Gene gbNAC8 for promoting DNA damage repair and flavonoid synthesis of ginkgo biloba and application thereof

Abstract

This invention discloses a gene, GbNAC8, that promotes DNA damage repair and flavonoid synthesis in Ginkgo biloba and its applications. The nucleotide sequence of the GbNAC8 gene is shown in SEQ ID NO.1. Through transcriptome analysis, gene cloning, and functional verification, this invention reveals that GbNAC8 participates in the DNA damage repair response process in Ginkgo biloba. Heterologous overexpression of GbNAC8 in Arabidopsis thaliana can reduce cell death and reactive oxygen species accumulation induced by ultraviolet radiation or DNA damage inducer treatment, thereby improving the plant's tolerance to DNA damage stress. Furthermore, GbNAC8 can regulate the total flavonoid content in Ginkgo biloba; overexpression of GbNAC8 increases the total flavonoid content in Ginkgo callus, while silencing GbNAC8 reduces the total flavonoid content in Ginkgo biloba leaves. This invention provides a new gene resource for cultivating Ginkgo biloba materials resistant to ultraviolet radiation and with high total flavonoid content.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and plant genetic engineering technology, specifically relating to a gene GbNAC8 that promotes DNA damage repair and flavonoid synthesis in Ginkgo biloba and its applications. Background Technology

[0002] Ginkgo is an important long-lived tree species and medicinal plant with strong environmental adaptability. Ginkgo leaves are rich in flavonoids and other active ingredients, possessing high medicinal and economic value. Plants are often affected by ultraviolet radiation, oxidative stress, and other environmental factors in the natural environment, leading to DNA damage, reactive oxygen species accumulation, and cell death. The ability to repair DNA damage is a crucial foundation for plants to maintain genome stability and adapt to adversity.

[0003] Currently, although some DNA damage repair-related genes have been reported in model plants such as Arabidopsis thaliana, rice, and poplar, there are still relatively few studies on functional genes involved in DNA damage repair and UV radiation resistance in Ginkgo biloba. As an ancient and long-lived tree species, Ginkgo biloba may have a strong DNA damage response and repair capacity, but the relevant molecular mechanisms are still unclear, and there is a lack of functional gene resources that can be used to improve the stress resistance and enhance the medicinal components of Ginkgo biloba.

[0004] NAC transcription factors are important transcriptional regulators unique to plants, participating in processes such as plant growth and development, stress response, and secondary metabolism regulation. However, the roles of Ginkgo NAC transcription factors in DNA damage repair, UV radiation resistance, and flavonoid synthesis have not been fully investigated. Flavonoids are not only important protective substances for plants against UV radiation and oxidative stress, but also one of the main medicinal active ingredients of Ginkgo. Therefore, identifying key genes that can simultaneously participate in the regulation of Ginkgo DNA damage repair response and total flavonoid accumulation is of great significance for stress-resistant breeding and enhancing the medicinal value of Ginkgo. Summary of the Invention

[0005] Objective: To address the shortcomings of existing technologies, this invention provides a gene, GbNAC8, that promotes DNA damage repair and flavonoid synthesis in Ginkgo biloba. Heterologous overexpression of this gene enhances the resistance to UV radiation and DNA damage stress in Arabidopsis thaliana, while transient silencing of this gene in Ginkgo biloba reduces its UV resistance. Furthermore, GbNAC8 can regulate the total flavonoid content in Ginkgo biloba; overexpression of GbNAC8 increases the total flavonoid content in Ginkgo callus, while silencing GbNAC8 decreases the total flavonoid content in Ginkgo biloba leaves. This invention provides a new gene resource for cultivating Ginkgo biloba materials with UV resistance and high total flavonoid content.

[0006] This invention also provides the application of the gene GbNAC8, which promotes DNA damage repair and flavonoid synthesis in Ginkgo biloba.

[0007] Technical solution: In order to achieve the above objectives, the present invention provides a gene GbNAC8 that promotes DNA damage repair and flavonoid synthesis in Ginkgo biloba, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The protein encoded by the gene GbNAC8 of this invention has the amino acid sequence shown in SEQ ID NO.2.

[0009] The biological material containing the gene GbNAC8 described in this invention includes a recombinant expression vector, a recombinant plasmid, a transgenic plant tissue, a transgenic callus or transgenic plant, and a host bacterium.

[0010] The application of the gene GbNAC8, the protein, or the biomaterial described in this invention in improving the DNA damage repair capacity of plants.

[0011] The application of the biomaterial in improving the plant's tolerance to DNA damage inducers and its resistance to DNA damage from ultraviolet radiation.

[0012] The application of the gene GbNAC8, the protein, or the biomaterial described in this invention in reducing the accumulation of reactive oxygen species in plants.

[0013] The biological material is either Arabidopsis thaliana or Ginkgo biloba.

[0014] The application of the gene GbNAC8, the protein, or the biomaterial described in this invention in increasing the total flavonoid content of Ginkgo biloba.

[0015] The application of the gene GbNAC8, the protein, or the biological material described in this invention in regulating the synthesis of ginkgo flavonoids.

[0016] Overexpression of the gene GbNAC8 increased the total flavonoid content of Ginkgo biloba, while silencing the gene GbNAC8 decreased the total flavonoid content.

[0017] Among them, overexpression of the gene GbNAC8 increased the total flavonoid content of Ginkgo callus, while silencing the gene GbNAC8 decreased the total flavonoid content of Ginkgo leaves.

