Citrus fruit anthocyanin synthesis regulation gene CsHY5.1 and application thereof

Abstract

The invention discloses a regulatory gene CsHY5.1 for synthesizing anthocyanin in citrus fruits and application of the regulatory gene CsHY5.1. The sequence of the regulatory gene CsHY5.1 is shown as SEQ ID No.1. The expression level of the CsHY5.1 in the fruits is in remarkable positive correlation with anthocyanin accumulation, instantaneous overexpression is performed in the citrus fruits by means of plant genetic engineering, the accumulation level of the anthocyanin can be promoted, and a good application prospect is provided for improvement of the nutritional quality of the citrus fruits.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a gene CsHY5.1 that regulates anthocyanin synthesis in citrus fruits and its applications. Background Technology

[0002] Anthocyanins are a class of flavonoids commonly found in plants. They are formed by the combination of anthocyanins and glycosides, and can give plant flowers, fruits, stems, leaves, and roots a variety of colors, from red and purple to blue. The anthocyanin content in fruits not only affects their color but is also an important nutritional component; therefore, anthocyanin content is a crucial indicator of the commercial value of fruits.

[0003] blood orange( Citrus sinensis Blood oranges (L. Osbeck) exhibit a distinctive red color due to their high anthocyanin content and are the only citrus fruit species capable of accumulating anthocyanins. This accumulation is influenced by both genetic and environmental factors. In blood oranges, cyanidin-3-glucoside and cyanidin-3(6”-malonyl)-β-glucoside are the main anthocyanins, accounting for over 70% of the total anthocyanin content. Their accumulation exhibits significant spatiotemporal specificity. During fruit ripening (200–324 days after flowering), anthocyanins are first synthesized in the pulp vesicles and flower tips, subsequently diffusing throughout the pulp. Trace amounts of anthocyanins can be detected in the pulp 261 days after flowering, reaching a content of up to 19.28% by 324 days. mg / L. It has been found that transcription factors Ruby and MYBF1 are important regulatory genes in anthocyanin synthesis in blood oranges. The Ruby gene, unique to blood oranges, encodes the R2R3-MYB transcription factor, which activates its expression by inserting a transposon into the promoter of CsRuby1, thereby promoting anthocyanin synthesis. Low temperatures (such as in winter) and light exposure (especially UVB) can induce anthocyanin accumulation in blood oranges. Low temperatures induce anthocyanin accumulation by activating the Ruby gene, which regulates the expression of anthocyanin synthase genes (such as ANS and UFGT), while high temperatures inhibit anthocyanin stability, leading to anthocyanin degradation.

[0004] HY5, a basic leucine zipper (bZIP) transcription factor, is considered a pivotal element in light signal transduction. Existing research indicates that the light signaling network regulated by HY5 is complex. The stability of the HY5 protein is dynamically regulated by light conditions, exhibiting both light intensity dependence and spectral specificity. In the visible light region, it effectively inhibits nucleoplasmic transport and assembly of the COP1-SPA1 complex by activating its respective photoreceptor system. This inhibition reduces the activity of the E3 ubiquitin ligase complex, thereby slowing down the 26S proteasome-dependent degradation of the HY5 protein. In the invisible light region, UVB radiation (280-315 nm) triggers a signal transduction cascade through its specific receptor UVR8. Activated UVR8 interacts with the WRKY36 transcription factor, inducing its dissociation from the W-box element in the HY5 gene promoter region. This dissociation relieves the transcriptional repression effect of WRKY36 on the HY5 gene, promoting HY5 mRNA synthesis and protein accumulation.

[0005] Plant adaptation is finely regulated at the molecular level, including post-transcriptional regulation such as alternative splicing. Alternative gene splicing is a crucial regulatory mechanism by which plants respond to endogenous and environmental signals. In eukaryotes, alternative splicing plays a vital role in regulating gene function by generating multiple different mRNA transcripts from a single gene. This process increases the complexity of the transcriptome, with different transcriptional isoforms potentially encoding protein variants with varying functions, stability, or subcellular localizations. In plants, approximately 40-63% of genes containing multiple exons undergo alternative splicing. Alternative splicing plays a key role in various plant biological processes, such as development, diurnal rhythms, metabolism, hormonal signaling, and resistance to biotic and abiotic stresses. In-depth research into the mechanisms of alternative splicing will help enhance the expression and accumulation of beneficial chemical components in plants, thereby improving plant growth, yield, and biomass, and strengthening their stress resistance. Currently, extensive alternative splicing has been observed in the HY5 gene of citrus plants, but its role and mechanism in light-regulated anthocyanin synthesis remain unclear. Summary of the Invention

