The sequencing results showed that the sequence of the SoUFGT1 gene was shown in sequence 1 in the sequence listing, the sequence of the SoMYB44 gene was shown in sequence 3 in the sequence listing, and the sequence of the SobHLH130 gene was shown in sequence 4 in the sequence listing.

[0036] The protein described in sequence 5 in the SoUFGT1 gene coding sequence table (consisting of 381 amino acids), the transcription factor protein described in sequence 6 in the SoMYB44 gene coding sequence table (consisting of 279 amino acids), and the transcription factor protein described in sequence 7 in the SobHLH130 gene coding sequence table (consisting of 340 amino acids).

[0037] qRT-PCR analysis of the SoUFGT1 gene in Syringa syringae ( Figure 1 Middle A) and the upstream transcription factor SoMYB44 ( Figure 1 Middle B), SobHLH130( Figure 1 C) expression pattern found (such as Figure 1 As shown in the figure, in the four stages of lilac petals, namely, the bud stage, the early flowering stage, the full flowering stage and the late flowering stage, the expression levels of the three genes showed a trend of first decreasing and then increasing. The expression level was the highest in the bud stage, then the expression level began to decrease, and then increased again in the late flowering stage. Among the different organs in the full flowering stage, SoUFGT1, SoMYB44 and SobHLH130 were expressed at the highest levels in the roots, followed by in the flowers, and the expression levels were lower in the stems and leaves.

[0038] 2.2 Subcellular localization and transient overexpression functional verification

[0039] After removing the terminator from the cloned SoUFGT1, SoMYB44, and SobHLH130 genes, the primers in Table 3 were used to connect them to the pRI101 vector (recorded in the literature Xi Zhang et al., MdWER interacts with MdERF109 andMdJAZ2 to mediatemethyl jasmonate-and light-induced anthocyaninbiosynthesisin apple fruit, The Plant Journal (2024) 118, 1327–1342, available to the public from Beijing Agricultural College) to obtain recombinant expression vectors pRI101-SoUFGT1, pRI101-SoMYB44, and pRI101-SobHLH130, respectively.

[0040] Table 3. Vector homology arm primer sequence 1

[0041]

[0042] The recombinant expression vectors pRI101-SoUFGT1, pRI101-SoMYB44, and pRI101-SobHLH130 were transformed into GV3101 Agrobacterium by liquid nitrogen transformation. After 3 days of incubation at 28°C, colony PCR verification was performed, and the bacterial solution with the correct band was shaken overnight. The cells were collected by centrifugation at room temperature and resuspended in infection solution to OD 600 The back of tobacco leaves was injected for infection, and the pRI101 empty vector was used as the control. After infection, the cells were kept in the dark for 24 hours, and then cultured normally for 2 days. The subcellular localization was observed using laser confocal microscopy.

[0043] like Figure 2 As shown in A, with the empty vector pRI101 as a negative control, under the excitation light of wavelength 488nm, it was found that the empty vector pRI101 was expressed in the nucleus, cell membrane and intracellular organelles without distribution specificity, while the green fluorescence signal of pRI101-SoUFGT1 was only observed in the cytoplasm, indicating that the SoUFGT1 protein was expressed in the cytoplasm. The green fluorescence signals of pRI101-SoMYB44 and pRI101-SobHLH130 were distributed in the nucleus, indicating that their proteins were expressed in the nucleus, which was consistent with the predicted results.

[0044] The infection solution containing pRI101-SoUFGT1 was injected into the leaves of lilac to verify transient overexpression, with pRI101 empty vector as control. The preparation method of infection solution and the experimental method were the same as those of subcellular localization experiment. Figure 2 As shown in B, compared with the uninjected leaves ( Figure 2Compared with the control group injected with pRI101 empty vector, the leaves of lilac after injection of pRI101-SoUFGT1 turned red. Figure 2 As shown in C, the gene was successfully expressed in the leaves of lilac, and the SoUFGT1 gene ( Figure 2 The expression level of pRI101-SoUFGT1 in the medium was significantly doubled. Weigh 0.1 g of leaves in a centrifuge tube, grind them in a grinder under liquid nitrogen conditions, add the extract, and extract anthocyanins by ultrasonic extraction (Beijing Solaibao Technology Co., Ltd., BC1350, China). Figure 2 As shown in D, transient overexpression of SoUFGT1 ( Figure 2 The total amount of anthocyanins in the leaves of the transfectant pRI101-SoUFGT1 increased 5-fold, indicating that the SoUFGT1 gene is involved in the biosynthesis of anthocyanins in plants.

