Application of CtGSTU32 gene in promoting accumulation of safflower flavonoids

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a CtGSTU32 gene in promoting accumulation of safflower flavonoid compounds, and the nucleotide sequence of the CtGSTU32 gene is shown as SEQ ID NO.1. The invention also relates to application of the CtGSTU32 gene in promoting accumulation of safflower flavonoid compounds. According to the invention, it is confirmed for the first time that overexpression of the CtGSTU32 gene can promote accumulation of safflower flavonoids compounds, a foundation is laid for cultivation of safflower varieties with high flavonoids compounds, and the CtGSTU32 gene has important economic value and social benefit.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and particularly relates to application of CtGSTU32 gene in promoting accumulation of safflower flavonoid compounds. Background Art

[0002] Safflower (Carthamus tinctorius L.), also known as red and blue flower, thorny safflower, belongs to the Asteraceae family, genus Carthamus. As a medicine, safflower has the effects of promoting blood circulation and menstruation, dispersing blood stasis and relieving pain, and is helpful in treating amenorrhea, dysmenorrhea, lochia retention, chest pain, abdominal pain due to blood stasis, chest and flank pain, traumatic injuries, and sores and swelling.

[0003] The study of bioactive components in safflower has a long history. At present, more than 200 compounds such as flavonoids, phenylethanoid glycosides, coumarins, fatty acids, sterols and safflower polysaccharides have been isolated and identified from different parts of safflower. Among them, flavonoids are secondary metabolites of safflower, mainly present in flowers, and are representative pharmacologically active substances of safflower, with great economic value. Flavonoids in safflower can relieve cardiovascular and cerebrovascular diseases, protect myocardial cells and brain cells, and play anti-inflammatory and anti-cancer effects. In addition, some flavonoids are also natural dyes and are widely used in food, cosmetics and industrial products. Safflower flavonoids can be divided into two categories: common and unique. Common flavonoids exist in most species and have multiple activities, represented by flavonoids, flavonols and dihydroflavonoids, including kaempferol, hyperoside, naringenin, quercetin and luteolin. The unique flavonoid compounds are currently only found in safflower. They have unique structures and important activities in treating cardiovascular and cerebrovascular diseases. Almost all of them are quinone chalcone compounds, such as safflower glycosides, safflower yellow A and hydroxysafflower yellow A (HSYA).

[0004] Mining the key genes in the biosynthesis pathway of flavonoids in safflower will help analyze its molecular regulatory network, provide a deeper understanding of the synthesis of flavonoids in safflower, regulate the synthesis of flavonoids in safflower at the genetic level, and more efficiently develop and utilize flavonoids in safflower. Glutathione-S-transferases (GSTs) are a class of soluble proteins widely present in organisms. Tau-type GSTs (GSTU) are plant-specific and play an important role in resisting stress, but the application of GSTU in promoting the accumulation of flavonoids in safflower has not been reported. Summary of the invention

[0005] In view of the above technical problems, the present invention provides the use of CtGSTU32 gene in promoting the accumulation of safflower flavonoids.

[0006] The present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the use of CtGSTU32 gene in promoting the accumulation of flavonoid compounds in safflower, wherein the nucleotide sequence of the CtGSTU32 gene is shown in SEQ ID NO. 1. The amino acid sequence of the protein encoded by the CtGSTU32 gene is shown in SEQ ID NO. 2.

[0008] The present invention uses the multifunctional economic crop safflower as plant material, combines bioinformatics and molecular biology methods, explores the key gene GST involved in the accumulation of safflower flavonoids, and confirms its function through genetic transformation. The study found that CtGSTU32 may be regulated by methyl jasmonate (MeJA) and participate in the accumulation of safflower flavonoids.

[0009] In a second aspect, the present invention provides biological materials containing the CtGSTU32 gene.

[0010] In some embodiments of the present invention, the biological material is an expression cassette, a recombinant vector or a recombinant bacterium.

[0011] In some embodiments of the present invention, the expression vector of the recombinant vector is pCAMBIA1302.

[0012] In some embodiments of the present invention, the recombinant vector is obtained by inserting the CtGSTU32 gene between the Nco I site and the Spe I site of pCAMBIA1302.

[0013] In some embodiments of the present invention, the host bacteria of the recombinant bacteria is Agrobacterium tumefaciens GV3101.

[0014] In a third aspect, the present invention provides the use of the biomaterial in promoting the accumulation of safflower flavonoid compounds.