[0006] The purpose of this invention is to provide a gene CsHY5.1 that regulates anthocyanin synthesis in citrus fruits and its applications. CsHY5.1 The expression level of anthocyanins in fruits is significantly positively correlated with anthocyanin accumulation. Transient overexpression of anthocyanins in citrus fruits using plant genetic engineering can promote anthocyanin accumulation, providing a promising application for improving the nutritional quality of citrus fruits.

[0007] The technical solution adopted by this invention to solve its technical problem is: A gene CsHY5.1 that regulates anthocyanin synthesis in citrus fruits, the sequence of which is shown in SEQ ID No. 1.

[0008] The fruit of the Citrus genus mentioned is the blood orange.

[0009] A recombinant plasmid containing the CsHY5.1 gene, which regulates anthocyanin synthesis in citrus fruits.

[0010] A citrus CsHY5.1 protein, encoded by the CsHY5.1 gene, which regulates anthocyanin synthesis in the fruits of the Citrus genus.

[0011] The application of the CsHY5.1 gene, which regulates anthocyanin synthesis in citrus fruits, in regulating anthocyanin synthesis in citrus fruits.

[0012] A method for increasing anthocyanin content in citrus fruits involves injecting Agrobacterium containing the CsHY5.1 gene recombinant vector into the fruit for overexpression 165 days after flowering. After injection, the fruit is bagged with a blue or white translucent bag, thereby significantly increasing the anthocyanin content in the fruit.

[0013] The sequence of the CsHY5.1 gene is shown in SEQ ID No. 1.

[0014] The beneficial effects of this invention are: this invention is the first to clone a regulator of anthocyanins from citrus fruits. CsHY5.1 The gene was confirmed by real-time quantitative PCR. CsHY5.1 The expression level in fruits is significantly positively correlated with anthocyanin accumulation. Transient overexpression in blood orange and strawberry fruits using plant genetic engineering can promote anthocyanin accumulation, providing a promising application for improving the nutritional quality of citrus fruits. Attached Figure Description

[0015] Figure 1 Citrus CsHY5.1 A full-length CDS clone of the gene.

[0016] Figure 2 The graph shows the relative expression level of the CsHY5.1 gene transiently expressed in 'Molo' blood oranges and the phenotypic changes in the fruit.

[0017] Figure 3 A graph showing the change in anthocyanin content after transient expression of the CsHY5.1 gene in 'Molo' blood oranges.

[0018] Figure 4 The graph shows the relative expression level of the CsHY5.1 gene transiently expressed in the 'Yellow Wonder 5AF7' strawberry and the changes in fruit phenotype.

[0019] Figure 5 The image shows the color difference changes in the 'Yellow Wonder 5AF7' strawberry after transient expression of the CsHY5.1 gene.

[0020] Figure 6 The graph shows the change in anthocyanin content after transient expression of the CsHY5.1 gene in the 'Yellow Wonder 5AF7' strawberry variety.

[0021] Figure 7 The graph shows the relative expression level of the CsHY5.1 gene transiently silenced in 'Molo' blood orange and the resulting changes in fruit phenotype.

[0022] Figure 8 A graph showing the change in anthocyanin content after transient silencing of the CsHY5.1 gene in 'Molo' blood oranges.

[0023] Figure 9 A graph showing the color difference changes after transiently silencing the CsHY5.1 gene in 'Moro' blood orange.

[0024] Figure 10 An expression diagram of anthocyanin metabolism-related genes after transient silencing of the CsHY5.1 gene in 'Molo' blood orange.

[0025] Figure 11 This is a map of the pNm-X-GFP vector. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0027] The reagents used in the examples mainly include molecular biology experimental reagents, consumables, and kits, all of which are commercially available. Specifically, these include: RNAprep Pure Plant Plus Kit (Tiangen Biotech, DP441); SanPA gel extraction kit (Sangon Biotech, B518131-0100); PrimeScript™ RT reagent Kit with gDNAEraser (Takara, Baori Biotech, Beijing), PrimeSTAR® Max DNA Polymerase (Takara, R045A), and T-Vector pMD™19 (Simple). Primer synthesis and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd. Unless otherwise specified, the methods provided in the examples of this invention are conventional methods.