[0045] SoMYB44 and SobHLH130 genes were overexpressed in lilac leaves, and the infection fluid containing pRI101-SoMYB44 or pRI101-SobHLH130 was injected into lilac leaves for transient overexpression verification, with pRI101 empty vector as control. The preparation method of infection fluid and the experimental method were the same as those of subcellular localization experiment. Figure 3 As shown in A, the leaves were found to turn red to varying degrees. After testing the anthocyanin content, it was found that the injection of infection solution containing pRI101-SoMYB44 or pRI101-SobHLH130 increased the total amount of anthocyanins by two to three times compared with the control group injected with pRI101 empty vector.

[0046] The callus tissue was placed in the infection solution containing pRI101-SoMYB44 or pRI101-SobHLH130, and the experiment was conducted according to the infection method of apple callus tissue (Li Y., Mao K., Zhao C., Zhao X., Zhang H., Shu H., Hao Y., 2012. MdCOP1 ubiquitin E3 ligases interact with MdMYB1 to regulate light-induced anthocyanin biosynthesis and red fruit coloration in apple. Plant Physiol. 160 (2), 1011-1022.). The callus tissues overexpressing transcription factors SoMYB44 and SobHLH130 were cultured in the dark for 20 days and then transferred to a 16°C light environment for culture. The phenotypic changes were observed and it was found that the callus tissue turned red significantly. Figure 3As shown in B, the expression levels of transcription factors SoMYB44 and SobHLH130 in callus tissue were significantly increased. The anthocyanins in the callus tissue were extracted and quantified. The results showed that the total content of anthocyanins increased significantly by 3-4 times. Figure 3 As shown in C, after overexpression of SoMYB44 ( Figure 3 pRI101-SoMYB44) and SobHLH130 ( Figure 3 The expression levels of SoUFGT1 gene in callus tissues of pRI101-SobHLH130 in the culture medium increased to varying degrees. After overexpression of SoMYB44, the expression level of SoUFGT1 gene increased by about 1.1 times, and after overexpression of SobHLH130, the expression level of SoUFGT1 gene increased by about 0.5 times. This indicates that the two transcription factors can regulate the expression level of SoUFGT1 gene, thereby affecting the biosynthesis of anthocyanins.

[0047] Example 2: Yeast one-hybrid and dual-luciferase assays

[0048] According to the genomic data, the first 2000bp of the CDS region of the SoUFGT1 gene was selected as the promoter sequence to design primers. The primers are shown in Table 4. Lilac DNA was extracted and the promoter was cloned after dilution. The amplified fragment was sequenced and named SoUFGT1pro. The sequence of the SoUFGT1 promoter is shown in Sequence 2 in the sequence table.

[0049] Table 4. Vector homology arm primer sequence 2

[0050]

[0051] The SoUFGT1pro binding site was predicted by the JASPAR website, and it was found that the promoter contained two types of transcription factor binding sites. The gene fragments SoMYB44 and SobHLH130 were constructed into the pNC-GADT7 vector (constructed according to the method disclosed in CN110863004A, which can be purchased from Hainan Nixing Biotechnology Co., Ltd.) and transformed into Escherichia coli. After culturing with DH5α for 12 hours, the bacteria were selected for sequencing. The pNC-GADT7-SoMYB44 and pNC-GADT7-SobHLH130 recombinant expression vectors were successfully obtained by sequencing and stored in a -20℃ refrigerator.

[0052] The SoUFGT1 promoter (sequence 2 in the sequence list) was constructed into the pNC-AbAi vector (available from Hainan Nixing Biotechnology Co., Ltd.), and the AbAi-SoUFGT1pro recombinant expression vector was obtained by successful sequencing.