[0015] In a fourth aspect, the present invention provides a method for promoting the accumulation of safflower flavonoids in safflower, comprising the step of transferring the CtGSTU32 gene into safflower.

[0016] Furthermore, the transfer method includes the pollen tube channel method, Agrobacterium-mediated method, plant virus vector method or direct DNA transformation method.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention reveals the biological process involved in the development of safflower tubular flowers, finds the core gene CtGSTU32 through WGCNA, and finds that MeJA has a regulatory effect on CtGSTU32 through whole genome analysis of the GST gene family, and confirms that CtGSTU32 is involved in the accumulation of safflower flavonoids through genetic transformation. The present invention lays a foundation for more efficient development and utilization of safflower flavonoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The phenotypes of different stages of tubular flower development of safflower are shown in Figure 2 .

[0020] Figure 2 This is a network diagram of the co-expression of core genes in four modules. Each color of the network diagram represents the corresponding module. The small circle in the middle represents the core gene, yellow represents transcription factors, and the others represent candidate genes.

[0021] Figure 3 The results of the total flavonoids content and CtGSTU32 gene expression in safflower suspension cells treated with MeJA are shown in Figure 2. Figure 3 A in the figure shows the phenotype of suspension cells treated with CK (without MeJA) and 50 μmol / L MeJA; Figure 3 B in the figure shows the results of the determination of the total flavonoids content in the suspension cells after safflower cells were treated with CK and MeJA; Figure 3 C in the figure represents the detection result of CtGSTU32 gene expression in safflower suspension cells after CK and MeJA treatment.

[0022] Figure 4 This is the result of the CtGSTU32 transgenic function verification; Figure 4 A in the figure represents the RT-qPCR verification results of wild-type (WT) and transgenic (CtGSTU32) plants; Figure 4 B in the figure shows the results of total flavonoid content determination in wild type (WT) and transgenic (CtGSTU32) plants. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0024] Experimental Materials:

[0025] The safflower variety used in the present invention is Anhui safflower.

[0026] Example 1: Screening of candidate genes in the transcriptome

[0027] Tubular flower samples from five safflower bud development stages, including bud stage I (BS1), bud stage II (BS2), initial bloom stage (IBS), full bloom stage (FBS), and fade stage (FS), were selected for transcriptome sequencing (e.g. Figure 1 The key genes involved in the regulation of flavonoid biosynthesis of safflower were mined by WGCNA method. The core gene GST screened from the MEblue module was named CtGSTU32 in the gene family. It may be regulated by MeJA and participate in the accumulation of flavonoid compounds in safflower. Therefore, it was used as a candidate gene (such as Figure 2 shown).

[0028] CtGSTU32 gene sequence (SEQ ID NO.1):

[0029] .

[0030] Amino acid sequence of CtGSTU32 protein (SEQ ID NO.2):

[0031] MKDKVILLDCWASSYGMRVKIALAEKGVEYDCREENFQEKSPLLLQSNPIHKTIPVLVHNGKPICESLIIVSYIDEVWNDKSPLLPSDPHQKSQALFWADYIDKKDQEIAKKELIEVLKNLERELGNKPYFGGENIGFVDVALVPFTSWFYTYETRGRFSIEAECPNLVAWTKRCIRERESVAKTLPHPHKIYDYALNLLHK.

[0032] Example 2: Effects of methyl jasmonate on flavonoid composition and CtGSTU32 expression in safflower suspension cells

[0033] 1. Construction of safflower suspension cell line:

[0034] (1) Sterile seedling culture

[0035] Select 20 safflower seeds with full grains and no damage on the surface, rinse them with tap water to clean the dust on the surface, and soak them at room temperature for more than 6 hours (preferably without exposing the hypocotyl), changing the water as needed during the period. After the seeds have absorbed enough water, use absorbent paper to dry the surface water and place them in a sterile clean bench.

[0036] First, rinse the safflower seeds with sterile water for 2-3 times, then use filter paper to absorb the water and place them in a sterilized centrifuge tube. Disinfect with 75% ethanol for 30 seconds, rinse with sterile water for 3-5 times, and place on filter paper to absorb the surface water of the seeds; then disinfect with 0.1% mercuric chloride (HgCl2) for 20 minutes, and rinse with sterile water for 3-5 times. After absorbing the surface water of the treated seeds with filter paper, inoculate them into Murashige and Skoog (MS) basic medium (L&M, PhytoTechLaboratories, HGW0519367A) with the big end facing down and the pointed end facing up. Cultivate in the dark at 25°C for 3 days, the hypocotyl of the seeds will elongate and penetrate into the culture medium, and then grow cotyledons. The seedlings in dark culture were taken out, the seed shells were removed in the clean bench, the seedlings were planted in the culture medium with their biological morphology facing upward, and transferred to a constant temperature light incubator for light culture (25°C, light intensity 5000lx, light time 16h / d).

