Application of lycium ruthenicum LrCHS2 gene and encoded protein thereof in promoting biosynthesis of lycium ruthenicum anthocyanin

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a lycium ruthenicum LrCHS2 gene and an encoded protein thereof in promoting biosynthesis of lycium ruthenicum anthocyanin. It is found for the first time that the LrCHS2 gene has a positive regulation effect on biosynthesis of anthocyanin, and accumulation of anthocyanin compounds can be remarkably or extremely remarkably promoted. The LrCHS2 gene is overexpressed in plants, and a new way for biosynthesis of anthocyanin compounds can be provided. According to the construction method of the recombinant plasmid and the recombinant bacterium for overexpressing the lycium ruthenicum LrCHS2 gene, a recombinant expression vector for overexpressing the LrCHS2 gene can be obtained, and the obtained recombinant expression vector can improve the content of anthocyanin in plants and can be applied to biosynthesis of anthocyanin compounds.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of a ruthenic wolfberry LrCHS2 gene and a protein encoded therein in promoting the biosynthesis of wolfberry anthocyanins. Background Art

[0002] Anthocyanins are a class of plant-specific water-soluble natural pigments that are found in a variety of plants. Anthocyanins have a variety of biological activities and can be used to prevent and treat a variety of diseases, with good medicinal value; they are also widely used in food as natural food pigments. In addition, anthocyanins also play an important role in promoting plant reproduction and protecting plants from biotic and abiotic stresses.

[0003] Anthocyanins are an important component of Lycium ruthenicum Murr. fruit, and the content of anthocyanins in Lycium ruthenicum Murr. is higher than that in blackcurrant or blueberry. At present, 37 anthocyanins have been identified in Lycium ruthenicum Murr. Different anthocyanins have different biological activities and other values. Therefore, it is necessary to continue to explore the structural genes related to anthocyanin synthesis to help the genetic improvement of Lycium ruthenicum and the biosynthesis of anthocyanin compounds. Summary of the invention

[0004] In view of the above technical problems, the present invention provides the application of the LrCHS2 gene of Lycium ruthenicum and its encoded protein in promoting the biosynthesis of anthocyanins in Lycium barbarum. The present invention firstly finds that the overexpression of the LrCHS2 gene can positively mediate the biosynthesis of anthocyanin compounds and improve the accumulation of anthocyanin compounds in plants.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an application of the LrCHS2 gene of lycium ruthenicum in increasing the anthocyanin content of plants. The nucleotide sequence of the LrCHS2 gene of lycium ruthenicum is shown in SEQ ID NO.1, or the encoded amino acid sequence is shown in SEQ ID NO.2.

[0007] The present invention identifies the LrCHS2 gene from wolfberry for the first time, and through research it is found that the LrCHS2 gene has a positive regulatory effect on the biosynthesis of anthocyanins, 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 (Cyanidin3-glucoside, the structural formula is shown in Formula I).

[0008]

[0009] Proanthocyanidin B2 and proanthocyanidin B1 are oligomeric proanthocyanidins with strong antioxidant and free radical scavenging effects. They can protect lipids from peroxidative damage and protect and stabilize vitamin C. They can restore collagen activity and make the skin smooth and elastic. They can inhibit the activity of tyrosinase and the Maillard reaction caused by protein amino groups and nucleic acid amino groups, thereby inhibiting the formation of skin spots. They are powerful metal chelators that can form inert compounds with metal ions in the body. They are rapidly absorbed orally and have a long half-life. Cyanidin-3-glucoside has a variety of biological activities, such as antioxidant, anti-tumor, protection of the nervous system, protection of retinal pigment epithelial cells, reduction of glomerular sclerosis and renal interstitial fibrosis in patients with diabetic nephropathy, etc. It can also be used as a plant protectant to protect cotton leaves from the damage of cotton bollworms. Overexpression of the LrCHS2 gene in plants can provide a new biosynthetic pathway for the above-mentioned anthocyanidin compounds.

[0010] Preferably, the plant is wolfberry.

[0011] Preferably, the anthocyanidins include procyanidin B2, procyanidin B1 and cyanidin-3-glucoside.

[0012] The second aspect of the present invention provides the use of a protein having an amino acid sequence as shown in SEQ ID NO.2 in increasing the anthocyanin content of a plant.

[0013] Preferably, the plant is wolfberry.

[0014] Preferably, the anthocyanidins include procyanidin B2, procyanidin B1 and cyanidin-3-glucoside.

[0015] The third aspect of the present invention provides a recombinant expression vector for overexpressing the above-mentioned Lycium ruthenicum LrCHS2 gene.

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

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

[0018] The fourth aspect of the present invention provides a method for constructing a recombinant plasmid that overexpresses the above-mentioned black wolfberry LrCHS2 gene: using the primer OE-LrCHS2-F with a sequence as shown in SEQ ID NO.5 and the primer OE-LrCHS2-R with a sequence as shown in SEQ ID NO.6, the black wolfberry LrCHS2 gene is transformed into a plasmid to obtain the recombinant plasmid that overexpresses the black wolfberry LrCHS2 gene.

