Use of CsC3'H gene to increase content of naringin, hesperidin and / or jaceosidin in citrus

Abstract

The application relates to the technical field of genetic engineering, and discloses application of a CsC3'H gene in increasing contents of citrus naringin, hesperidin and / or jaceosidin, a CDS sequence of the CsC3'H gene is shown as SEQ ID NO. 1, and a protein sequence coded by the CsC3'H gene is shown as SEQ ID NO. 2. It is found for the first time that the CsC3'H gene is positively correlated with the contents of citrus naringin, hesperidin and / or jaceosidin, and the highest content of citrus naringin, hesperidin and / or jaceosidin in a citrus transiently transformed with a CsC3'H gene overexpression vector can be 69.38% higher than that in a citrus transformed with a blank vector; therefore, the CsC3'H gene can be used as a candidate gene for breeding of a new citrus variety with high contents of citrus naringin, hesperidin and / or jaceosidin, and has important significance for cultivating a functional new citrus variety by using a genetic engineering method, accelerating a breeding process, reducing a breeding workload and improving comprehensive utilization of citrus.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the CsC3'H gene in increasing the content of rutin, hesperidin and / or lemon balm glycoside in citrus peel. Background Technology

[0002] Citrus fruits are the largest category of fruits in China, ranking first in the world in both cultivation area and output. Citrus fruits are rich in nutrients and bioactive secondary metabolites. These components not only give them unique flavor and health benefits but also play a significant role in antioxidation, anti-inflammation, and anti-cancer activity. Currently known substances in citrus fruits with important anti-inflammatory, antioxidant, and anti-tumor effects include phenolic acids, flavonoids, carotenoids, essential oils, limonene, and synephrine.

[0003] Naringin, hesperidin, lemon balm glycoside, and sweet orange flavonoids are all typical flavanone glycosides with significant biological activities, including: 1) plant stress resistance and defense, protecting against pathogens (bacteria, fungi) and pests; 2) antioxidant and photoprotective effects, scavenging intracellular reactive oxygen species (ROS) and excess free radicals, reducing oxidative damage; 3) anti-inflammatory effects, exhibiting anti-inflammatory activity by inhibiting pro-inflammatory cytokines and reducing leukocyte infiltration; 4) cardiovascular protection, studies have shown that dietary intake of naringin and hesperidin can effectively reduce the risk of cardiovascular disease death in adults; 5) anti-cancer and anti-tumor effects, studies have shown that dietary intake of naringin and hesperidin can reduce the risk of breast cancer, lung cancer, colon cancer, prostate cancer, and pancreatic cancer; 6) metabolic regulation and anti-obesity: regulating gut microbiota balance, inhibiting the growth of pathogenic bacteria, promoting the proliferation of probiotics (such as Bifidobacteria), and improving intestinal barrier function.

[0004] Naringin, hesperidin, and / or lemon balm glycoside are widely distributed in various vegetables and fruits and are used in food processing to reduce the use of synthetic chemicals and improve human health. Identifying the key enzymes in their biosynthetic pathways and elucidating their molecular mechanisms of function can provide a theoretical basis for molecular breeding of functional citrus fruits with high ringin, hesperidin, and / or lemon balm glycoside content, which is of great significance for improving the comprehensive utilization rate of citrus fruits. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the CsC3'H gene in increasing the content of rutin, hesperidin and / or lemon balm in citrus peel, so as to provide a candidate gene related to the synthesis of rutin, hesperidin and / or lemon balm in citrus peel, and contribute to the cultivation of new functional citrus varieties by using genetic engineering methods, accelerate the breeding process, and reduce the workload of breeding.

[0006] To achieve the above objectives, the present invention provides the application of the CsC3'H gene in increasing the content of rutin, hesperidin and / or lemon balm in citrus peel, wherein the CDS sequence of the CsC3'H gene is shown in SEQ ID NO.1 and the protein sequence encoded by the CsC3'H gene is shown in SEQ ID NO.2.

[0007] Furthermore, overexpression of the CsC3'H gene in citrus increases the content of naringin, hesperidin and / or lemon balm glycoside in citrus peel.

[0008] Furthermore, the expression level of the CsC3'H gene was positively correlated with the content of naringin, hesperidin and / or lemon balm glycoside in citrus peel.

