Application of CsCYP75B1 gene in breeding of new citrus varieties

Abstract

The application discloses application of a CsCYP75B1 gene in breeding of new citrus varieties, belongs to the technical field of genetic engineering, and particularly relates to application of the CsCYP75B1 gene in breeding of new citrus varieties, wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1. The application improves the content of naringin, hesperidin and / or jaceosid II in citrus pericarp by overexpressing the CsCYP75B1 gene, provides the CsCYP75B1 gene related to synthesis of naringin, hesperidin and / or jaceosid II, and contributes to breeding of functional new citrus varieties by using a genetic engineering method, accelerates a breeding process, and reduces a breeding workload.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to application of a CsCYP75B1 gene in breeding of new citrus varieties. BACKGROUND

[0002] Citrus is one of the fruit species with large cultivation area and high yield in China. Citrus fruits contain rich nutritional functional components and bioactive secondary metabolites, which not only endow them with unique flavor and health value, but also have significant effects on oxidation resistance, inflammation resistance and cancer resistance. It is known that the substances with important effects such as anti-inflammatory, antioxidant and anti-tumor in citrus include phenolic acids, flavonoids, carotenoids, essential oils, limonin and synephrine.

[0003] Rutin, hesperidin and melilotoside belong to flavanone glycosides in flavonoids, and have significant biological activities, including: 1) plant stress resistance, which can resist the invasion of pathogenic bacteria, fungi and pests; 2) antioxidant and light protection, which can scavenge intracellular reactive oxygen species (ROS) and excessive free radicals and reduce oxidative damage; 3) anti-inflammatory, which shows anti-inflammatory activity by inhibiting pro-inflammatory cytokines and reducing leukocyte infiltration; 4) cardiovascular protection, studies have shown 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, studies have shown that the intake of naringin and hesperidin in diet can reduce the risk of breast cancer, lung cancer, colon cancer, prostate cancer and pancreatic cancer; 6) metabolic regulation and anti-obesity: regulating the balance of intestinal flora, inhibiting the growth of pathogenic bacteria, promoting the proliferation of probiotics such as bifidobacteria, and improving the intestinal barrier function.

[0004] Rutin, hesperidin and melilotoside are widely distributed in various vegetables and fruits and are used in food processing to reduce the use of synthetic chemicals, improve human health, and excavate the key enzymes of their biosynthetic pathways and clarify the molecular mechanisms of their functions, which can provide a theoretical basis for functional citrus molecular breeding with high rutin, hesperidin and melilotoside content, and have important significance for improving the comprehensive utilization rate of citrus. SUMMARY

[0005] To solve the above technical problems, the application provides application of a CsCYP75B1 gene in breeding of new citrus varieties, which improves the content of rutin, hesperidin and / or melilotoside in citrus pericarp by overexpressing the CsCYP75B1 gene, provides the CsCYP75B1 gene related to synthesis of rutin, hesperidin and / or melilotoside, and contributes to the breeding of functional new citrus varieties by using genetic engineering method, accelerates the breeding process and reduces the workload of breeding.

[0006] In order to achieve the above-mentioned purpose, the application provides application of the CsCYP75B1 gene in breeding of new citrus varieties, wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1.

[0007] Preferably, the CsCYP75B1 gene is overexpressed to increase contents of naringin, hesperidin and / or jaceosid in the citrus peel, so that the new citrus variety with high contents of naringin, hesperidin and / or jaceosid is obtained.

[0008] The application further provides an overexpression vector containing the CsCYP75B1 gene, wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1.

[0009] The application further provides a recombinant strain containing the CsCYP75B1 gene, wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1.

[0010] The application further provides application of the overexpression vector or the recombinant strain in breeding of new citrus varieties, wherein the CsCYP75B1 gene is overexpressed to increase contents of naringin, hesperidin and / or jaceosid in the citrus peel, so that the new citrus variety with high contents of naringin, hesperidin and / or jaceosid is obtained.

