Application of CaCHS02 gene in regulation and control of alpha-glucosidase inhibitory activity of capsicum

Abstract

The invention belongs to the field of plant genetic engineering, and discloses an application of a pepper chalcone synthetase CaCHS02 gene in regulation and control of pepper flavonoid content and alpha-glucosidase inhibitory activity (AGI activity). The full-length nucleotide sequence of the capsicum CaCHS02 gene is shown as SEQ ID NO: 1, and the encoding protein of the capsicum CaCHS02 gene is shown as SEQ ID NO: 2. A large amount of expression of an over-expression vector of the gene in pepper finds that over-expression of the gene promotes significant increase of the chalcone synthetase activity and the total flavone content in pepper leaves, and the AGI activity of the pepper leaves is also significantly enhanced, so that the invention provides a good research basis for breeding functional pepper rich in AGI activity.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to application of the CaCHS02 gene in regulating the inhibitory activity of pepper alpha-glucosidase. Background Art

[0002] Functional vegetables refer to vegetable products that have other benefits to human health in addition to their basic functions through bio-nutrition fortification or other biotechnological means. For example, vegetable product ingredients or phytochemicals of plant origin, such as flavonoids and allyl sulfides, have the function of regulating human health physiological functions. α-Glucosidase inhibitors (AGI) are a group of widely used anti-diabetic drugs that effectively reduce high blood sugar and insulin levels in the intestine after meals. Many studies have developed plant-derived substances with natural AGI activity and no side effects or lower side effects. It is reported that both pepper fruits and leaves have AGI activity, and the AGI activity in pepper leaves is related to the synthesis of flavonoid compounds. However, there have been no reports on the functional studies of genes that regulate the synthesis and accumulation of flavonoid glucosides in pepper leaves. Summary of the invention

[0003] In view of the above problems, the present invention provides the use of the CaCHS02 gene in regulating the inhibitory activity of pepper α-glucosidase.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] The application of the CaCHS02 gene in regulating the inhibitory activity of pepper α-glucosidase, the full-length nucleotide sequence of the pepper CaCHS02 gene is shown in SEQ ID NO: 1.

[0006] As a preferred embodiment of the above scheme, the pepper CaCHS02 protein sequence is shown as SEQ ID NO.2.

[0007] As a preferred option of the above scheme, CaCHS02 mainly regulates the functionality of peppers by regulating the chalcone synthase activity, total flavonoid content and AGI activity in pepper leaves.

[0008] Due to the above structure, the beneficial effects of the present invention are:

[0009] The present invention discloses for the first time the application of the pepper CaCHS02 gene in hypoglycemic functions such as increased chalcone synthase activity, total flavonoid content and AGI activity. The transgenic results using pepper as a receptor confirmed that overexpression of the CaCHS02 gene increased chalcone synthase activity, total flavonoid content and AGI activity. The experimental results of the present invention show that the expression level of the CaCHS02 gene increased, the chalcone synthase activity increased, and the total flavonoid content and AGI activity were significantly increased compared with the control group receptor. Therefore, the present invention proposes that the overexpression of CaCHS02 can be used to regulate the hypoglycemic activity of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0011] Figure 1 This is a schematic diagram of the detection of CaCHS02 overexpression in pepper leaves transiently transformed with the CaCHS02 overexpression vector of the present invention;

[0012] Figure 2 Schematic diagram of the detection of chalcone synthase activity in pepper leaves transiently transformed with the CaCHS02 overexpression vector of the present invention;

[0013] Figure 3 Schematic diagram of the detection of total flavonoid content in pepper leaves transiently transformed with the CaCHS02 overexpression vector of the present invention;

[0014] Figure 4 This is a schematic diagram of the detection of AGI activity in pepper leaves transiently transformed with the CaCHS02 overexpression vector of the present invention;

[0015] The data in the figure are the mean ± SD of at least 3 biological replicates, ** indicates a significant difference at the P < 0.01 level, and **** indicates a significant difference at the P < 0.0001 level. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0017] Example 1: Cloning of the full-length pepper CaCHS02 gene

[0018] The specific region of the CaCHS02 gene was amplified by PCR using the genomic DNA of pepper (Capsicum annuum L.) as a template, the CaCHS02 forward primer F1: 5'-TTAAGCCTAGGACGCGTATGTACTACGTAACTCC-3', the reverse primer R1: 5'-GTATGGGTACATACTAGTAGTAGACACACTATGGAGC-3', and finally the nucleotide sequence shown in SEQ ID NO: 3 was obtained.

