Application of CsCSE2 gene in increasing content of citrus vicenin-2 and / or hesperidin

Abstract

The invention relates to the technical field of gene engineering, and discloses an application of a CsCSE2 gene in increasing the content of citrus vicenin-2 and / or hesperidin, the CDS sequence of the CsCSE2 gene is shown as SEQ ID NO.1, and the protein sequence coded by the CsCSE2 gene is shown as SEQ ID NO.2. The invention also discloses a method for preparing the CsCSE2 gene for improving the content of citrus vicenin-2 and / or hesperidin. According to the invention, the CsCSE2 gene is found to be positively correlated with the content of citrus vicenin-2 and / or hesperidin for the first time, and in an experiment, the highest value of citrus of which the CsCSE2 gene overexpression vector is transformed can be 69.38% higher than that of citrus of which an empty vector is transformed; therefore, the CsCSE2 gene can be used as a candidate gene for breeding a new citrus variety with high-dimensioning-2 and / or hesperidin content, and has important significance in breeding a new functional citrus variety by adopting a genetic engineering method, accelerating the breeding process, reducing the breeding workload and improving the comprehensive utilization rate of citrus.

Description

TECHNICAL FIELD

[0001] The application relates to the field of genetic engineering, and particularly to application of a CsCSE2 gene in increasing content of vitamin C and / or hesperidin in citrus. BACKGROUND

[0002] Citrus fruits contain rich nutritional functional components and bioactive secondary metabolites, which not only endow them with unique flavors and health values, but also have significant effects on oxidation resistance, inflammation resistance and cancer resistance. It is known that the substances with important effects of inflammation resistance, oxidation resistance and tumor resistance contained in citrus include phenolic acids, flavonoids, carotenoids, essential oils, limonin and synephrine.

[0003] Vitamin C belongs to flavonoid glycosides, and hesperidin is a typical flavanone glycoside compound, which has significant biological activities, including: 1) plant stress resistance and defense, which can resist the invasion of pathogens (bacteria and fungi) and pests; 2) oxidation resistance and light protection, which can remove active oxygen (ROS) and excessive free radicals in cells and reduce oxidative damage; 3) inflammation resistance, which can exhibit anti-inflammatory activity by inhibiting pro-inflammatory cytokines and reducing leukocyte infiltration; 4) cardiovascular protection, researches show that the intake of naringin and hesperidin in diet can effectively reduce the risk of cardiovascular disease death in adults; 5) anti-cancer and anti-tumor, researches show that the intake of naringin and hesperidin in diet can reduce the incidence of breast cancer, lung cancer, colon cancer, prostate cancer and pancreatic cancer; 6) metabolism regulation and anti-obesity: regulating intestinal flora balance, inhibiting the growth of pathogenic bacteria, promoting the proliferation of probiotics (such as bifidobacteria) and improving intestinal barrier function.

[0004] Vitamin C and / or hesperidin are widely distributed in various vegetables and fruits and are used in food processing to reduce the use of synthetic chemicals, improve human health, excavate key enzymes of the biosynthetic pathway and clarify the molecular mechanism of the function, which can provide a theoretical basis for functional citrus molecular breeding with high vitamin C and / or hesperidin content, and has important significance for improving the comprehensive utilization rate of citrus. SUMMARY

[0005] The application aims to provide application of a CsCSE2 gene in increasing content of vitamin C and / or hesperidin in citrus, so as to provide a candidate gene related to vitamin C and / or hesperidin synthesis, and contribute to the cultivation of functional new citrus varieties by using the method of genetic engineering, accelerate the breeding process and reduce the workload of breeding.

[0006] To achieve the above objectives, the present invention provides the application of the CsCSE2 gene in increasing the content of veselcin-2 and / or hesperidin in citrus fruits. The CDS sequence of the CsCSE2 gene is shown in SEQ ID NO.1, and the protein sequence encoded by the CsCSE2 gene is shown in SEQ ID NO.2.

[0007] Furthermore, overexpression of the CsCSE2 gene in citrus increases the content of vescenin-2 and / or hesperidin in citrus peel.

[0008] Furthermore, the expression level of the CsCSE2 gene was positively correlated with the content of veselin-2 and / or hesperidin in citrus peel.