[0011] The application further provides a breeding method of the new citrus variety with high contents of naringin, hesperidin and / or jaceosid, comprising the following steps: amplifying the CDS sequence of the CsCYP75B1 gene, constructing an overexpression vector of the CsCYP75B1 gene, transforming Agrobacterium, infecting the citrus fruit, and obtaining the new citrus variety; wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1.

[0012] The application further provides application of the CsCYP75B1 gene in breeding of the new citrus variety with high content of naringin, wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1; the CsCYP75B1 gene is overexpressed to increase the content of naringin in the citrus peel, so that the new citrus variety with high content of naringin is obtained.

[0013] The application further provides application of the CsCYP75B1 gene in breeding of the new citrus variety with high content of hesperidin, wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1; the CsCYP75B1 gene is overexpressed to increase the content of hesperidin in the citrus peel, so that the new citrus variety with high content of hesperidin is obtained.

[0014] The application also provides application of the CsCYP75B1 gene in breeding of a new citrus variety with high content of erioglaucine, wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1; the content of erioglaucine in the citrus peel is increased by overexpressing the CsCYP75B1 gene, and the new citrus variety with high content of erioglaucine is obtained.

[0015] The application also provides application of the CsCYP75B1 gene in increasing contents of naringin, hesperidin and erioglaucine in the citrus peel, wherein the CDS sequence of the CsCYP75B1 gene is shown as SEQ ID NO. 1; the contents of naringin, hesperidin and erioglaucine in the citrus peel are increased by overexpressing the CsCYP75B1 gene.

[0016] Compared with the prior art, the application has the following advantages and technical effects:

[0017] The application provides application of the CsCYP75B1 gene in breeding of a new citrus variety, and the specific technical effects are as follows:

[0018] (1) The application first finds that the expression amount of the CsCYP75B1 gene is positively correlated with the contents of naringin, hesperidin and / or erioglaucine in the citrus, the higher the expression amount of the CsCYP75B1 gene, the higher the contents of naringin, hesperidin and / or erioglaucine in the citrus peel, and the citrus with the CsCYP75B1 gene overexpression vector in the experiment is 37.41% higher than the citrus with the empty vector;

[0019] (2) The CsCYP75B1 gene can be used as a candidate gene for breeding of a new citrus variety with high contents of naringin, hesperidin and / or erioglaucine, and has important significance for cultivating functional new citrus varieties by using a genetic engineering method, accelerating the breeding process, reducing the breeding workload and improving the comprehensive utilization rate of the citrus. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only show some embodiments of the application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0021] Figure 1 Figure 1 is a structural diagram of the CsCYP75B1 gene of the late-jin orange, wherein A is the chromosomal localization of the CsCYP75B1 gene, bp represents base, B is the structure of the CsCYP75B1 gene, Exon1 and Exon2 represent exons, and C is the conserved domain of the CsCYP75B1 gene.

[0022] Figure 2 Figure 2 is a diagram of the agarose gel electrophoresis result of the amplification of the CDS sequence of the CsCYP75B1 gene, wherein M represents Marker, and CDS represents the CDS sequence of the CsCYP75B1 gene;

[0023] Figure 3 Figure 3 is a structural diagram of the overexpression vector pLGNe-CsCYP75B1 of the CsCYP75B1 gene, wherein GUS:NPTII represents the β-glucuronidase gene, P 35S represents the plant constitutive promoter derived from the cauliflower mosaic virus, T NOS represents the opine synthase gene terminator;

[0024] Figure 4 Figure 4 is a photograph of the latekumquat fruits of the experimental group and the control group on the day of transient transformation and after 5 days of culture, wherein A is a photograph of the latekumquat fruits of the experimental group and the control group on the day of transient transformation, B is a photograph of the latekumquat fruits of the experimental group and the control group after 5 days of culture, and C is a photograph of part of the samples of the latekumquat fruits of the experimental group and the control group after 5 days of culture for total RNA extraction, wherein pLGNe represents the control group, and pLGNe-CsCYP75B1-1, pLGNe-CsCYP75B1-2 and pLGNe-CsCYP75B1-3 represent the experimental group;