[0009] Furthermore, this invention also provides the application of the CsC3'H gene in citrus variety breeding, wherein the CDS sequence of the CsC3'H gene is shown in SEQ ID NO.1, and the protein sequence encoded by the CsC3'H gene is shown in SEQ ID NO.2; the citrus variety breeding involves selecting citrus varieties with increased content of naringin, hesperidin and / or lemon balm glycoside in the peel.

[0010] The present invention also provides a plant overexpression vector containing the CsC3'H gene, wherein the CDS sequence of the CsC3'H gene is shown in SEQ ID NO.1, and the protein sequence encoded by the CsC3'H gene is shown in SEQ ID NO.2.

[0011] Furthermore, this invention also provides the application of plant overexpression vectors containing the CsC3'H gene in citrus breeding.

[0012] Furthermore, the present invention also provides a method for cultivating citrus peel with increased content of naringin, hesperidin and / or lemon balm glycoside, by introducing the CsC3'H gene or a plant overexpression vector containing the CsC3'H gene into citrus plants, so that the CsC3'H gene is overexpressed in citrus plants, thereby cultivating citrus plants with increased content of naringin, hesperidin and / or lemon balm glycoside;

[0013] The CDS sequence of the CsC3'H gene is shown in SEQ ID NO.1, and the protein sequence encoded by the CsC3'H gene is shown in SEQ ID NO.2.

[0014] Therefore, the advantages and positive effects of the CsC3'H gene provided by this invention in increasing the content of rutin, hesperidin and / or lemon balm in citrus peel are as follows:

[0015] (1) This invention first discovered that the expression level of CsC3'H gene is positively correlated with the content of rutin, hesperidin and / or lemon balm in citrus peel. The higher the expression level of CsC3'H gene, the higher the content of rutin, hesperidin and / or lemon balm in citrus peel. In the experiment, the citrus transformed with the CsC3'H gene overexpression vector had a maximum content 69.38% higher than that of citrus transformed with the empty vector.

[0016] (2) The CsC3'H gene can be used as a candidate gene for breeding new citrus varieties with high content of rutin, hesperidin and / or lemon balm glycoside in citrus peel. It is of great significance for breeding new functional citrus varieties by using genetic engineering methods, accelerating the breeding process, reducing the breeding workload and improving the comprehensive utilization rate of citrus.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The following is the bioinformatics analysis result of the CsC3'H gene in Example 1 of the present invention, where A is the chromosomal location of the CsC3'H gene of Late Orange, and bp is the base; B is the structure of the CsC3'H gene of Late Orange, and Exon1-3 are exons; C is the conserved domain of the CsC3'H gene of Late Orange, and aa is an amino acid;

[0020] Figure 2 This is an electrophoresis diagram of the PCR amplification product of the CsC3'H gene coding sequence cloned in Example 2 of the present invention, where CDS represents the CsC3'H gene coding sequence and M represents the DNA molecular weight standard.

[0021] Figure 3 This is a structural diagram of the CsC3'H gene overexpression vector in Example 3 of the present invention, where GUS:NPTII represents the β-glucosidase gene; P 35S This indicates a plant constitutive promoter derived from cauliflower mosaic virus; T NOS This indicates the terminator of the crown gall alkaloid synthase gene;

[0022] Figure 4These are photos of fruits taken on the day of transient transformation and 5 days after transient transformation in Example 4 of the present invention, where A is a photo of the fruit on the day of transient transformation; B is a photo of the fruit 5 days after transient transformation; and C is a partial sample photo taken when total RNA was extracted.

[0023] Figure 5 The results of the relative expression level analysis of the CsC3'H gene in Example 4 of this invention;

[0024] Figure 6 The results of the detection of the contents of naringin, hesperidin and / or lemon balm glycoside in the peel of the late-ripened orange in Example 5 of the present invention are shown. A is the content of naringin; B is the content of hesperidin; and C is the content of lemon balm glycoside. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

[0028] The Late-maturing Jincheng Orange is a superior late-maturing Jincheng Orange strain bred by the Citrus Research Institute of the Chinese Academy of Agricultural Sciences from the early 1980s to 2010. Variety Approval Number: Yu Shen Citrus 2011001.

[0029] Example 1: Bioinformatics analysis of the CsC3'H gene in Late Orange

[0030] The structure of the CsC3'H gene is as follows: Figure 1 As shown, the CsC3'H gene is located between 3,752,127 bp and 3,756,838 bp on chromosome 1 of Late Orange, contains 3 exons, and encodes 509 amino acids. The CDS sequence of the CsC3'H gene is shown in SEQ ID NO.1:

[0031] SEQ ID NO.1:

[0032]

[0033] The CsC3'H protein sequence is shown in SEQ ID NO.2.