[0028] Example 1 CsHY5.1 Cloning of gene cDNA sequences (1) Extraction of total RNA from citrus fruits 'Moro' blood orange fruit (provided by the Citrus Research Institute of Southwest University) was thoroughly ground into powder in liquid nitrogen for total RNA extraction. Total RNA was extracted according to the instructions of the RNAprep Pure Plant Total RNA Extraction Kit (DP441) manufactured by TIANGEN. The obtained RNA products were quality assessed using an Agilent 2100 bioanalyzer, focusing on concentration and purity parameters.

[0029] A reverse transcription system was constructed using the Takara PrimeScript™ RT kit (containing gDNA Eraser, RR047A). The RNA concentration of each sample was first standardized to 200 ng / μL, and then cDNA first strand was synthesized using it as a template.

[0030] (2) CsHY5.1 Full-length cloning of genes PCR amplification was performed using PrimeSTAR® Max DNA Polymerase (Takara). The procedure is as follows: Using the sweet orange genome (Citrus sinensis v3.0, https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_018105775.1 / )... CsHY5 Primers were designed based on the sequence information, and PCR amplification was performed using 'Molo' blood orange cDNA as a template.

[0031] Amplification system: PrimeSTAR® Max DNA polymerase 12.5 μL, CsHY5.1-F 0.5 μL, CsHY5.1-R 0.5 μL, cDNA template 2 μL, ddH2O to 25 μL. Upstream specific primer CsHY5.1-F: 5'-ATGCAAGAACAAGCAACGAG-3' (SEQ ID No.2), downstream specific primer CsHY5.1-R: 5'-TTAAAAGGACCCATCGGCAT-3' (SEQ ID No.3), amplification program: (1) 98 ℃, 3 min; (2) 98 ℃, 10 sec; (3) 60 ℃, 10 sec; (4) 72 ℃, 5 sec / kb; (5) 72 ℃, 5 min; 2-4 steps, 38 cycles.

[0032] After PCR amplification products were separated by agarose gel electrophoresis, gel bands containing the target DNA fragments were precisely excised under UV light. Subsequently, the DNA fragments were purified and recovered according to the instructions of the SanPrep DNA Gel Extraction Kit (B518131, Sangon Biotech) to ensure high-purity, structurally intact DNA products for subsequent experiments. Following this, 1% agarose gel electrophoresis was performed. Figure 1 The DNA was recovered according to the instructions of the SanPrep Column DNA Gel Recovery Kit (B518131, Sangon Biotech). The recovered PCR product was ligated into the pMD19-T vector, transformed into E. coli competent cells, and single clones were picked and sent for sequencing. CsHY5.1 The full-length sequence is shown in SEQ ID No. 1.

[0033] Example 2 CsHY5.1 Gene expression vector pNm- CsHY5.1- GFP and transient silencing vector pTRV2- CsHY5.1 Construction The pNm-X-GFP vector was provided by the Citrus Research Institute of Southwest University (plasmid map can be found here). Figure 11 The pNm-X-GFP vector plasmid and its purified PCR product were subjected to double digestion with KpnI and XbaI; simultaneously, the pTRV2 vector plasmid and its purified PCR product were subjected to double digestion with XbaI and KpnI. After digestion, the products were recovered, purified, and stored at -20℃ for later use.

[0034] The fragments recovered from double enzyme digestion were directionally ligated into the linearized vectors pNm-X-GFP and pTRV2, and introduced into *E. coli* DH5α competent cells via heat shock. Recombinant plasmids that were verified by sequencing were extracted and stored at -20°C for later use. The recombinant plasmids were then introduced into *Agrobacterium* GV3101 competent cells via heat shock. After single-cell growth, PCR was performed for positive identification. Following PCR in the bacterial culture, agarose gel electrophoresis was performed to confirm the amplification of the target band. Finally, positive bacterial cultures were sequenced. Successful sequencing results indicate successful construction of the expression vector pNm-X-GFP. CsHY5.1 -GFP and pTRV2- CsHY5.1 .

