Lycium ruthenicum Murr. LrCHS2 gene and application of the encoded protein in promoting anthocyanin biosynthesis

Abstract

The present application belongs to the field of biotechnology, and particularly relates to Lycium ruthenicum Murr LrCHS2 gene and application of the coded protein in promoting Lycium barbarum L. anthocyanin biosynthesis. The present application first finds that LrCHS2 gene has a positive regulation on anthocyanin biosynthesis, and can significantly or extremely significantly promote the accumulation of anthocyanin compounds. Overexpression of LrCHS2 gene in plants can provide a new biosynthesis way for anthocyanin compounds. The present application provides a construction method of recombinant plasmid and recombinant bacteria overexpressing LrCHS2 gene of Lycium ruthenicum Murr, and the recombinant expression vector overexpressing LrCHS2 gene can be obtained, the obtained recombinant expression vector can improve the content of anthocyanin in plants, and can be applied to anthocyanin compound biosynthesis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of the LrCHS2 gene and its encoded protein in promoting the biosynthesis of anthocyanins in wolfberry. Background Technology

[0002] Anthocyanins are a class of water-soluble natural pigments unique to plants and found in a wide variety of plants. Anthocyanins possess various biological activities and can be used to prevent and treat a variety of diseases, exhibiting good medicinal value. They are also widely used as natural food colorings. Furthermore, anthocyanins play an important role in promoting plant reproduction and protecting plants from biotic and abiotic stresses.

[0003] Anthocyanins are an important component of black goji berry (Lycium ruthenicum Murr.) fruit, and their content is higher in black goji berries than in blackcurrants or blueberries. Currently, 37 anthocyanins have been identified in black goji berries. Different anthocyanins possess different biological activities and other values. Therefore, it is necessary to continue exploring structural genes related to anthocyanin synthesis to aid in the genetic improvement of goji berries and the biosynthesis of anthocyanin compounds. Summary of the Invention

[0004] To address the above technical problems, this invention provides the application of the LrCHS2 gene and its encoded protein in promoting anthocyanin biosynthesis in wolfberry. This invention is the first to discover that overexpression of the LrCHS2 gene can positively mediate the biosynthesis of anthocyanin compounds, thereby increasing the accumulation of anthocyanin compounds in plants.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides the application of the LrCHS2 gene of black goji berries in increasing the anthocyanin content of plants, wherein the nucleotide sequence of the LrCHS2 gene of black goji berries is shown in SEQ ID NO.1, or encodes a protein with an amino acid sequence shown in SEQ ID NO.2.

[0007] This invention is the first to identify the LrCHS2 gene in wolfberry, and through research, it was found that the LrCHS2 gene has a positive regulatory effect on anthocyanin biosynthesis, and can significantly or extremely significantly promote the accumulation of anthocyanin compounds procyanidin B2 (CAS No.: 29106-49-8), procyanidin B1 (CAS No.: 20315-25-7), and cyanidin 3-glucoside (structural formula shown in Formula I).

[0008]

[0009] Proanthocyanidins B2 and B1 are oligomeric proanthocyanidins with strong antioxidant and free radical scavenging effects. They protect lipids from oxidative damage and protect and stabilize vitamin C. They can restore collagen activity, making skin smooth and elastic. They can inhibit tyrosinase activity and the Maillard reaction caused by protein amino groups and nucleic acid amino groups, thus inhibiting the formation of skin pigmentation. They are also powerful metal chelators, forming inert compounds with metal ions in vivo. Oral absorption is rapid and has a long half-life. Cyanidin-3-glucoside has various biological activities, such as antioxidant, antitumor, nervous system protection, protection of retinal pigment epithelial cells, and reduction of glomerular sclerosis and interstitial fibrosis in patients with diabetic nephropathy. It can also act as a plant protectant, protecting cotton leaves from bollworm damage. Overexpression of the LrCHS2 gene in plants can provide a new biosynthetic pathway for the above-mentioned anthocyanin compounds.

[0010] Preferably, the plant is wolfberry.

[0011] Preferably, the anthocyanins include proanthocyanidin B2, proanthocyanidin B1, and cyanidin-3-glucoside.

[0012] A second aspect of the present invention provides the application of a protein with an amino acid sequence as shown in SEQ ID NO.2 in increasing the anthocyanin content of plants.