[0034] SEQ ID NO.2:

[0035] MALPLIPLSIIFIILAYKLYQRLRFNLPPGPRPLPIVGNLYDIKPVRFRCFAEWAQQYGPVISVWFGSTLNVIVSNTELAREVLKEHDQQLADRHRSRSAAKFSRDGKDLIWADYGPHYVKVRKVCT LELFTPKRLEALRPIREDEVTAMVESIFKDCTDPQNYGKSVLVKKYLGAVAFNNITRLLAFGKRFVNSEDVMDEQGKEFKAIVANGLKLGASLAMAEHIPWLRWMFPLEEGAFAKHGERRDRLTRAIM EEHTLARQKSGGTKQHFVDALLTLQEKYDLSEDTIIGLLWDMITAGMDTTAISTEWGMAELIKNPRVQQKAQEELDRVIGFERVMTETDFSNLPYLQAVAKEALRLHPPTPLMLPHRANANVKIGGY DVPKGSNIHVNVWAVARDPAVWKDPLEFRPERFFEEDVDMKGHDFRLLPFGAGRRVCPGAQLGINLVTSMLGHLLHHFAWAPPEGVKPEEIDMSENPGLVTYMKTPLQAVPTPRLPSHLYKRVAADM.

[0036] Example 2: Cloning of the CsC3'H gene coding sequence of Late Orange

[0037] 2.1 RNA extraction and cDNA synthesis:

[0038] Total RNA was extracted from leaves of *Citrus aurantiacus* using a plant total RNA extraction kit (Adley, CAT: RN09). RNA quality was verified by agarose gel electrophoresis, and RNA concentration was determined using a concentration meter. cDNA was then synthesized using a reverse transcription kit (PrimeScript RT Master Mix, TaKaRa, CAT: RR036A) according to the accompanying instructions.

[0039] 2.2 Amplification of the CsC3'H coding sequence:

[0040] Using primers OE-CsC3'HF (sequence shown in SEQ ID NO.3), OE-CsC3'HR (sequence shown in SEQ ID NO.4), and the high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, CAT: R045Q), and with the late-ripening orange cDNA obtained in 2.1 as a template, the amplification system was prepared according to the instructions accompanying the PrimeSTAR Max DNA Polymerase. The PCR amplification program was: 98℃, 5 min; 98℃, 30 s, 56℃, 30 s, 72℃, 1.5 min, 35 cycles; extension at 72℃ for 10 min. A DNA fragment encoding the CsC3'H sequence was obtained, with a fragment length of 1536 bp (1527 bp CDS sequence - 3 bp terminator + 12 bp of restriction enzyme sites).

[0041] Agarose gel electrophoresis results are as follows Figure 2 As shown, the amplified fragment size results were as expected. Under UV light, the agarose gel block containing the target fragment was cut off with a clean blade, and the DNA fragment was recovered using a kit (BioFlux, CAT: BSC02M1). A portion of the recovered product was sent to the company for sequencing. The sequencing results, after comparative analysis, confirmed that the obtained DNA fragment was the coding sequence of the CsC3'H gene of Late Orange (SEQ ID NO.1).

[0042] SEQ ID NO.3:GGTACCATGGCTCTCCCACTCATCCC;

[0043] SEQ ID NO. 4: GAATTCCATATCAGGCGCCACACG.

[0044] Example 3: Construction of CsC3'H overexpression vector and transformation of Agrobacterium tumefaciens

[0045] 3.1 Construction of CsC3'H overexpression vector:

[0046] The recovered DNA fragments and overexpression vector pLGNe obtained in Example 2 were double-digested with restriction endonucleases KpnⅠ and EcoRI (Thermo Fisher), then gel-recovered and ligated overnight at 16°C. The digestion system and reaction conditions were performed according to the accompanying instructions. The ligation was performed using the T4 DNA Ligase kit (Promega, CAT: M1801), and the ligation system and reaction conditions were performed according to the instructions accompanying the T4 DNA Ligase kit.