[0025] Figure 5 Figure 5 is the expression amount of the CsCYP75B1 gene in the fruit peel of the injection area of the latekumquat of the experimental group and the control group after 5 days of culture, wherein pLGNe represents the control group, and pLGNe-CsCYP75B1-1, pLGNe-CsCYP75B1-2 and pLGNe-CsCYP75B1-3 represent the experimental group, and P<0.0001 represents that the data of the two groups have a significant difference;

[0026] Figure 6 Figure 6 is a diagram of the detection result of the content of naringin, hesperidin and jaceosidin in the fruit peel of the injection area of the latekumquat of the experimental group and the control group after 5 days of culture, wherein A is the detection result of the content of naringin, B is the detection result of the content of hesperidin, and C is the detection result of the content of jaceosidin, wherein pLGNe represents the control group, and pLGNe-CsCYP75B1-1, pLGNe-CsCYP75B1-2 and pLGNe-CsCYP75B1-3 represent the experimental group, and P<0.0001 represents that the data of the two groups have a significant difference;

[0027] Figure 7 Figure 7 is a structural formula of naringin, hesperidin and jaceosidin, wherein A is naringin, B is hesperidin, and C is jaceosidin. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but rather as a description of certain aspects, features and embodiments of the present application.

[0029] The source of the material used in the present application: late Jin orange is derived from the late-maturing Jin orange elite line selected and obtained by the Citrus Research Institute of Chinese Academy of Agricultural Sciences, and the variety approval number is YuShen citrus 2011001; the overexpression vector pLGNe is purchased from Baosai Biology, and the Agrobacterium tumefaciens EHA105 is purchased from Weidi Biology.

[0030] Example 1

[0031] I. Bioinformatics analysis of CsCYP75B1 gene

[0032] The structure of the CsCYP75B1 gene of late Jin orange is as shown in Figure 1 The structure of the CsCYP75B1 gene of late Jin orange is as shown in

[0033]

[0034] II. Cloning of CsCYP75B1 gene CDS sequence.

[0035] 1. RNA extraction and cDNA synthesis:

[0036] Total RNA was extracted from the leaves of Citrus sinensis cv. Valencia by using the plant total RNA extraction kit (Aidley, CAT: RN09). The quality of the obtained RNA was verified by agarose gel electrophoresis, and the concentration of the obtained RNA was determined by using a concentration meter. Then, cDNA was synthesized by using the reverse transcription kit PrimeScript RT Master Mix (TaKaRa, CAT: RR036A) according to the attached instructions.

[0037] 2. Amplification of CsCYP75B1 gene CDS sequence:

[0038] The primers OE-CsCYP75B1-F (SEQ ID NO. 2), OE-CsCYP75B1-R (SEQ ID NO. 3) and high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, CAT: R045Q) were used to amplify the obtained cDNA of Citrus sinensis cv. Valencia as a template, and the amplification system was prepared according to the instructions attached to the high-fidelity enzyme PrimeSTAR Max DNA Polymerase. The PCR amplification program was as follows: 98°C, 5 min; 98°C, 30 s, 56°C, 30 s, 72°C, 1.5 min, 35 cycles; 72°C, 10 min. The CsCYP75B1 gene CDS sequence was amplified, and the fragment length was 1560 bp (CDS sequence 1551 bp-terminator 3 bp + 12 bp of enzyme cutting sites before and after a total of 12 bp).

[0039] The nucleotide sequence of primer OE-CsCYP75B1-F is SEQ ID NO. 2: GGTACCATGTCTACTTTACCACTACTGATACTG.