[0053] The AbAi-SoUFGT1pro recombinant expression vector was linearized and digested with Bstb I enzyme, and then transformed into Y1HGold competent cells (the method of transforming yeast competent cells is described in the instructions of the kit, Weidi Biotechnology, China), and coated on SD / -Ura plates, and inverted and cultured at 28°C for 48 to 96 hours. After the growth of plaques, the bacteria were picked for colony PCR identification. After successful identification by electrophoresis, a single colony was picked and cultured in 5 mL SD / -Ura liquid culture medium at 28°C and 200 rpm overnight until OD 600 When the concentration is about 0.5, start preparing competent cells and store them at -20℃.

[0054] According to the instructions of the kit, pNC-GADT7 was transferred into the AbAi-SoUFGT1pro yeast competent medium prepared in the previous step, and spread on the defective SD / -Leu / -Ura solid medium, and inverted at 28°C for 48-96h. After the plaques grow, pick a single colony in 10μL water, mix well, and then draw 2μL to spot on SD / -Leu / -Ura screening medium containing different concentrations of AbA (Aureobasidin A) (200ng / ml, 400ng / ml, 600ng / ml), repeat three times, inverted at 28°C for 48-96h, observe the growth of the colonies, and determine the AbA concentration that inhibits the self-activation of the promoter.

[0055] Using pNC-SoMYB44 and pNC-SobHLH130 series vector primers (JT-SoMYB44 and JT-SobHLH130 in Table 4), and using lilac cDNA as a template, the cloned gene fragment was constructed into the pNC-GADT7 vector to obtain the pNC-GADT7-SoMYB44 and pNC-GADT7-SobHLH130 recombinant expression vectors, respectively. According to the instructions of the kit, they were transferred into the prepared AbAi-SoUFGT1pro yeast competent medium, spread on SD / -Leu / -Ura plates, and inverted and cultured at 28°C for 48 to 96 hours. After the growth of plaques, pick a single colony and dilute it in 10μL of water, pipette 2μL and spot it on the SD / -Leu / -Ura plate containing 600ng / ml AbA, and culture it in a constant temperature incubator at 28°C for observation. Figure 4 As shown in A, it was found that the yeast colonies transformed with pNC-GADT7-SoMYB44 and pNC-GADT7-SobHLH130 could grow normally, while the colonies transformed with pNC-GADT7 empty vector could not grow, indicating that both transcription factors can bind to SoUFGT1pro of lilac and regulate the expression of SoUFGT1 gene.

[0056] In addition, SoUFGT1pro was connected to the pNC-Green-LUC vector (the vector is recorded in the literature Yan P., Tuo D., Shen W., Deng H., Zhou P., and Gao XA Nimble Cloning-compatibl e vector systemfor high-throughput gene functional analysis in plants. Plant Co mmunications (2023) 4, 100471., and the public can obtain it from the Chinese Academy of Tropical Agricultural Sciences). After successful sequencing, the LUC:SoUFGT1pro recombinant plasmid was obtained and transformed into Agrobacterium. The transformation method is the same as above. Mix equal volumes of LUC:SoUFGT1pro and pRI101 infection solution (control group) ( Figure 4 pRI101+LUC:SoUFGT1pro), LUC:SoUFGT1pro and pRI101-SoMYB44 infection solution were mixed in equal volumes ( Figure 4 pRI101-SoMYB44+LUC:SoUFGT1pro), LUC:SoUFGT1pro and pRI101-SobHLH 130 infection solution were mixed in equal volumes ( Figure 4 pRI101-SobHLH130+LUC:SoUFGT1pro) was used to infect tobacco after 3 h in darkness, using the same infection method as above. After 24 h in darkness and 2 days of light culture, the fluorescence intensity of tobacco leaves was observed in a bioluminescence instrument (Tianneng, China). Figure 4 As shown in B, it was found that the fluorescence intensity of tobacco leaves transformed with both SoUFGT1pro and transcription factors was stronger than that of the control group. This further indicates that both transcription factors can bind to the SoUFGT1 promoter and positively regulate the expression of the SoUFGT1 gene.