[0009] Furthermore, this invention also provides the application of the CsCSE2 gene in citrus variety breeding, wherein the CDS sequence of the CsCSE2 gene is shown in SEQ ID NO.1, and the protein sequence encoded by the CsCSE2 gene is shown in SEQ ID NO.2; the citrus variety breeding is to select citrus varieties with increased content of veselin-2 and / or hesperidin in the peel.

[0010] The present invention also provides a plant overexpression vector containing the CsCSE2 gene, wherein the CDS sequence of the CsCSE2 gene is shown in SEQ ID NO.1, and the protein sequence encoded by the CsCSE2 gene is shown in SEQ ID NO.2.

[0011] Furthermore, this invention also provides the application of plant overexpression vectors containing the CsCSE2 gene in citrus breeding.

[0012] Furthermore, the present invention also provides a method for cultivating citrus peel with increased content of vescenin-2 and / or hesperidin, by introducing the CsCSE2 gene or a plant overexpression vector containing the CsCSE2 gene into citrus plants, thereby overexpressing the CsCSE2 gene in the citrus plants and cultivating citrus plants with increased content of vescenin-2 and / or hesperidin.

[0013] The CDS sequence of the CsCSE2 gene is shown in SEQ ID NO.1, and the protein sequence encoded by the CsCSE2 gene is shown in SEQ ID NO.2.

[0014] Therefore, the specific technical effects of the CsCSE2 gene provided by this invention in increasing the content of veselcin-2 and / or hesperidin in citrus are as follows:

[0015] (1) This invention first discovered that the expression level of CsCSE2 gene is positively correlated with the content of vescenin-2 and / or hesperidin in citrus. The higher the expression level of CsCSE2 gene, the higher the content of vescenin-2 and / or hesperidin in citrus peel. In the experiment, the citrus transformed with CsCSE2 gene overexpression vector can be up to 34.5% higher than that of citrus transformed with empty vector.

[0016] (2) The CsCSE2 gene can be used as a candidate gene for breeding new citrus varieties with high content 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 results of bioinformatics analysis of the CsCSE2 gene in Example 1 of this invention are shown below; where A is the chromosomal location of the CsCSE2 gene of Late Orange, and bp is a base; B is the structure of the CsCSE2 gene of Late Orange, and Exon1-2 are exons; C is the conserved domain of the CsCSE2 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 CsCSE2 gene coding sequence cloned in Example 2 of the present invention; where CDS represents the CsCSE2 gene coding sequence; M represents the DNA molecular weight standard;

[0021] Figure 3 This is a structural diagram of the CsCSE2 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 4 These 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 CsCSE2 gene in Example 4 of this invention;

[0024] Figure 6 The results show the detection of the content of vesine-2 and / or hesperidin in the peel of the late-ripened orange in Example 5 of this invention. 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 descriptions 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 CsCSE2 gene in Late Orange

[0030] The structure of the CsCSE2 gene is as follows: Figure 1 As shown, the CsCSE2 gene is located between 19,477,432 bp and 19,480,761 bp on chromosome 1 of Late Orange, contains two exons, and encodes 324 amino acids. The CDS sequence of the CsCSE2 gene is shown in SEQ ID NO.1.

[0031] SEQ ID NO.1:

[0032] ATGCCTCCAGAAACGCAAACCGAAACGCCTCCGAATTTCTGGGGCGACATGCCGGAAGAGGAGTACTACACGTCTCAGGGAGTGCGAAACGGTAAAAAGTACTTCGAAACGCCCAACGGGAAGCTCTTTACGCAGAGTTTCCTGCCGTTGGATCAAAAGGTGAAGGCCACGGTGTACATGACCCACGGCTATGGATCGGACACCGGCTGGATGTTTCAGAAGATTTGCATCAGCTTTGCTACGTGGGGTTACGCTGTCTTTGCCGCTGATCTTCTCGGGCACGGCCGATCGGACGGCATACGTTGCTATCTAGGTGACATGGAGAAGGTCGCTGCTTCGTCCCTATCATTCTTCAAGCATGTGCGCGACAGCGAACCATACAGGGACTTGCCCGGTTTCTTATTTGGCGAGTCCATGGGTGGGGCCGCAACAATGCTGATGTACTTCCAGTCAGAGCCCAATACGTGGACGGGTCTGATCTTCTCGGCCCCACTCTTCGTCATCCCAGAAAACATGAAACCTAGTAAGCTCCATCTCTTCATGTACGGTCTCCTATTTGGACTGGCCGACACGTGGGCGGCGATGCCCGATAACAAAATGGTCGGAAAAGCCATAAAAGACCCTGAAAAATTGAAGGTCATAGCATCAAATCCGAGGAGATATACGGGAAAGCCGAGGGTGGGGACCATGAGAGAAATTGCTAGAGTGTGTCAGTATATACAGGATAACTTCTCCAAAGTAACGGTGCCGTTTTTGACAGTGCATGGGACGGCTGACGGGGTCACGTGCCCTACATCATCCAAGTTGTTGTATGAGAAGGCATCCAGTGCGGACAAGAGTATCAAAATTTATGATGGGATGTACCATTCTTTGATTCAAGGAGAGCCTGATGAGAATGCTAATCTTGTGTTGAAGGATATGAGGGAGTGGATTGATGAGAGAGTTGAGAGGTATGGGCCTAAAAATTGTTAA。

[0033] The CsCSE2 protein sequence is shown in SEQ ID NO.2.

[0034] SEQ ID NO.2:

[0035] MPPETQTETPPNFWGDMPEEEYYTSQGVRNGKKYFETPNGKLFTQSFLPLDQKVKATVYMTHGYGSDTGWMFQKICISFATWGYAVFAADLLGHGRSDGIRCYLGDMEKVAASSLSFFKHVRDSEPYRDLPGFLFGESMGGAATMLMYFQSEPNTWTGLIFS APLFVIPENMKPSKLHLFMYGLLFGLADTWAAMPDNKMVGKAIKDPEKLKVIASNPRRYTGKPRVGTMREIARVCQYIQDNFSKVTVPFLTVHGTADGVTCPTSSKLLYEKASSADKSIKIYDGMYHSLIQGEPDENANLVLKDMREWIDERVERYGPKNC.

[0036] Example 2: Cloning of the CsCSE2 gene coding sequence of Late Orange

[0037] 2.1 RNA extraction and cDNA synthesis:

[0038] Total RNA was extracted from leaves of *Citrus aurantiacus* (Late Orange) 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 CsCSE2 coding sequence:

[0040] Using primers OE-CsCSE2-F (sequence shown in SEQ ID NO.2), OE-CsCSE2-R (sequence shown in SEQ ID NO.3), 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 CsCSE2 sequence was obtained, with a fragment length of 981 bp (CDS sequence 972 bp - terminator 3 bp + 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 Late Orange CsCSE2 gene (SEQ ID NO.1).

[0042] SEQ ID NO.3:GGTACCATGCCTCCAGAAACGCAAACC

[0043] SEQ ID NO.4:GAATTCACAATTTTTAGGCCCATACCTCTCAAC

[0044] Example 3: Construction of CsCSE2 overexpression vector and transformation of Agrobacterium tumefaciens

[0045] 3.1 Construction of CsCSE2 overexpression vector:

[0046] The recovered DNA fragments and overexpression vector pLGNe obtained in Example 2 were double-digested with restriction endonucleases KpnⅠ and EcoRI (ThermoFisher), then gel-recovered and ligated overnight at 16°C. The digestion system and reaction conditions were performed according to the accompanying instructions. 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 CsCSE2 overexpression vector pLGNe-CsCSE2. The vector structure is shown below. Figure 3 As shown.

[0048] 3.2 Transform the CsCSE2 overexpression vector into Agrobacterium:

[0049] The overexpression vector pLGNe-CsCSE2 obtained was introduced into Agrobacterium tumefaciens EHA105 using the 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-CsCSE2-F (sequence shown in SEQ ID NO.4) and ID-CsCSE2-R (sequence shown in SEQ ID NO.5) and the high-fidelity enzyme PrimeSTAR Max DNAPolymerase (TaKaRa, CAT: R045Q). The amplification system was prepared according to the instructions for the high-fidelity enzyme PrimeSTAR Max DNAPolymerase. 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-CsCSE2.