[0035] Example 3 Containing pNm- CsHY5.1- GFP Agrobacterium was transferred into blood orange fruit Take pNm- CsHY5.1 The specific steps for transforming the GFP expression plasmid with Agrobacterium are as follows: 1. Absorb and store pNm- CsHY5.1Add 3 µL of -GFP plasmid to 50 µL of Agrobacterium competent cells, mix well, and incubate on ice for 30 min.

[0036] 2. After ice bath, quick freeze with liquid nitrogen for 1 min, then thaw in a 37℃ water bath for 3 min.

[0037] 3. Add 1 mL of YEP liquid medium without any antibiotics and incubate at 28°C and 220 rpm for 3 h until the bacterial culture becomes turbid.

[0038] 4. Centrifuge at 8000 rpm for 1 min to collect bacterial cells, discard the supernatant, and keep 100 µL for plating on YEP solid medium containing antibiotics (50 mg / L rifampin, 50 mg / L gentamicin, 50 mg / L kanamycin) and incubate for 3 days.

[0039] 5. The transformed Agrobacterium colonies were identified by PCR. Positive single colonies were shaken and incubated with glycerol at -80℃.

[0040] Contains pTRV2- CsHY5.1 and pNm- CsRuby1 The preparation method for -GFP using Agrobacterium is the same as above. pTRV2- CsHY5.1 Upstream specific primer TRV2-HY5.1-F: 5'-AAGGTTACCGAATTCTCTAGAGTATGGAGAGCGATGAAGAGATCA-3' (SEQ ID No. 4), downstream specific primer TRV2-HY5.1-R: 5'-CGTGAGCTCGGTACCGGATCCGTGTCTCAGCATCTGATTCTCATTTT-3' (SEQ ID No. 5). pNm- CsRuby1 -GFP upstream specific primer CsRuby1-F: 5'-GCCATGGAGGCCAGTGAATTCATGGCGGATTCCTTAGGAGTTC-3' (SEQ ID No. 6), downstream specific primer CsRuby1-R: 5'-ACGATTCATCTGCAGCTCGAGTTACTTTGCATTGAGAAGATCCCA-3' (SEQ ID No. 7).

[0041] The material used for transient transformation was the 'Moro' blood orange variety. Transient expression was performed 165 days after flowering. The constructed pNm- CsHY5.1 -GFP, pTRV2- CsHY5.1The vector, *Agrobacterium*, invaded the pulp of blood oranges. Four injection points were evenly selected at the equatorial region of the blood orange fruit, with each vector being injected repeatedly into 45 fruits. After injection, the fruits were bagged using either blue or white translucent bags, with 45 fruits in each type of bag. Unbagged fruits served as a control. The fruits were harvested after 7 days, and the color difference was measured. The blood oranges were then peeled, the pulp was cut into small pieces, immediately frozen in liquid nitrogen, and stored at -80°C for later use.

[0042] Three to four days before transient expression, Agrobacterium tumefaciens bacterial suspension was plated on YEP solid medium containing antibiotics (50 mg / L rifampin, 50 mg / L gentamicin, 50 mg / L kanamycin) for activation. After spot development, a single colony was picked and inoculated into 5 mL of YEP liquid medium containing antibiotics (50 mg / L rifampin, 50 mg / L gentamicin, 50 mg / L kanamycin) and cultured overnight. The next day, 2 mL of bacterial suspension was added to 20 mL of liquid medium (MgCl2 20 mg / L, MES buffer 100 mg / L, acetylsuccione 5 mg / L, YEP containing antibiotics (50 mg / L rifampin, 50 mg / L gentamicin, 50 mg / L kanamycin) and cultured overnight. After centrifugation at 6000 rpm for 10 min, the culture was transferred to MS + MgCl2 + MES + As liquid medium (MgCl2 20 mg / L, MES buffer 100 mg / L, acetylsuccione 5 mg / L, ... (mg / L, remaining MS liquid medium) resuspended to OD 600 =1.0, let stand for 3 hours before use.