[0013] Preferably, the plant is wolfberry.

[0014] Preferably, the anthocyanins include proanthocyanidin B2, proanthocyanidin B1, and cyanidin-3-glucoside.

[0015] A third aspect of the present invention provides a recombinant expression vector for overexpressing the LrCHS2 gene of black goji berries.

[0016] Preferably, the recombinant expression vector comprises a recombinant plasmid and a recombinant bacterium.

[0017] More preferably, the recombinant bacteria is Agrobacterium.

[0018] The fourth aspect of this invention provides a method for constructing a recombinant plasmid overexpressing the LrCHS2 gene of *Lycium barbarum*: using primer OE-LrCHS2-F with the sequence shown in SEQ ID NO.5 and primer OE-LrCHS2-R with the sequence shown in SEQ ID NO.6, the LrCHS2 gene of *Lycium barbarum* is transformed into a plasmid, thereby obtaining the recombinant plasmid overexpressing the LrCHS2 gene of *Lycium barbarum*.

[0019] Preferably, the plasmid is pCambia 1300-35s.

[0020] The fifth aspect of the present invention provides a method for constructing a recombinant bacterium overexpressing the LrCHS2 gene of black goji berries: a recombinant plasmid is constructed using primer OE-LrCHS2-F with the sequence shown in SEQ ID NO.5 and primer OE-LrCHS2-R with the sequence shown in SEQ ID NO.6, and the recombinant plasmid is transformed into Agrobacterium to obtain the recombinant bacterium overexpressing the LrCHS2 gene of black goji berries.

[0021] Preferably, the plasmid is pCambia 1300-35s.

[0022] Preferably, the Agrobacterium is Agrobacterium GS115, Agrobacterium LBA4404, Agrobacterium GV3101 or Agrobacterium EHA105.

[0023] The sixth aspect of this invention provides the application of the recombinant bacteria overexpressing the LrCHS2 gene of black goji berries in increasing the anthocyanin content of plants.

[0024] Preferably, the recombinant bacteria is Agrobacterium GV3101.

[0025] Preferably, the plant is wolfberry.

[0026] Preferably, the anthocyanins include proanthocyanidin B2, proanthocyanidin B1, and cyanidin-3-glucoside.

[0027] Preferably, the bacterial suspension of the above-mentioned recombinant bacteria is injected into the plant.

[0028] The beneficial effects of this invention are as follows: This invention is the first to identify the LrCHS2 gene from wolfberry, and through homologous cloning, transient transformation of wolfberry, and quantitative fluorescence detection, it is the first to discover that the LrCHS2 gene can positively regulate the biosynthesis of anthocyanin compounds proanthocyanidins B1, B2, and cyanidin-3-glucoside. This provides an effective technical means for improving the quality of wolfberry and a new pathway for obtaining these anthocyanin compounds. The recombinant plasmid and recombinant bacteria overexpressing the LrCHS2 gene in black wolfberry provided by this invention can obtain a recombinant expression vector overexpressing the LrCHS2 gene. The obtained recombinant expression vector can increase the content of proanthocyanidins B1, B2, and cyanidin-3-glucoside in plants and can be applied to the biosynthesis of these anthocyanin compounds. Attached Figure Description

[0029] Figure 1 This refers to the expression level of the LrCHS2 gene in the OE-LrCHS2 overexpressing plants and the control in Example 1 of this invention;

[0030] Figure 2This refers to the anthocyanin compound content in the OE-LrCHS2 overexpressing plants and the control in Example 1 of this invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0032] Anthocyanins are one of the most important nutritional components of black goji berries. This invention, through the comprehensive application of homologous cloning, transcriptome differential expression, and transient transformation detection techniques, identified the important structural gene LrCHS2 that regulates the biosynthesis of anthocyanins in goji berries. Experimental studies have shown that this gene can effectively promote the biosynthesis of procyanidin B2, procyanidin B1, and cyanidin 3-glucoside, all anthocyanin-like compounds in goji berries.

[0033] Based on the above research findings, this invention provides the application of the LrCHS2 gene from black goji berries in increasing anthocyanin content in plants. The nucleotide sequence of the LrCHS2 gene is shown in SEQ ID NO.1, or it encodes a protein with an amino acid sequence shown in SEQ ID NO.2. Without affecting the protein structure and activity, the amino acid sequences obtained by substituting, deleting, or adding one or more amino acids, or by terminal modification of the amino acid sequence shown in SEQ ID NO.2, and the nucleotide sequences encoding them, are also within the scope of protection of this invention.