[0047] The obtained ligation product was transformed into *E. coli* DH5α using the method described in the *E. coli* DH5α instruction manual. Plasmids from positive clones were extracted using a plasmid extraction kit (Omega, CAT: D6942) to obtain the CsC3'H overexpression vector pLGNe-CsC3'H. The vector structure is shown below. Figure 3 As shown.

[0048] 3.2 Transform Agrobacterium tumefaciens into the CsC3'H overexpression vector:

[0049] The overexpression vector pLGNe-CsC3'H obtained was introduced into Agrobacterium tumefaciens EHA105 using a heat shock method. The specific steps are as follows:

[0050] Thaw 50 μL of frozen Agrobacterium tumefaciens competent cells EHA105 in a 2 mL centrifuge tube on ice. Add 2 μL of the plasmid overexpression vector to the competent cells, mix well by pipetting, and then place the mixture on ice for 5 min, flash freeze in liquid nitrogen for 5 min, incubate at 37 °C for 5 min, and place on ice for 5 min. Then add 800 μL of LB liquid medium to a 2 mL centrifuge tube, mix well by pipetting, and incubate at 28 °C with shaking at 260 rpm for 2 h. After the designated time, centrifuge the bacterial culture at 6000 rpm for 1 min, discard the supernatant, resuspend the bacterial cells in 50 μL of LB liquid medium, and then spread the resuspended cells onto LB solid medium containing 50 mg / L kanamycin. Incubate in the dark at 28°C for 2 days. Once plaques have grown, pick colonies and perform PCR verification on single colonies using primers ID-CsC3'HF (sequence shown in SEQ ID NO.5) and ID-CsC3'HR (sequence shown in SEQ ID NO.6) and the high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, CAT: R045Q). The amplification system was prepared according to the instructions for PrimeSTAR Max DNA Polymerase. The PCR amplification conditions were: 94°C for 3 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0051] The PCR amplification products were subjected to agarose gel electrophoresis. Colonies with the correct band size were positive clones containing the overexpression vector pLGNe-CsC3'H.

[0052] SEQ ID NO.5:TCGTTGAAGATGCCTCTGCCGACAG;

[0053] SEQ ID NO. 6: CATATCAGCGGCCACACG.

[0054] Example 4: Transient conversion of the CsC3'H overexpression vector pLGNe-CsC3'H

[0055] 4.1 Agrobacterium infection:

[0056] Select late-ripening orange fruits of uniform growth and sterilize them with 75% alcohol in a clean bench for later use. Add 500 μL of Agrobacterium tumefaciens containing pLGNe and pLGNe-CsC3'H plasmids to 50 mL of liquid LB medium (containing 50 mg / L kanamycin) and incubate at 28℃ and 200 rpm until OD500. 600 =0.5. Centrifuge and collect the precipitate. Resuspend Agrobacterium in 1 / 2 MS liquid medium. Then, randomly select four injection points on the diagonal of the equatorial plane of the sterilized Late Jin Orange fruit and mark them. Inject the Agrobacterium resuspension containing pLGNe-CsC3'H plasmid into the peel of the Late Jin Orange using a 1 mL syringe. Inject 1 mL into each area, and record them as the experimental group (pLGNe-CsC3'H-1, pLGNe-CsC3'H-2, pLGNe-CsC3'H-3). The Late Jin Orange fruit injected with the same method and the same amount of Agrobacterium resuspension containing pLGNe plasmid is the control group (pLGNe). The experimental group and the control group are repeated three times. The Late Jin Orange fruit injected with Agrobacterium resuspension are placed in a 28℃ incubator and incubated in the dark for 5 days. The fruit photos on the day of transient transformation and after 5 days of incubation are shown below. Figure 4 As shown.

[0057] 4.2 qRT-PCR analysis of transiently transformed late-ripening orange fruits:

[0058] Total RNA (Adelai, CAT No: RN09) was extracted from the peel of the injected area of ​​*Citrus reticulata* var. *mairei* (Citrus reticulata) cultured in the dark at 28℃ for 5 days in section 4.1. cDNA was synthesized using the PrimeScript RT Master Mix reverse transcription kit (TaKaRa, CAT: RR036A), and the expression level of the target gene was detected by qRT-PCR. The detection primers were RT-CsC3'HF (SEQ ID NO.7) and RT-CsC3'HR (SEQ ID NO.8). Two... -△△Ct The relative expression level of the CsC3'H gene in the experimental and control groups of late-ripening oranges was calculated as follows: the control group sample was defined as the reference factor, i.e., its CsC3'H expression level was 1. Then, the fold increase in gene expression in the experimental group sample relative to the reference factor was calculated as 2. -△△Ct , which is its relative expression level. The results are as follows: Figure 5 As shown, the expression level of the CsC3'H gene in the experimental group was significantly higher than that in the control group, with the highest level being more than 8 times that of the control.