[0043] Gene expression levels detected using real-time quantitative PCR showed that transient overexpression was significantly higher than that of the control group injected with empty vector (EV). CsHY5.1 In the fruit, the expression level of the target gene was significantly upregulated ( p <0.05), the expression fold under white light transmission was 1.8 times, and the expression fold under blue light transmission was 3.3 times. Figure 2 This proves that the instantaneous transformation fruit was successfully obtained.

[0044] Subsequently, CsHY5.1 The total anthocyanin content of fruits that showed successful transient expression was determined. The results showed that under white transmitted light treatment, the anthocyanin content was significantly higher. CsHY5.1 The anthocyanin content in the -OE group was significantly higher than that in the control group, increasing by 42.7%. Simultaneously, under blue light transmission treatment, the anthocyanin content was significantly higher than that under white light transmission treatment. Figure 3 ).

[0045] Example 4: Containing pNm- CsHY5.1- GFP Agrobacterium was transferred into strawberry fruit Using diploid forest strawberry 'Yellow Wonder 5AF7' (provided by the Institute of Modern Agriculture, Peking University) as the test material, transient expression was performed at the green-ripe stage. The constructed pNm- CsHY5.1 The -GFP vector was injected into strawberry fruits via Agrobacterium tumefaciens, along the vascular bundles at the pedicel. 100 μL of bacterial solution was injected into each fruit, and each vector was injected repeatedly into 15 fruits. After injection, the fruits were kept in the dark for 24 h, and then the culture conditions were adjusted to 16 h of light exposure, a light intensity of 20,000 lx, a temperature of 25 °C, and an air humidity of 70%. Fruits were harvested after 5 days, color differences were measured, and fruit phenotypes were photographed and recorded. The pulp was then cut into small pieces, rapidly frozen in liquid nitrogen, and stored in an ultra-low temperature freezer at -80 °C for later use. The preparation method for the transient expression bacterial solution was the same as in Example 3.

[0046] qPCR results showed that CsHY5.1 expression increased by 12.2-fold, indicating successful transformation. Figure 4 Transient overexpression of CsHY5.1 failed to produce red fruit in the strawberry, but after co-injection with CsRuby1 bacterial solution, the strawberries transiently overexpressing CsHY5.1 showed a distinct red color. Figure 4 The CIRG value of strawberries injected with a mixture of CsHY5.1 and CsRuby1 bacterial cultures (at a volume ratio of 1:1) was also significantly higher than that of the control group. Figure 5 Further analysis of anthocyanin content in transgenic fruits revealed anthocyanin accumulation in strawberry fruits injected with a mixture of empty vector and CsRuby1 bacterial suspension, but the accumulation effect was not as good as that in fruits injected with a mixture of CsHY5.1 and CsRuby1 bacterial suspensions. Further determination of total anthocyanin content in transiently expressed strawberry fruit samples showed that strawberries injected with a mixture of CsHY5.1 and CsRuby1 bacterial suspensions had the highest anthocyanin content, significantly higher than the control group. p <0.05%, which is 120.8% higher than the control group ( Figure 6 ).

[0047] Example 5 CsHY5.1 Gene-mediated blue light transmission treatment positively regulates anthocyanin synthesis To further clarify CsHY5.1 To investigate the regulatory function of genes in anthocyanin synthesis in blood orange fruit, this study used the virus-induced gene silencing (VIGS) technology system to silence the CsHY5 gene.

[0048] Virus-induced gene silencing (VIGS) technology involves constructing a viral vector carrying a specific fragment of a target gene. After introduction into a host plant, this vector efficiently and specifically degrades endogenous homologous mRNA, thereby achieving rapid inhibition of gene expression. Using cDNA as a template, the upstream specific primer CsHY5-F: 5'-GTATGGAGAGCGATGAAGAG-3' (SEQ ID No. 8) and the downstream specific primer CsHY5-R: 5'-GTGTCTCAGCATCTGATTCTC-3' (SEQ ID No. 9) were used to amplify... CsHY5 A 181 bp specific interference fragment of the gene was amplified, and the amplified product was cloned into the pTRV2 vector. After sequencing verification, pTRV2- was obtained. CsHY5 The recombinant vector was introduced into Agrobacterium tumefaciens GV3101 competent cells via electroporation.