[0037] (2) Callus culture

[0038] Select safflower seedlings that have grown for about a week and are growing well, pick them up with tweezers and place them on a sterile inoculation plate for cutting, cut the stem segments and cotyledons as callus induction materials, and cut the stem segments and cotyledons with a knife. Cut the stem segments into 0.5 cm long, and the cotyledons into 0.5 cm × 0.5 cm in size and inoculate them on the surface of the callus induction medium. The above materials are cultured under light and dark culture conditions, and subculture is performed every 15 days.

[0039] (3) Cell suspension culture

[0040] Select callus tissue with good growth and loose structure after three subcultures, and use tweezers to fully loosen it and inoculate it into 40mL liquid culture medium. The inoculation amount is about 0.04g / mL (wet weight). -1 Under dark conditions, shake the culture. After 7 days, pass the cell suspension through a 100-mesh sieve to filter out larger cell clusters, so that the cells in the suspension remain uniform in size. Then continue shaking the culture, use the suspended cells in good growth state as the stock solution, and subculture every 18 days. Add 40 mL of fresh culture medium to the stock solution each time, shake well, and evenly divide it into two parts, and continue shaking culture.

[0041] 2. Effect of MeJA on CtGSTU32

[0042] In the analysis of cis-acting elements of the safflower GST gene family, it was found that the upstream cis-acting element region of most GST genes was identified to have abundant methyl jasmonate (MeJA) regulatory elements (TGACG-motif and CGTCA-motif), indicating that MeJA may be involved in the transcriptional regulation of some GST genes. To explore whether MeJA regulates CtGSTU32, 4 μL of 0.5 mol / L MeJA solution was added to 40 mL of safflower cell suspension under sterile conditions. After 7 days of co-culture on a shaker, phenotypic changes were observed, the total flavonoid content of the cells was determined, and RT-qPCR was performed to verify the expression of the CtGSTU32 gene. The EF-1α gene was used as an internal reference, and the primers are as follows:

[0043] Primers for RT-qPCR verification of CtGSTU32 gene expression:

[0044] qRT-CtGSTU32-F (SEQ ID NO. 3): 5′-AAGCCTTGTTTTGGGCTGAC-3′.

[0045] qRT-CtGSTU32-R (SEQ ID NO. 4): 5′-GCCTAACTCCCTCTCCAAGT-3′.

[0046] Primers for RT-qPCR verification of EF-1α gene expression:

[0047] qRT-EF-1α-F (SEQ ID NO. 5): 5′-TCAGCATTGTCGTCATCGGA-3′.

[0048] qRT-EF-1α-R (SEQ ID NO. 6): 5′-ACGTTCGATCACACGCTTGTC-3′.

[0049] RT-qPCR reaction system: 1 μL cDNA solution (final concentration 50 ng / μL), 5 μL 2×Universal BlueSYBR Green qPCR Master Mix, 0.4 μL each of upstream and downstream primers (10 μM), and 10 μL of nuclease-free water. (Note: All operations were performed in an ice water bath)

[0050] RT-qPCR reaction conditions: 95°C for 30 sec; 95°C for 15 sec, 58°C for 10 sec, 72°C for 30 sec, and the number of cycles was 40.

[0051] The results showed that compared with the control suspension cells (CK) without MeJA, the color of the suspension cells treated with MeJA deepened and the total flavonoid content increased significantly. It is speculated that MeJA induced an increase in total flavonoids, which led to the color change (e.g. Figure 3 RT-qPCR results showed that the expression of CtGSTU32 gene in cells treated with MeJA increased significantly, suggesting that MeJA has a positive regulatory effect on CtGSTU32 (as shown in A and C in Figure 2). Figure 3 (as shown in B in the figure).

[0052] Example 3: Effect of overexpression of CtGSTU32 on the synthesis of safflower total flavonoids

[0053] (1) CtGSTU32 target gene amplification

[0054] The CDS sequence of CtGSTU32 was extracted from the safflower genome, and the nucleotide sequence was shown in SEQ ID NO. 1. Specific amplification primers CtGSTU32-F / CtGSTU32-R with restriction sites and homology arms were designed. The cDNA of tubular flowers in bloom of Anhui safflower material was used as a template and PCR amplification was performed using 2×Phanta Max Master Mix.