[0059] qRT-PCR reaction conditions: 95℃ for 3 min, 94℃ for 10 s; 56℃ for 10 s, 72℃ for 10 s, 40 cycles; 72℃ for 10 min.

[0060] SEQ ID NO.7: TAATGGGCTGAAGCTTGGGG;

[0061] SEQ ID NO. 8: GCTCGAGTAAGACGGTCTCG.

[0062] Example 5: Determination of the content of naringin, hesperidin and / or lemon balm glycoside in fruit.

[0063] The contents of naringin, hesperidin, and / or lemon balm glycosides in the peels of the experimental and control groups after 5 days of transient transformation in Example 4 were determined using UPLC-MS. Statistical results are shown below. Figure 6 As shown, compared with the control group fruit transiently transformed with pLGNe, the contents of naringin, hesperidin and / or lemon balm glycoside in the peel of the fruit transiently transformed with pLGNe-CsC3'H increased by 66.34%–69.38%, 9.36%–9.49% and 44.95%–46.89%, respectively, indicating that transient overexpression of the CsC3'H gene can significantly increase the contents of naringin, hesperidin and / or lemon balm glycoside in the peel of the late-ripening orange.

[0064] Therefore, this invention is the first to discover a positive correlation between the expression level of the CsC3'H gene and the content of rutin, hesperidin, and / or lemon balm glycosides in citrus peel. The higher the expression level of the CsC3'H gene, the higher the content of rutin, hesperidin, and / or lemon balm glycosides in the citrus peel. In the experiment, citrus transformed with the CsC3'H gene overexpression vector showed up to 69.38% higher levels of these compounds compared to citrus transformed with the empty vector. The CsC3'H gene can serve as a candidate gene for breeding new citrus varieties with high rutin, hesperidin, and / or lemon balm glycoside content. This is of great significance for using genetic engineering methods to cultivate functional new citrus varieties, accelerate the breeding process, reduce the breeding workload, and improve the comprehensive utilization rate of citrus.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. CsC3'H The application of genes in increasing the content of rutin, hesperidin and / or lemon balm glycoside in citrus peel is characterized by: The CsC3'H The CDS sequence of the gene is shown in SEQ ID NO.

1. CsC3'H The protein sequence encoded by the gene is shown in SEQ ID NO.2; it is overexpressed in citrus. CsC3'H Genes that increase the content of naringin, hesperidin and / or lemon balm glycoside in citrus peel.

2. As described in claim 1 CsC3'H The application of genes in increasing the content of rutin, hesperidin and / or lemon balm glycoside in citrus peel is characterized by: CsC3'H The expression level of the gene was positively correlated with the content of naringin, hesperidin and / or lemon balm glycoside in citrus peel.

3. CsC3'H The application of genes in citrus variety breeding is characterized by: The CsC3'H The CDS sequence of the gene is shown in SEQ ID NO.

1. CsC3'H The protein sequence encoded by the gene is shown in SEQ ID NO.2; citrus variety breeding involves selecting citrus varieties with increased content of naringin, hesperidin, and / or lemon balm glycosides in the peel, among which, citrus varieties with increased content of naringin, hesperidin, and / or lemon balm glycosides in the peel... CsC3'H Increased gene expression levels.

4. Includes CsC3'H The application of plant overexpression vectors for genes in citrus breeding is characterized by: The CsC3'H The CDS sequence of the gene is shown in SEQ ID NO.

1. CsC3'H The protein sequence encoded by the gene is shown in SEQ ID NO.

2.

5. A method for increasing the content of naringin, hesperidin, and / or lemon balm glycoside in citrus peel, characterized in that: Will CsC3'H Genes or containing CsC3'H The gene overexpression vector was introduced into citrus plants, enabling... CsC3'H The gene was overexpressed in citrus plants to cultivate citrus plants with increased contents of rutin, hesperidin and / or lemon balm glycoside in citrus peel; The CsC3'H The CDS sequence of the gene is shown in SEQ ID NO.

1. CsC3'H The protein sequence encoded by the gene is shown in SEQ ID NO.2.

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