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

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

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

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

[0023] A sixth aspect of the present invention provides the use of the above-mentioned recombinant bacteria overexpressing the LrCHS2 gene of Lycium ruthenicum in increasing the anthocyanin content of plants.

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

[0025] Preferably, the plant is wolfberry.

[0026] Preferably, the anthocyanidins include procyanidin B2, procyanidin B1 and cyanidin-3-glucoside.

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

[0028] The beneficial effects of the present invention are as follows: the present invention identifies the LrCHS2 gene from wolfberry for the first time, and discovers for the first time through homologous cloning, wolfberry transient transformation, fluorescence quantitative detection and other technologies that the LrCHS2 gene can positively regulate the biosynthesis of anthocyanidin compounds proanthocyanidin B1, proanthocyanidin B2 and cyanidin-3-glucoside, which can provide effective technical means for improving the quality of wolfberry and provide a new way to obtain the above-mentioned anthocyanidin compounds. The construction method of the recombinant plasmid and recombinant bacteria for overexpressing the LrCHS2 gene of black wolfberry provided by the present invention can obtain a recombinant expression vector for overexpressing the LrCHS2 gene, and the obtained recombinant expression vector can increase the content of proanthocyanidin B1, proanthocyanidin B2 and cyanidin-3-glucoside in plants, and can be applied to the biosynthesis of the above-mentioned anthocyanidin compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the expression level of the LrCHS2 gene in the OE-LrCHS2 overexpressing plant and the control in Example 1 of the present invention;

[0030] Figure 2It is the anthocyanin compound content of the OE-LrCHS2 overexpressing plant and the control in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0032] Anthocyanin is one of the most important nutrients of black wolfberry. The present invention uses a combination of homologous cloning, transcriptome differential expression, wolfberry transient transformation detection and other technologies to find the important structural gene LrCHS2 that regulates the biosynthesis of wolfberry anthocyanin. Experimental studies have found that this gene can be effectively used to promote the biosynthesis of wolfberry anthocyanin compounds procyanidin B2, procyanidin B1 and cyanidin 3-glucoside.

[0033] Based on the above research results, the embodiment of the present invention provides the use of the LrCHS2 gene of black wolfberry in increasing the anthocyanin content of plants, and the nucleotide sequence of the LrCHS2 gene of black wolfberry is shown in SEQ ID NO.1, or the protein encoded by the amino acid sequence is shown in SEQ ID NO.2. Under the premise of not affecting the structure and activity of the protein, the amino acid sequence and the nucleotide sequence encoding the amino acid sequence obtained by substituting, deleting or adding one or more amino acids or terminally modifying the amino acid sequence shown in SEQ ID NO.2 also fall within the protection scope of the present invention.

[0034] The embodiment of the present invention also provides a recombinant expression vector for overexpressing the above-mentioned Lycium ruthenicum LrCHS2 gene and a construction method thereof.

[0035] The following is further described with reference to specific embodiments.

[0036] Unless otherwise specified, the raw materials, reagents, drugs or instruments used in the following examples are all conventional commercial products obtained from commercial channels. The methods used in the following examples are all conventional methods in the art unless otherwise specified.

[0037] Example 1

[0038] This example provides the cloning and functional verification of the LrCHS2 gene of Lycium ruthenicum.

[0039] 1. Cloning of LrCHS2 gene from Lycium ruthenicum

[0040] Total RNA from Lycium ruthenicum fruit was isolated using the RNA extraction kit from Takara (Takara, Dalian, China). Single-stranded cDNA of the LrCHS2 gene was prepared using the 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 components were as follows: 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 Science, Osaka, Japan), and 10 μL Millipore H2O. The PCR reaction program was set as follows: 98°C pre-denaturation for 3 minutes, followed by 30 cycles, each cycle including denaturation for 10 seconds, 58°C annealing for 30 seconds, and 68°C extension for 2 minutes; and finally 68°C extension 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 LrCHS2 open reading frame (CDS) is 1170 bp in length, and the nucleotide sequence is shown in SEQ ID NO.1:

[0043]

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

[0045] 2. Functional verification of LrCHS2 gene in Lycium ruthenicum

[0046] 2.1 Transient overexpression transformation

[0047] The cloned LrCHS2 sequence was constructed into the pCambia 1300-35s vector using primers OE-LrCHS2-F and OE-LrCHS2-R, and the OE-LrCHS2 overexpression vector was obtained. The OE-LrCHS2 overexpression vector was transformed into Agrobacterium GV3101 to obtain the OE-LrCHS2 overexpression engineered bacteria. The engineered bacteria were adjusted to an OD of 0.05 in infection buffer (5 g / L D-glucose, 50 mM MES, 2 mM Na3PO4·12H2O and 0.1 mM acetobutanone). 600 The concentration of 0.5-0.6 was injected into the leaves of Ningxia wolfberry seedlings that had been cultured for 30 days in advance, and 5-10 mL was injected per seedling; pCambia1300-35 s-GFP was used as a negative control. The leaves injected with the engineered bacterial suspension were collected 72 hours after injection, and subsequent real-time fluorescence quantitative PCR (qRT-PCR) and anthocyanin content detection were performed.