[0019] In the above scheme, in order to achieve connection with the vector, Mlu I restriction site (5'-ACGCGT-3') and Spe I restriction site (5'-ACGCGT-3') and corresponding protective bases were added to the 5' end of the forward and reverse primers, respectively.

[0020] Example 2: Construction of recombinant vector

[0021] The specific fragment PCR product of the cloned pepper CaCHS02 gene was recovered and double-enzyme digested with the gene overexpression vector PMV2. The reaction system was: Mlu I: 2μL, Spe I: 2μL, 10*cut smart buffer: 5μL, DNA: 25μL, sterile water: 16μL; a total of 50μL. Reaction at 37℃ for 2h. The double-enzyme digested plasmid and PCR product were purified and recovered using the kit according to the instructions, and the recovery results were detected by 1% agarose gel.

[0022] The specific steps for the recombination and transformation of the target gene and the expression plasmid vector are as follows:

[0023] (1) Prepare the recombination reaction system (10 μL)

[0024] 10×T4 DNA ligase Buffer: 1μL; T4 DNA ligase: 1μL; DNA fragment (the molar number of DNA fragment is controlled to be 3-10 times that of vector DNA); ddH2O to 10μL. Incubate at 16℃ for 12h;

[0025] (2) All the above ligation products were added to 100 μL DH5α competent cells and placed on ice for 25 min. After heat shock at 42°C for 45 s, the cells were placed in an ice water bath for 3 min.

[0026] (3) Add 400 μL LB liquid medium and culture at 37°C with shaking at 150 rpm / min for 60 min; then incubate in a humidified medium containing spectinomycin (Spe + , 100 mg / L) on LB solid medium overnight;

[0027] (4) Pick a single colony and place it on LB solid medium (Spe + , 100 mg / L) for about 12 h, and positive clones were detected by PCR colony detection.

[0028] Example 3: Recombinant mass transformation of Agrobacterium GV3101

[0029] The plasmid was extracted using a plasmid extraction kit, and the specific instructions were followed. The sequence of the target fragment recombined into the vector was further confirmed to be accurate by first-generation sequencing. The above recombinant plasmid was then transformed into Agrobacterium GV3101, and the method steps were as follows:

[0030] (1) Add 1 μg of purified plasmid to 100 μL competent Agrobacterium GV3101 and gently shake to mix; place on ice for 5 min, and immediately place in liquid nitrogen for 5 min; place in a 37°C water bath for 5 min; add 350 μL of LB medium and shake and culture at 200 rpm / min on a shaker at 28°C for 1 h;

[0031] (2) Centrifuge to remove most of the supernatant, precipitate, mix gently by pipetting, take 100 μL of the bacterial solution, apply it on an LB plate containing 50 mg / L kanamycin and 12.5 mg / L rifampicin, and incubate at 28°C for 48 h;

[0032] (3) When resistant colonies are visible, pick a single colony and inoculate it into 2 mL of LB medium (containing kanamycin and rifampicin) and culture it at 28°C overnight with shaking; finally, perform PCR to identify positive colonies.

[0033] Example 4: Agrobacterium-mediated transient genetic transformation of pepper leaves

[0034] (1) Select positive Agrobacterium monoclonal clones (pTF101-6HA, CaCHS02-pTF101-6HA) and place them in a culture medium containing spectinomycin (Spe + , 100mg / L) and rifampicin (Rif + , 50 mg / mL) in liquid LB medium. Culture on a 28°C constant temperature shaker with a speed set to 220 rpm / min until the bacterial solution is saturated.

[0035] (2) The saturated bacterial suspension was added to a liquid LB medium (50 ml LB medium) containing 100 mg / mL Spe, 50 mg / mL Rif, 10 μM pH 5.6 morpholineethanesulfonic acid (MES) and 40 μM acetosyringone (AS) at a ratio of 1:40, and cultured on a 28°C constant temperature shaker with a rotation speed set to 220 rpm / min for 16 h.

[0036] (3) Collect Agrobacterium at a speed of 5000 rpm / min, discard the supernatant, and resuspend Agrobacterium in ddH2O containing 200 mM magnesium chloride (MgCl2) and 150 μM AS (i.e., 1 ml of the prepared resuspension solution + 50 ml of water). Adjust the bacterial solution to an appropriate concentration (OD600 = 0.6), then wrap it with tin foil and let it stand at room temperature for 2-3 hours before injection infection.