[0034] This invention also provides a recombinant expression vector for overexpressing the LrCHS2 gene of black goji berries and its construction method.

[0035] The following detailed description, using specific examples, will further illustrate this point.

[0036] Unless otherwise specified, the raw materials, reagents, pharmaceuticals, or instruments used in the following embodiments are all commercially available products. The methods used in the following embodiments are, unless otherwise specified, conventional methods in the art.

[0037] Example 1

[0038] This embodiment provides the cloning and functional verification of the LrCHS2 gene in black goji berries.

[0039] 1. Cloning of the LrCHS2 gene in black goji berries

[0040] Total RNA was isolated from black goji berry fruits using an RNA extraction kit from Takara (Takara, Dalian, China). Single-stranded cDNA of the LrCHS2 gene was prepared using a Reverse Aid First-strand cDNA synthesis kit from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). The open reading frame (CDS) of the LrCHS2 gene was amplified using primers LrCHS2-F and LrCHS2-R. The amplification system consisted of the following components: 25 μL 2×PCR buffer, 10 μL dNTP (2 mM), 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 5 μL single-stranded cDNA, 1 μL KOD FX Neo (Toyo Life Sciences, Osaka, Japan), and 10 μL Millipore H2O. The PCR reaction program was set as follows: pre-denaturation at 98°C for 3 minutes, followed by 30 cycles, each cycle consisting of denaturation for 10 seconds, annealing at 58°C for 30 seconds, extension at 68°C for 2 minutes, and a final extension at 68°C for 5 minutes. The amplified products were purified and independently cloned into the pMD18-T vector (Toyobo Life Science, Osaka, Japan) for Sanger sequencing.

[0041] LrCHS2-F (as shown in SEQ ID NO.3): ATGGTCACCGTGGAGGAGTATC; LrCHS2-R (as shown in SEQ ID NO.4): CTAAGCAGCAACACTGTGGA.

[0042] The full-length open reading frame (CDS) of LrCHS2 is 1170 bp, and its nucleotide sequence is shown in SEQ ID NO.1:

[0043]

[0044] The LrCHS2 gene encodes 389 amino acids, with the sequence shown in SEQ ID. Shown in NO.2: MTVVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTYPDYYFRITDSEHKTELKEKFKRMCDKSMIKKRYMHLTEEILKENPNMCAYMAPSLD ARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLAKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNKGARVLVVCS EITAVTFRGPSESHLDSLVGQALFGDGAAAIIMGSDPIPGVERPLFELVSAAQTLLPDSEGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLVEAFQPLGISDWNSLFWIAHPGGPAILDQVELKLALKPEKLRATRDVLSNYGNMSSACVLFILDEMRKASAKEGLGTTGEGLEWGVLFGFGPGLTVETVVLHSVAA.

[0045] 2. Functional verification of the LrCHS2 gene in black wolfberry

[0046] 2.1 Transient overexpression transformation

[0047] Using primers OE-LrCHS2-F and OE-LrCHS2-R, the cloned LrCHS2 sequence was constructed into the pCambia 1300-35s vector, resulting in the OE-LrCHS2 overexpression vector. This OE-LrCHS2 overexpression vector was transformed into Agrobacterium GV3101 to obtain the OE-LrCHS2 overexpressing engineered bacterium. The concentration of this engineered bacterium was adjusted to OD in infection buffer (5 g / L D-glucose, 50 mM MES, 2 mM Na3PO4·12H2O, and 0.1 mM acetobutanone). 600 The concentration of the bacterium was 0.5–0.6 mL, and 5–10 mL was injected into the leaves of Ningxia wolfberry seedlings that had been cultured for 30 days prior. pCambia1300-35 s-GFP was used as a negative control. Leaves injected with the engineered bacterial suspension were collected 72 h after injection for subsequent real-time quantitative PCR (qRT-PCR) and anthocyanin content detection.

[0048] OE-LrCHS2-F (as shown in SEQ ID NO.5): TCCTGGGCTGATCTCGAAAA; OE-LrCHS2-R (as shown in SEQ ID NO.6): TCCTTGTGGCCCTAAGTTT.