[0040] The nucleotide sequence of primer OE-CsCYP75B1-R is SEQ ID NO. 3: GAATTCAGCTTGATAAGCATTGGGCG.

[0041] The results of agarose gel electrophoresis are shown in Figure 1. Figure 2As shown, the amplified fragment size results are as expected. Under UV light, the agarose gel block containing the target fragment was cut with a clean blade, and the DNA fragment was recovered using a kit (BioFlux, CAT: BSC02M1). Part of the recovered product was sent to the company for sequencing. The sequencing results were analyzed by comparison, and it was determined that the obtained DNA fragment was the CDS sequence of the CsCYP75B1 gene of the late Jin orange (SEQ ID NO. 1).

[0042] III. Construction of CsCYP75B1 gene overexpression vector and transformation of Agrobacterium tumefaciens EHA105.

[0043] 1. Construction of CsCYP75B1 gene overexpression vector:

[0044] The obtained recovered DNA fragment, i.e., the CDS sequence of the CsCYP75B1 gene of the late Jin orange, and the overexpression vector pLGNe were double-digested with restriction endonucleases Kpn I and EcoR I (ThermoFisher) and then gel-recovered, and connected at 16°C for 12h. The enzyme digestion system and reaction conditions were prepared according to the attached instructions. The connection used T4 DNA Ligase kit (Promega, CAT: M1801), and the connection system and reaction conditions were prepared according to the attached instructions of T4 DNA Ligase kit.

[0045] The obtained connection product was transformed into Escherichia coli DH5α, and the transformation method used the method recorded in the attached instructions of Escherichia coli DH5α (purchased from Weidi Biology). The plasmid extraction kit (Omega, CAT: D6942) was used to extract the plasmid of the positive clone, and the CsCYP75B1 gene overexpression vector pLGNe-CsCYP75B1 was obtained.

[0046] As shown in Figure 3 , it is the structure of the CsCYP75B1 gene overexpression vector pLGNe-CsCYP75B1.

[0047] 2. Transformation of CsCYP75B1 gene overexpression vector into Agrobacterium tumefaciens EHA105:

[0048] The obtained overexpression vector pLGNe-CsCYP75B1 was introduced into Agrobacterium tumefaciens EHA105 by heat shock method, and the specific steps were as follows: 2 mL centrifuge tube containing frozen Agrobacterium tumefaciens EHA105 (50 μL) was thawed on ice; 2 μL overexpression vector plasmid was added to the competent cells, and the mixture was mixed by blowing and then placed on ice for 5 min, frozen in liquid nitrogen for 5 min, incubated at 37°C for 5 min, and placed on ice for 5 min. Then 800 μL LB liquid medium was added to the 2 mL centrifuge tube, mixed by blowing with a pipette, and cultured at 260 r / min, 28°C for 2 h. After the time, the bacterial solution was centrifuged at 6000 r / min for 1 min, the supernatant was discarded, and the bacterial body was resuspended with 50 μL LB liquid medium. After resuspension, it was spread on LB solid medium containing 50 mg / L kanamycin, and cultured at 28°C for 2 days. After the bacterial colonies grew, the colonies were picked and PCR verified using primers ID-CsCYP75B1-F (SEQ ID NO. 4), primers ID-CsCYP75B1-R (SEQ ID NO. 5) and high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, CAT: R045Q). The amplification system was prepared according to the instructions of high-fidelity enzyme PrimeSTAR Max DNA Polymerase, and the PCR amplification conditions were as follows: 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.

[0049] The nucleotide sequence of primer ID-CsCYP75B1-F is SEQ ID NO. 4: TCGTTGAAGATGCCTCTGCCGACAG.

[0050] The nucleotide sequence of primer ID-CsCYP75B1-R is SEQ ID NO. 5: AGCTTGATAAGCATTGGGCG.

[0051] The PCR amplification product was subjected to agarose gel electrophoresis, and the colony with correct band size was the positive clone containing the overexpression vector pLGNe-CsCYP75B1.