[0037] (4) The pepper seedlings to be injected are at the 6-8 leaf stage. During the injection, the bacterial solution is injected into the leaves through a 1 mL syringe on the back of the cotyledons or true leaves. The injected pepper plants are placed in a dark culture at 25°C for 48 hours, and leaf samples are taken for testing.

[0038] Example 5: Real-time fluorescence quantitative PCR detection of transformed gene expression

[0039] The leaves of a single pepper plant were selected for RNA extraction and reverse transcription and real-time fluorescence quantitative PCR were performed to detect the expression of the gene. The pepper gene CaUbi3 was used as an internal reference gene, and the primer sequences are as follows: CaUbi3-F: CCCTGGAATTGCTGACCGTA, CaUbi3-R: TGGAAAGTGCTGAGGGATGC as the internal reference gene. The forward primer of the CaCHS02 gene is 5'-CAGCAATCCTGGACCAAGTTG-3', and the reverse primer is 5'-GAGAACACAAGCACTAGACATG-3'. RNA extraction from pepper leaves, reverse transcription of the first chain of cDNA, and fluorescence quantitative PCR were all performed using the kit according to the instructions. The results are shown in the figure. Figure 1 As shown in A, the expression level of CaCHS02 in the leaves of these overexpressing peppers was higher than that in the control group.

[0040] Example 6: Determination of chalcone synthase activity in pepper leaves

[0041] Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, set up standard wells and sample wells, add 50 μL of standards of different concentrations to each standard well, add 10 μL of the sample to be tested to the sample well first, and then add 40 μL of sample diluent; nothing is added to the blank wells. Except for the blank wells, 100 μL of detection antibody labeled with horseradish peroxidase (HRP) is added to each well of the standard well and the sample well, and the reaction wells are sealed with a sealing film. Incubate in a 37°C water bath or incubator for 60 minutes, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times (you can also use a plate washer to wash the plate), add 50 μL of substrate A and B to each well, incubate at 37°C in the dark for 15 minutes, add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes (such as Figure 2 shown).

[0042] Example 7: Determination of total flavonoid content in pepper leaves

[0043] After the sample is ground into powder with liquid nitrogen, weigh about 0.1g of the sample, add 1mL of 60% ethanol solution, homogenize, and extract ultrasonically at 60℃ for 30min. Centrifuge at 10000g, 25℃ for 10min, and take the supernatant for testing. Take 20μL of the supernatant in a 96-well plate, add 6μL of 5% sodium nitrite, mix well, and let stand at room temperature for 6min; then add 6μL of 10% Al(NO3)3 solution, mix well, let stand at room temperature for 6min, then add 80μL of 4% NaOH solution and 88μL of distilled water, mix well, let stand at room temperature for 15min, and then measure the absorbance value at a wavelength of 510nm (such as Figure 3 shown).

[0044] Example 8: Determination of α-glucosidase inhibitory activity in pepper leaves

[0045] The harvested pepper leaf samples were dried at 55°C for 2 days and ground into fine powder using a mortar and pestle. 250 mg of dried pepper leaves were mixed with 2 mL of 70% ethanol and kept at 55°C for 16 hours. The evaporated samples were concentrated in a fast vacuum centrifuge (CVE-2200, Eyela, Japan) to obtain dry particles. The dried particles were dissolved in 500 μL of 20 mM sodium phosphate buffer (pH = 6.9), and each sample was extracted in triplicate. 50 μL of the extract was used to analyze AGI activity, and 200 μL of α-glucosidase (1 unit·mL -1 ) and incubate at 37°C for 10 minutes; next, 400 μL of substrate (3.0 mM pNPG dissolved in 20 mM phosphate buffer) was added to start the reaction, the total reaction volume was 650 μL, and the mixture was incubated at 37°C for 20 min; 4.35 mL of 0.1 M Na2CO3 was added to stop the reaction, and the absorbance was measured. Using a micro-spectrophotometer, the absorbance value was detected at 405 nm using the instrument Epoch (BioTek Instruments, Inc., Winooski, VT, USA). At the same time, 4.95 mL of Na2CO3 was added to 50 μL of the unreacted extract sample, the absorbance value of this control sample was measured, and the AGI activity was calculated according to the formula (such as Figure 4 shown).