[0049] 2.2 qRT-PCR detection

[0050] Using Lycium barbarum Ef1a as an internal reference gene, the overexpression of LrCHS2 gene in Lycium barbarum leaves was detected and analyzed using the SYBR Green dye method and BIO-RAD real-time PCR instrument (primers: CCATACCAGCATCACCATTCTTC, as shown in SEQ ID NO.7).

[0051] GTCACACTTCCCACATTGCC, as shown in SEQ ID NO.8).

[0052] The qRT-PCR reaction system was: 12 μL Power Green PCR Master mixture, 1 μL qRTF (10 μM), 5 μL cDNA template, and 20 μL ddH2O; reaction conditions: 95.0℃ pre-denaturation for 5 min; 95.0℃ denaturation for 10 s, 55℃ annealing for 30 s, and 72℃ extension for 20 s (40 cycles); melting curve was calculated from 65℃ to 95℃, with each reading increasing by 0.5℃. 2 -ΔΔCT The relative expression level of the target gene was determined by a method.

[0053] Analysis of OE-LrCHS2 overexpressing plants and controls showed that LrCHS2 gene expression was significantly higher than GFP in OE-LrCHS2 overexpressing plants. Figure 1 As shown, this construction method achieves overexpression of the LrCHS2 gene.

[0054] 2.3 Anthocyanin content detection,

[0055] The anthocyanin content of OE-LrCHS2 overexpressing plants was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0056] 2.3.1 Sample Pretreatment

[0057] Accurately weigh 0.2g of sample into a 2ml centrifuge tube, add 1ml of 5% formic acid, extract on ice for 30min, centrifuge at 12000rpm for 10min, and take the supernatant for LC-MS / MS analysis.

[0058] 2.3.2 Chromatographic and Mass Spectrometry Acquisition Conditions

[0059] The chromatographic system used was a Waters ultra-high performance liquid chromatography system (AcQuity UPLC, Waters, USA), using a Waters HSS T3 (100*2.1mm, 1.8um) liquid chromatography column, with an injection volume of 2μL and a column temperature of 40℃; mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (0.1% formic acid-acetonitrile).

[0060] The mass spectrometry system employed a Q exactive high-resolution mass spectrometry detection system from Thermo Fisher Scientific, equipped with an electrospray ionization (ESI) source and an Xcalibur workstation. The key mass spectrometry conditions included: electrospray ionization (ESI) with analytes analyzed in single-ion detection (SIM) mode under simultaneous positive ion scanning, which significantly improves sensitivity; sheath gas pressure 40 arb; auxiliary gas pressure 10 arb; ion spray voltage +3000 V; temperature 350 °C; and ion transmission tube temperature 320 °C.

[0061] The results were obtained by examining OE-LrCHS2 overexpressing plants and controls (e.g.) Figure 2 As shown in the figure, overexpression of LrCHS2 significantly or extremely significantly promoted the accumulation of anthocyanin compounds procyanidin B2, procyanidin B1 and cyanidin 3-glucoside, indicating that LrCHS2 positively mediates anthocyanin biosynthesis.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Black goji berries LrCHS2 The application of genes in increasing anthocyanin content in plants is characterized by, The black goji berries LrCHS2 The nucleotide sequence of the gene is shown in SEQ ID NO. 1; the plant is Ningxia wolfberry; the anthocyanins are proanthocyanidin B2, proanthocyanidin B1 and cyanidin-3-glucoside.

2. The application of a protein with an amino acid sequence as shown in SEQ ID NO. 2 in increasing the anthocyanin content of plants; wherein the plant is Ningxia wolfberry; and the anthocyanins are proanthocyanidin B2, proanthocyanidin B1 and cyanidin-3-glucoside.

3. Overexpression of the black goji berry as described in claim 1 LrCHS2 Application of recombinant bacteria in increasing anthocyanin content in plants; the plant is Ningxia wolfberry; the anthocyanins are proanthocyanidin B2, proanthocyanidin B1 and cyanidin-3-glucoside.

4. The application according to claim 3, characterized in that, The recombinant bacteria is Agrobacterium GV3101; and / or The method of application is as follows: injecting the bacterial suspension of the recombinant bacteria into the plant.

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