[0055] CtGSTU32-F (SEQ ID NO. 7): 5′-acgggggactcttgaccatggATGAAGGATAAAGTAATCTTATTGGATTG-3′.

[0056] CtGSTU32-R (SEQ ID NO. 8): 5′-aagttcttctcctttactagtTTTGTGTAAAAGATTCAAGGCATAG-3′.

[0057] Reaction system: 2×Phanta Max Master Mix, 5 μL; upstream primer, 0.4 μL; downstream primer, 0.4 μL; safflower tubular flower cDNA, 1 μL; nuclease-free water, 3.2 μL.

[0058] PCR amplification system: pre-denaturation, 95°C, 3 min; denaturation, 95°C, 15 s; annealing, 59°C, 15 s; extension, 72°C, 50 s; complete extension, 72°C, 5 min.

[0059] (2) Plasmid digestion and purification

[0060] The overexpression vector pCAMBIA1302 was double-digested with Nco I / Spe I. The specific digestion reaction system (50 μL): Nco I 1 μL; Spe I 1 μL; pCAMBIA1302 1 μg; 10×K Buffer 5 μL; 0.1% BSA 5 μL; nuclease-free water 29 μL. After mixing, the reaction was carried out at 37°C on a PCR instrument for 4 hours.

[0061] The target fragment and the linearized vector amplification products were subjected to DNA agarose gel electrophoresis, and then the products were purified using the FastPure Gel DNA Extraction Mini Kit of Novazonic Biotech Co., Ltd. For specific steps, please refer to the kit instructions.

[0062] (3) Gene transformation

[0063] The linearized plasmid after enzyme digestion was connected with the target fragment by seamless cloning. The reaction system (20μL): 1μL linearized vector, 2μL target fragment, 4μL 5×CE II Buffer, 2μL Exnase II, 11μL sterile water. After mixing, react at 37℃ for 30min on a PCR instrument, and immediately cool on ice after the reaction. The reconnected vector was transformed into Escherichia coli (E.coli) competent cells. After sequencing verification, the plasmid with correct sequencing was transformed into Agrobacterium tumefaciens GV3101 competent cells for use.

[0064] (3) Verification of CtGSTU32 gene function

[0065] Overexpression plants were obtained by the pollen tube method, and after the T0 generation safflowers set seeds, T1 generation transgenic seeds were collected and T1 generation plants were planted. T1 generation positive plants were identified by PCR.

[0066] The results showed that the expression level of CtGSTU32 gene in transgenic plants was significantly higher than that in wild-type plants (Figure Figure 4 Finally, the total flavonoid content in the leaves of the plants was determined to explore whether the increased expression of the CtGSTU32 gene would affect the accumulation of flavonoid compounds. The results showed that the total flavonoid content in the leaves of the transgenic plants was significantly higher than that of the wild type. It is speculated that the CtGSTU32 gene may be involved in regulating the accumulation of safflower flavonoid compounds, thereby leading to an increase in the total flavonoid content (as shown in Figure 2A). Figure 4 (as shown in B in the figure).

[0067] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0068] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The application of CtGSTU32 gene in promoting the accumulation of flavonoid compounds in safflower, characterized in that: The nucleotide sequence of the CtGSTU32 gene is shown in SEQ ID NO.

1.

2. A biological material containing the CtGSTU32 gene according to claim 1.

3. The biomaterial according to claim 2, characterized in that The biological material is an expression cassette, a recombinant vector or a recombinant bacterium.

4. The biomaterial according to claim 3, characterized in that The expression vector of the recombinant vector is pCAMBIA1302.

5. The biomaterial according to claim 4, characterized in that The recombinant vector is obtained by inserting the CtGSTU32 gene between the Nco I site and the Spe I site of pCAMBIA1302.

6. The biomaterial according to claim 3, characterized in that The host bacteria of the recombinant bacteria is Agrobacterium tumefaciens GV3101.

7. Use of the biomaterial according to any one of claims 2 to 6 in promoting the accumulation of flavonoid compounds in safflower.

8. A method for promoting the accumulation of safflower flavonoids in safflower, characterized in that: The method comprises the step of transferring the CtGSTU32 gene described in claim 1 into safflower.

9. The method for promoting the accumulation of safflower flavonoids in safflower according to claim 8, characterized in that: The transfer method includes pollen tube channel method, Agrobacterium-mediated method, plant virus vector method or direct DNA transformation method.

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