[0046] SEQ ID NO:1

[0047] ATGTACTACGTAACTCCAACACGACCCTCAATCAAACGACTCATGATGTACCAACAAGGTTGGTTTGCTGGTGGAACCGTTATCCGACTAGCAAAGGACTTAGCTGAAAACAACAAGGGAGCTCGAGTCCTTGTTGTTTGCTTAGAAATAACTGCAGTTACTTTTCGTGGCCCAAGTGATACACA CTTAGATAGTATGGTTGGACAAGCGCTCTTTGGTGATGGGACAGCTGCACTCATTGTAGGTTCTAATCCATTACCAGGAGTTGAAAAACCTTTGTTTGAGCTTGACTCTGCGGCCCAAACACTTCTCCCTAACAGCAAGGGCGCTATAGATGGTCACCTTCATGAAGCTGGGCTTACATTTCACT TACTCAAAGATGATCCTGGATTGATCTCAAAGAACATTGAGAAGAGTTTGATAGAAGCATTCCAACCATTGGGGATTTCTAATTGGAACTCTATCTTTTGGATTGCTCACCCCGGTGGGCCAGCAATCCTGGACCAAGTTGAATTAAAGTTGGGCCTAAAGCCCAAAAAACTCTGGGCTACTAAGAAAGTCTTGAGTGACTATGAAAACATGTCTAGTGCTTGTGTTCTCTTAATTTTGGATGAAATTAGAAAGGCTTCAGTCAAAGAAGGGTTTAGTAGTACTGGTGAAGGCCTTGATTGGGGTGTACTTTTTGGATTTAGACCTGGGCTTACGGTTGAGACTGTTGTGCTCCATAGTGTGTCTACTTAA

[0048] SEQ ID NO:2

[0049] MYYVTPTRPSIKRLMMYQQGWFAGGTVIRLAKDLAENNKGARVLVVCLEITAVTFRGPSDTHLDSMVGQALFGDGTAALIVGSNPLPGVEKPLFELDSAAQTLLPNSKGAIDGHLHEAGLTFHLLKDDPGLISKNIEKSLIEAFQPLGISNWNSIFWIAHPGGPAILDQVELKLGLKPKKLWATKKVLSDYENMSSACVLLILDEIRKASVKEGFSSTGEGLDWGVLFGFRPGLTVETVVLHSVST

[0050] SEQ ID NO:3

[0051] TTAAGCCTAGGACGCGTATGTACTACGTAACTCCAACACGACCCTCAATCAAACGACTCATGATGTACCAACAAGGTTGGTTTGCTGGTGGAACCGTTATCCGACTAGCAAAGGACTTAGCTGAAAACAACAAGGGAGCTCGAGTCCTTGTTGTTTGCTTAGAAATAACTGCAGTTACTTTTCGTGGCCCAAG TGATACACACTTAGATAGTATGGTTGGACAAGCGCTCTTTGGTGATGGGACAGCTGCACTCATTGTAGGTTCTAATCCATTACCAGGAGTTGAAAAACCTTTGTTTGAGCTTGACTCTGCGGCCCAAACACTTCTCCCTAACAGCAAGGGCGCTATAGATGGTCACCTTCATGAAGCTGGGCTTACATTTCAC TTACTCAAAGATGATCCTGGATTGATCTCAAAGAACATTGAGAAGAGTTTGATAGAAGCATTCCAACCATTGGGGATTTCTAATTGGAACTCTATCTTTTGGATTGCTCACCCCGGTGGGCCAGCAATCCTGGACCAAGTTGAATTAAAGTTGGGCCTAAAGCCCAAAAAACTCTGGGCTACTAAGAAAGTCT TGAGTGACTATGAAAACATGTCTAGTGCTTGTGTTCTCTTAATTTTGGATGAAATTAGAAAGGCTTCAGTCAAAGAAGGGTTTAGTAGTACTGGTGAAGGCCTTGATTGGGGTGTACTTTTTGGATTTAGACCTGGGCTTACGGTTGAGACTGTTGTGCTCCATAGTGTGTCTACTACTAGTATGTACCCCATC

[0052] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The application of CaCHS02 gene in regulating the inhibitory activity of pepper α-glucosidase is characterized by: The full-length nucleotide sequence of the pepper CaCHS02 gene is shown in SEQ ID NO:

1.

2. The use of the CaCHS02 gene according to claim 1 in regulating the inhibitory activity of pepper α-glucosidase, characterized in that: The pepper CaCHS02 protein sequence is shown in SEQ ID NO.

2.

3. The use of the CaCHS02 gene according to claim 1 or 2 in regulating the inhibitory activity of pepper α-glucosidase, characterized in that: CaCHS02 mainly regulates the functionality of peppers by regulating the chalcone synthase activity, total flavonoids content and AGI activity in pepper leaves.

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