[0052] SEQ ID NO.5:TCGTTGAAGATGCCTCTGCCGACAG;

[0053] SEQ ID NO.6:ACAATTTTTAGGCCCATACCTCTCAAC.

[0054] Example 4: Transient conversion of the CsCSE2 overexpression vector pLGNe-CsCSE2

[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-CsCSE2 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-CsCSE2 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-CsCSE2-1, pLGNe-CsCSE2-2, pLGNe-CsCSE2-3). 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-CsCSE2-F (SEQ ID NO.6) and RT-CsCSE2-R (SEQ ID NO.7). Two... -△△Ct The relative expression levels of the CsCSE2 gene in the experimental and control groups of Late Jin oranges were calculated as follows: the control group sample was defined as the reference factor, i.e., its CsCSE2 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 CsCSE2 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:GAATTTCTGGGGCGACATGC;

[0061] SEQ ID NO. 8: TGCGTAAAGAGCTTCCCGTT.

[0062] Example 5: Determination of the content of veseldin-2 and / or hesperidin in the fruits of the experimental and control groups in Example 4.

[0063] The contents of veselin-2 and / or hesperidin components in the pericarps 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 vesernin-2 and / or hesperidin in the peel of fruit transiently transformed with pLGNe-CsCSE2 increased by 26.75%–34.5% and 6.6%–7.3%, respectively, indicating that transient overexpression of the CsCSE2 gene can significantly increase the contents of vesernin-2 and / or hesperidin in the fruit of Wanjin orange.

[0064] Therefore, this invention is the first to discover a positive correlation between the expression level of the CsCSE2 gene and the content of vescenin-2 and / or hesperidin in citrus. The higher the expression level of the CsCSE2 gene, the higher the content of vescenin-2 and / or hesperidin in the citrus peel. In the experiment, citrus transiently transformed with the CsCSE2 gene overexpression vector showed up to 34.5% higher content than citrus transformed with the empty vector. The CsCSE2 gene can be used as a candidate gene for breeding new citrus varieties with high vescenin-2 and / or hesperidin content, which is of great significance for using genetic engineering methods to cultivate new functional 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. Use of CsCSE2 gene for increasing content of citrus Vincin-2 and / or hesperidin in citrus, characterized in that: The CDS sequence of the CsCSE2 gene is shown as SEQ ID NO. 1, and the protein sequence encoded by the CsCSE2 gene is shown as SEQ ID NO.

2.

2. The use of the CsCSE2 gene according to claim 1 for increasing the content of citrus Vincin-2 and / or hesperidin, characterized in that: Overexpressing the CsCSE2 gene in citrus increases the content of vitamin-2 and / or hesperidin in the citrus peel.

3. The use of the CsCSE2 gene in improving the content of vitamin C-2 and / or hesperidin in citrus according to claim 1, characterized in that: The expression amount of the CsCSE2 gene is positively correlated with the content of vitamin-2 and / or hesperidin in the citrus peel.

4. Use of the CsCSE2 gene in the breeding of citrus varieties, characterized in that: The CDS sequence of the CsCSE2 gene is shown as SEQ ID NO. 1, and the protein sequence encoded by the CsCSE2 gene is shown as SEQ ID NO.

2.

5. A plant overexpression vector comprising the CsCSE2 gene, characterized by: The CDS sequence of the CsCSE2 gene is shown as SEQ ID NO. 1, and the protein sequence encoded by the CsCSE2 gene is shown as SEQ ID NO.

2.

6. The application of the plant overexpression vector containing the CsCSE2 gene in the breeding of citrus according to claim 5.

7. A method for breeding citrus fruits with increased content of vitamin E and / or hesperidin in the fruit peel, characterized in that: The CsCSE2 gene or the plant overexpression vector containing the CsCSE2 gene is introduced into a citrus plant to overexpress the CsCSE2 gene in the citrus plant, and a citrus plant with increased content of vitamin-2 and / or hesperidin is obtained through cultivation; The CDS sequence of the CsCSE2 gene is shown as SEQ ID NO. 1, and the protein sequence encoded by the CsCSE2 gene is shown as SEQ ID NO. 2.

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