[0049] Real-time quantitative PCR results showed that in fruits treated with VIGS technology, the transcription level of the target gene in the CsHY5 gene silencing group (TRV2-HY5) was significantly lower than that in the empty vector control group (EV). p <0.05, proving that the genetically modified fruit was successfully obtained ( Figure 7 Trait testing revealed that under white light treatment, the anthocyanin content in the TRV2-HY5 group significantly decreased to 5.5 μg / gFW, a 28.1% decrease compared to the control group. However, under blue light treatment, the anthocyanin content in the TRV2-HY5 group significantly increased, a 16.2% decrease compared to the control group. Figure 8 These results indicate that transient silencing of CsHY5 can lead to a reduction in anthocyanin accumulation in blood oranges, while blue light can partially restore the reduction in anthocyanin accumulation caused by CsHY5 silencing.

[0050] After measuring the color difference index of 'Molo' fruit under various treatments, it was found that the blue light transmission treatment significantly increased the CIRG value of the control group fruit, but the CIRG value of the TRV2-HY5 group was not significantly different from that of the white-bagged fruit. However, the a* value of the TRV2-HY5 peel showed a significant difference from the control group under both white and blue bagging conditions. p <0.05), compared to the control group, TRV2-HY5 significantly reduced the a* value of peel color difference ( Figure 9 ).

[0051] This study used qRT-PCR to analyze anthocyanin synthesis-related genes in the peel of 'Molo' blood oranges treated with transient white and blue light. The results showed significant differences in the relative expression levels of some genes under white and blue light treatments. In the control group injected with the empty vector, the expression level of Ruby1 was significantly increased under blue light treatment compared to white light treatment, consistent with the fact that blue light promotes anthocyanin accumulation in blood oranges. Silencing HY5 significantly reduced Ruby1 expression in both white and blue light-treated blood orange peels, indicating that HY5 can positively regulate Ruby1 expression. The expression level of phenylalanine ammonia-lyase (PAL) was slightly increased in the empty vector treated with blue light, while its expression was significantly reduced under both light treatments after silencing HY5. Cinnamyl-4-hydroxylase C4H, 4-coumarate-CoA ligase 4CL, and chalcone isomerase CHI showed no significant difference in expression levels between the blue-light-transmitted and white-light-transmitted empty vectors. Furthermore, C4H expression remained unchanged even with HY5 silencing. Chalcone synthase CHS also showed no significant difference in expression levels between the blue-light-transmitted and white-light-transmitted empty vectors; however, CHS expression was significantly reduced with HY5 silencing. Anthocyanin synthase ANS exhibited similar expression patterns to PAL and CHS; however, ANS expression was slightly reduced in the peel of the white-light-transmitted bag after HY5 silencing. Overall, TRV2-HY5 treatment significantly inhibited the expression of PAL, CHS, and ANS, particularly in the PAL and CHS genes, suggesting that overexpression of the CsHY5 gene may affect anthocyanin synthesis by promoting the expression of these anthocyanin synthesis-related genes. Figure 10 ).

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A citrus fruit anthocyanin synthesis regulating gene CsHY5.1, characterized in that, The sequence of the CsHY5.1 gene is shown in SEQ ID No.

1.

2. The Citrus fruit anthocyanin synthesis regulating gene CsHY5.1 according to claim 1, characterized in that, The citrus fruit is blood orange.

3. A recombinant plasmid, characterized in that, The citrus fruit anthocyanin synthesis regulation gene CsHY5.1 comprises the CsHY5.1 gene shown in claim 1.

4. A citrus CsHY5.1 protein, characterized in that, The citrus fruit anthocyanin synthesis regulation gene CsHY5.1 is encoded by the CsHY5.1 gene shown in claim 1.

5. Application of the citrus fruit anthocyanin synthesis regulation gene CsHY5.1 shown in claim 1 in regulating anthocyanin synthesis of citrus fruit.

6. A method of increasing anthocyanin content in a citrus fruit, characterized by, At 165 days after flowering of the citrus plant, Agrobacterium containing the recombinant vector of the CsHY5.1 gene is injected into the fruit for overexpression, and after injection, the fruit is bagged with blue or white light-transmitting bags, so as to significantly improve the anthocyanin content in the fruit.

7. The method of claim 6, wherein, The sequence of the CsHY5.1 gene is shown in SEQ ID No.

1.

8. The method of claim 6, wherein, The citrus fruit is blood orange.

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