[0052] Four, the overexpression vector pLGNe-CsCYP75B1 of CsCYP75B1 gene was transiently transformed.

[0053] 1. Agrobacterium tumefaciens EHA105 infection:

[0054] Select the late Jin orange fruit with consistent growth state, in the clean bench, with volume concentration of 75% ethanol solution disinfection for standby; respectively, 500 μL containing pLGNe and pLGNe-CsCYP75B1 plasmid of Agrobacterium tumefaciens EHA105 bacteria liquid is added to 50 mL liquid LB medium (containing 50 mg / L kanamycin), at 28°C, 200 r / min culture to OD 600 =0.5. Centrifugal precipitation, using 1 / 2MS liquid medium to resuspend Agrobacterium tumefaciens EHA105, then randomly select four injection points on the diagonal of the equatorial surface of the sterilized late Jin orange fruit and mark, use 1 mL syringe to inject Agrobacterium tumefaciens EHA105 containing pLGNe-CsCYP75B1 plasmid resuspension into the late Jin orange peel, 1 mL per area, recorded as experimental group. The same method is used, and the late Jin orange injected with equal amount of Agrobacterium tumefaciens EHA105 containing pLGNe plasmid resuspension is used as control group. The experimental group and the control group are repeated three times, and the late Jin orange fruit injected with Agrobacterium tumefaciens EHA105 resuspension is placed in a 28°C incubator for dark culture for 5d.

[0055] As Figure 4 shown, the photos of the experimental group and the control group of late Jin orange fruit transient transformation on the same day and after 5d culture, Figure 4 A is the photo of the control group (pLGNe) and the experimental group (pLGNe-CsCYP75B1-1, pLGNe-CsCYP75B1-2 and pLGNe-CsCYP75B1-3) of late Jin orange fruit on the same day of transient transformation, Figure 4 B is the photo of the control group (pLGNe) and the experimental group (pLGNe-CsCYP75B1-1, pLGNe-CsCYP75B1-2 and pLGNe-CsCYP75B1-3) of late Jin orange fruit after 5d culture, Figure 4 C is the photo of part of the sample when extracting total RNA.

[0056] 2, qRT-PCR analysis of transiently transformed late Jin orange fruit:

[0057] The total RNA of the injection area of the experimental group and the control group of late Jin orange fruit cultured in a 28°C incubator for 5d in the dark was extracted (Aidley, CAT No: RN09), and cDNA was synthesized using reverse transcription kit PrimeScript RT Master Mix (TaKaRa, CAT: RR036A). The expression amount of the target gene was detected by qRT-PCR. The detection primer is RT-CsCYP75B1-F (SEQ ID NO. 6) and RT-CsCYP75B1-R (SEQ ID NO. 7).

[0058] qRT-PCR reaction conditions: 95℃ 3min, 94℃ 10s; 56℃ 10s, 72℃ 10s, 40 cycles; 72℃ 10min.

[0059] Nucleotide sequence of primer RT-CsCYP75B1-F SEQ ID NO. 6: ACGTTCGCCAGGAAGAGATG.

[0060] Nucleotide sequence of primer RT-CsCYP75B1-R SEQ ID NO. 7: GGCATTCACAACGCACAAGT.

[0061] The relative expression of CsCYP75B1 gene in the experimental group and the control group was calculated by the method of 2-ΔΔCt. , and the relative expression of the experimental group sample was calculated as the relative expression of the reference factor gene.

[0062] The results are shown in Table 1, Table 2 and Table 3. Figure 5 The expression of CsCYP75B1 gene in the experimental group (pLGNe-CsCYP75B1-1, pLGNe-CsCYP75B1-2 and pLGNe-CsCYP75B1-3) was significantly higher than that in the control group (pLGNe), and the highest was more than 30 times of the control.

[0063] V. Determination of hesperidin, hesperidin and melilotoside content in fruits of the experimental group and the control group.