[0057] Example 3: Yeast two-hybrid and bimolecular fluorescence complementation assay

[0058] SobHLH130 was connected to the pNC-GBKT7 vector (constructed according to the method disclosed in CN110863004A, and can be purchased from Hainan Nixing Biotechnology Co., Ltd.) to obtain the recombinant expression vector pNC-GBKT7-SobHLH130. At the same time, pNC-GADT7-SoMYB44 and pNC-GBKT7-SobHLH130 were co-transformed into yeast competent cells Y2Hgold (the method for transforming yeast competent cells is shown in the instructions of the kit, Weidi Biotechnology, China), spread on SD / -Leu / -Trp solid culture medium, and inverted at 28°C for 48 to 96 hours. At the same time, pNC-GADT7 empty vector and pNC-GBKT7 empty vector were co-transformed as a control group. After plaques grow on the plate, perform colony PCR on the plaques. The single colony with the correct band is diluted in 10 μL of water and spotted on SD / -Ade / -His / -Leu / -Trp medium. After drying, invert it in a 28°C constant temperature incubator and culture it for 48 to 96 hours to observe the growth of the colonies. The grown colonies are stained with X-α-gal and cultured in the dark at 28°C for 3 to 4 hours to observe whether they change color, and take photos and record them at the same time.

[0059] like Figure 5 As shown in A, compared with the control group co-transformed with pNC-GADT7+pNC-GBKT7 empty vector, the yeast co-transformed with pNC-GADT7-SoMYB44+pNC-GBKT7-SobHLH130 can grow normally in both the two-deficiency and four-deficiency culture media, and will turn blue after being stained with X-α-gal for 3 to 4 hours, indicating that SoMYB44 and SobHLH130 interact in vitro.

[0060] Using pNC-SoMYB44 and pNC-SobHLH130 series vector primers (JT-SoMYB44 and JT-SobHLH130 in Table 4) and cDNA as a template, the cloned gene fragments were constructed into pNC-BiFC-Enn and pNC-BiFC-Ecn vectors (pNC-BiFC-Enn and pNC-BiFC-Ecn vectors are recorded in the literature Yan P., Tuo D., Shen W., Deng H., Zhou P., and Gao XA Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants. Plant Communications (2023) 4, 100471., and are available to the public from the Chinese Academy of Tropical Agricultural Sciences) to obtain pNC-BiFC-Enn-SoMYB44 and pNC-BiFC-Ecn-SobHLH130 recombinant expression vectors, respectively.

[0061] The recombinant expression vectors were transformed into Agrobacterium competent GV3101 (Weidi Biotechnology, China) with pSoup and p19 tags, respectively. When preparing the infection solution, the infection solution containing pNC-BiFC-Enn-SoMYB44 and pNC-BiFC-Ecn-SobHLH130 bacterial solution was mixed in equal volumes, and the tobacco leaves were infected after 3 hours in the dark. After culturing for 2 to 3 days, the tobacco leaves near the infection holes were cut to prepare slides, and the YFP signal was observed under a laser confocal microscope.

[0062] like Figure 5 As shown in B, YFP signals were present in tobacco plants injected with the pNC-BiFC-Enn-SoMYB44+pNC-BiFC-Ecn-SobHLH130 recombinant vector, and occurred in the nucleus, indicating that SoMYB44 interacts with SobHLH130 in the nucleus.

[0063] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all within the scope defined by the claims of this application.

Claims

1. A transcription factor for regulating the synthesis of anthocyanins in lilac, characterized in that: The amino acid sequence of the transcription factor is shown in Sequence 6 or Sequence 7 in the sequence listing.

2. The transcription factor according to claim 1, characterized in that The nucleotide sequence of the transcription factor is sequence 3 or sequence 4 in the sequence listing.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the transcription factor according to claim 1 or 2.

4. A recombinant host bacterium, characterized in that: The recombinant host bacteria contains the recombinant expression vector according to claim 1 or 2.

5. Use of the transcription factor according to claim 1 or 2 in regulating the expression of the Syringa syringa SoUFGT1 gene, the nucleotide sequence of the Syringa syringa SoUFGT1 gene being as shown in Sequence 1 in the sequence listing.

6. Use of the transcription factor according to claim 1 or 2 in activating the SoUFGT1 gene promoter, the nucleotide sequence of the SoUFGT1 gene promoter is as shown in Sequence 2 in the sequence listing.

7. Use of the transcription factor according to claim 1 or 2 in cultivating lilac germplasm.

8. The method according to claim 7, characterized in that The lilac germplasm contains higher anthocyanins than the wild type.

9. A method for cultivating lilac germplasm, comprising overexpressing the transcription factor according to claim 1 or 2 in lilac by genetic engineering technology to obtain lilac plants containing higher anthocyanins than wild type.

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