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

[0049] 2.2qRT-PCR detection

[0050] Using Lycium barbarum Ef1a as the internal reference gene, the SYBR Green dye method and BIO-RAD fluorescence quantitative PCR instrument were used to detect and analyze the overexpression of the LrCHS2 gene in Lycium barbarum leaves (primers: CCATACCAGCATCACCATTCTTC, as shown in SEQ ID NO.7;

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

[0052] qRT-PCR reaction system: 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, 72℃ extension for 20 s, (40 cycles); melting curve from 65℃ to 95℃, each reading increased by 0.5℃. 2 -ΔΔCT The relative expression level of the target gene was determined by the method.

[0053] The results of testing the OE-LrCHS2 overexpression plants and the control showed that in the OE-LrCHS2 overexpression plants, the expression of the LrCHS2 gene was significantly higher than that of GFP. Figure 1 As shown, this construction method achieved overexpression of the LrCHS2 gene.

[0054] 2.3 Anthocyanin content detection,

[0055] Liquid chromatography tandem mass spectrometry (LC-MS / MS) was used to determine the anthocyanin content in OE-LrCHS2 overexpressing plants.

[0056] 2.3.1 Sample pretreatment

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

[0058] 2.3.2 Chromatographic mass spectrometry acquisition conditions

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

[0060] The mass spectrometry system uses the Q exactive high-resolution mass spectrometry detection system of Thermo Corporation, USA, equipped with an electrospray ionization (ESI) source and an Xcalibur workstation. The mass spectrometry conditions mainly include: using an electrospray ionization (ESI) source, the analyte is analyzed in single ion detection (SIM) mode under positive ion simultaneous scanning, which can greatly improve the sensitivity; sheath gas pressure 40arb; auxiliary gas pressure 10arb; ion spray voltage +3000V; temperature 350℃; ion transfer tube temperature 320℃.

[0061] The results (such as Figure 2 The results showed that 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 mediated 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 in the protection scope of the present invention.

Claims

1. The application of the LrCHS2 gene of black wolfberry in increasing the anthocyanin content of plants, characterized in that: The nucleotide sequence of the LrCHS2 gene of Lycium ruthenicum is shown as SEQ ID NO.1, or the encoded amino acid sequence is shown as a protein as SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The plant is wolfberry; and / or The anthocyanidins include procyanidin B2, procyanidin B1 and cyanidin-3-glucoside.

3. Application of the protein with the amino acid sequence as shown in SEQ ID NO.2 in increasing the anthocyanin content of plants.

4. The use according to claim 3, characterized in that: The plant is wolfberry; and / or The anthocyanidins include procyanidin B2, procyanidin B1 and cyanidin-3-glucoside.

5. A recombinant expression vector for overexpressing the LrCHS2 gene of Lycium ruthenicum according to claim 1.

6. A method for constructing a recombinant plasmid for overexpressing the LrCHS2 gene of Lycium ruthenicum according to claim 1, characterized in that: The primer OE-LrCHS2-F with a sequence as shown in SEQ ID NO.5 and the primer OE-LrCHS2-R with a sequence as shown in SEQ ID NO.6 are used to transform the LrCHS2 gene of Lycium ruthenicum into a plasmid, thereby obtaining the recombinant plasmid overexpressing the LrCHS2 gene of Lycium ruthenicum.

7. The construction method according to claim 6, characterized in that: The plasmid is pCambia1300-35s.

8. A method for constructing a recombinant bacterium overexpressing the LrCHS2 gene of Lycium ruthenicum according to claim 1, characterized in that: The recombinant plasmid was constructed using the primer OE-LrCHS2-F with a sequence as shown in SEQ ID NO.5 and the primer OE-LrCHS2-R with a sequence as shown in SEQ ID NO.6, and the recombinant plasmid was transformed into Agrobacterium to obtain the recombinant bacteria overexpressing the LrCHS2 gene of Lycium ruthenicum.

9. Use of a recombinant bacterium overexpressing the LrCHS2 gene of Lycium ruthenicum according to claim 1 in increasing the anthocyanin content of a plant.

10. The use according to claim 9, characterized in that: The recombinant bacterium is Agrobacterium GV3101; and / or The plant is wolfberry; and / or The anthocyanidins include procyanidin B2, procyanidin B1 and cyanidin-3-glucoside; and / or The application method is: injecting the bacterial suspension of the recombinant bacteria into plants.

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