[0064] The contents of hesperidin, hesperidin and melilotoside in the fruit peels of the experimental group and the control group after 5d culture in 28℃ incubator were determined by UPLC-MS.

[0065] The results are shown in Table 4, Table 5 and Table 6. Figure 6 Table 4, Table 5 and Table 6. Figure 6 Table 4, Table 5 and Table 6. Figure 6 Compared with the control group of pLGNe after 5d culture, the contents of hesperidin, hesperidin and melilotoside in the fruit peels of pLGNe-CsCYP75B1 after 5d culture were increased by 36.05%~36.92%, 6.59%~6.69% and 35.97%~37.41% respectively, indicating that overexpression of CsCYP75B1 gene could significantly increase the contents of hesperidin, hesperidin and melilotoside in the fruits of late Jin orange, as shown in Table 4, Table 5 and Table 6. Figure 7 Table 4, Table 5 and Table 6. Figure 7 Table 4, Table 5 and Table 6. Figure 7 Table 4, Table 5 and Table 6.

[0066] ​In conclusion, the application firstly finds that the expression amount of CsCYP75B1 gene is positively correlated with the contents of naringin, hesperidin and eriocitrin in citrus, the higher the expression amount of CsCYP75B1 gene is, the higher the contents of naringin, hesperidin and eriocitrin in citrus are, and the citrus with CsCYP75B1 gene overexpression vector in the experiment is 37.41% higher than the citrus with empty vector; the CsCYP75B1 gene can be used as a candidate gene for breeding new citrus varieties with high naringin, hesperidin and eriocitrin contents, and has important significance for cultivating functional new citrus varieties by using genetic engineering method, speeding up the breeding process, reducing the workload of breeding and improving the comprehensive utilization rate of citrus.

[0067] The above-described embodiments are only used to describe the preferred modes of the application, and do not limit the scope of the application, and various modifications and improvements of the technical solutions of the application made by those skilled in the art without departing from the design spirit of the application shall fall within the protection scope of the application defined by the claims.

Claims

1. The application of the CsCYP75B1 gene in the breeding of new citrus varieties, characterized by, The CDS sequence of the CsCYP75B1 gene is shown in SEQ ID NO.

1. By overexpressing the CsCYP75B1 gene, the content of rutin, hesperidin and / or lemon balm glycoside in citrus peel is increased, resulting in a new citrus variety with high content of rutin, hesperidin and / or lemon balm glycoside.

2. The application of an overexpression vector containing the CsCYP75B1 gene in the breeding of new citrus varieties, characterized in that, The CDS sequence of the CsCYP75B1 gene is shown in SEQ ID NO.

1. The overexpression vector increases the content of rutin, hesperidin and / or lemon balm in citrus peel by overexpressing the CsCYP75B1 gene, thereby obtaining a new citrus variety with high content of rutin, hesperidin and / or lemon balm.

3. The application of a recombinant strain containing the CsCYP75B1 gene in the breeding of new citrus varieties, characterized in that, The CDS sequence of the CsCYP75B1 gene is shown in SEQ ID NO.

1. The recombinant strain increases the content of rutin, hesperidin and / or lemon balm glycoside in citrus peel by overexpressing the CsCYP75B1 gene, thereby obtaining a new citrus variety with high content of rutin, hesperidin and / or lemon balm glycoside. The recombinant strain was prepared by Agrobacterium tumefaciens EHA105.

4. A method for breeding a new citrus variety with high content of naringin, hesperidin, and / or lemon balm glycoside, characterized in that, Includes the following steps: The CDS sequence of the CsCYP75B1 gene was amplified, an overexpression vector of the CsCYP75B1 gene was constructed, Agrobacterium was transformed, and citrus fruits were infected to obtain a new citrus variety. The CDS sequence of the CsCYP75B1 gene is shown in SEQ ID NO.1.

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