[0018] The application of the gene GbNAC8, the protein, or the biological material described in this invention in cultivating Ginkgo biloba varieties resistant to ultraviolet radiation and / or with high flavonoid content.

[0019] The method for improving the resistance of plants to ultraviolet radiation according to the present invention includes increasing the expression level of the gene GbNAC8 in the target plant, or introducing the gene GbNAC8 into the target plant and causing it to be expressed.

[0020] The method for improving the DNA damage repair capacity of plants according to the present invention includes increasing the expression level of the gene GbNAC8 in the target plant, or introducing the gene GbNAC8 into the target plant and causing it to be expressed.

[0021] The method for increasing the total flavonoid content of Ginkgo biloba according to the present invention includes increasing the expression level of the gene GbNAC8 in Ginkgo biloba materials.

[0022] The ginkgo materials mentioned above are ginkgo plants, ginkgo leaves, ginkgo callus, ginkgo cells, or ginkgo tissue cultures.

[0023] The method for cultivating plant materials with resistance to ultraviolet radiation and / or high flavonoid content according to the present invention includes introducing the gene GbNAC8 into the target plant material and expressing it, and screening to obtain plant materials with improved resistance to ultraviolet radiation and improved flavonoid content.

[0024] The present invention relates to an expression vector containing the Ginkgo DNA damage repair gene GbNAC8.

[0025] Preferably, in the expression vector, the 5' end of the GbNAC8 gene is connected to a constitutive promoter CaMV 35S, and the 3' end is connected to a NOS terminator. The CaMV 35S promoter drives the expression of the GbNAC8 gene in plant cells, and the NOS terminator terminates the transcription of the GbNAC8 gene.

[0026] Furthermore, the expression vector is assembled with an NPTⅡ gene expression cassette, which serves as a screening marker for transgenic Ginkgo biloba, and kanamycin is used to screen for transgenic Ginkgo biloba.

[0027] Furthermore, the expression vector is assembled with LB (T-Border left) and RB (T-Border right) sequences, which promote the integration of the GbNAC8 gene expression framework and the selection marker gene NPTⅡ assembled therein into the Ginkgo chromosome.

[0028] Preferably, the primer pairs used to amplify the GbNAC8 gene to construct the overexpression vector PRI-101 are SEQ ID NO.3: gaccccgggggtaccggatccATGGTGGCTCAAGAGTGGCTAG and SEQ ID NO.4: tttacccatgaattcggatccTGTCCAATCTGGATTGTCCCTT. The primer pairs used to construct the virus-induced gene silencing vector pTRV2 are SEQ ID NO.6: gtgagtaaggttaccgaattcGGAGATGTTCGCTGGCACAA and SEQ ID NO.7: gagacgcgtgagctcggtaccGATCTCCTTTAGGAATTAATGCTTCC.

[0029] The host cell containing the Ginkgo DNA damage repair gene GbNAC8 or its expression vector described in this invention is generally Agrobacterium.

[0030] This invention relates to the application of the Ginkgo DNA damage repair gene GbNAC8, the protein, the expression vector, or the host bacterium in Ginkgo DNA damage repair. The Ginkgo DNA damage repair process is as follows: using Ginkgo and transgenic Arabidopsis as materials, the GbNAC8 gene is cloned and constructed into the overexpression vector PRI-101 to obtain a recombinant vector. This recombinant vector is then transformed into Agrobacterium, and the Agrobacterium resuspension is used to infect Arabidopsis. Driven by the CaMV35S promoter, GbNAC8 can be efficiently expressed in Arabidopsis, thereby improving its tolerance to DNA damage stress. Furthermore, a virus-induced gene silencing vector pTRV2 targeting GbNAC8 was constructed, and specific silencing of this gene was achieved through Agrobacterium-mediated injection, thereby verifying its DNA damage repair function in Ginkgo.

[0031] The application of the Ginkgo DNA damage repair gene GbNAC8, the protein, the expression vector, or the host bacterium described in this invention in the cultivation of high-resistance Ginkgo varieties and Ginkgo with high total flavonoid content.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0033] This invention marks the first time that GbNAC8 has been cloned from Ginkgo biloba. By transferring the GbNAC8 gene into Arabidopsis thaliana, overexpression of the GbNAC8 gene enhances the DNA damage tolerance of Arabidopsis. Furthermore, after specifically inhibiting this gene using virus-induced gene silencing technology, the silenced plants showed significantly higher levels of DNA damage after UV irradiation treatment compared to the control group. This indicates that GbNAC8 is a key gene for DNA damage repair in Ginkgo biloba, and therefore, GbNAC8 has significant application value in improving plant resistance to stress, especially UV irradiation.

[0034] This invention, through transcriptome analysis, gene cloning, and functional verification, reveals that GbNAC8 participates in the DNA damage repair response process in Ginkgo biloba. Heterologous overexpression of GbNAC8 in Arabidopsis thaliana reduces cell death and reactive oxygen species accumulation induced by UV irradiation or DNA damage inducer treatment, thereby improving the plant's tolerance to DNA damage stress. Transient silencing of GbNAC8 in Ginkgo biloba increases the degree of UV irradiation-induced DNA damage. Furthermore, GbNAC8 can regulate the total flavonoid content in Ginkgo biloba; overexpression of GbNAC8 increases the total flavonoid content in Ginkgo callus, while silencing GbNAC8 decreases the total flavonoid content in Ginkgo biloba leaves. This invention provides a new gene resource for cultivating Ginkgo biloba materials resistant to UV irradiation and with high total flavonoid content.

[0035] This invention, through the cloning and functional analysis of the GbNAC8 gene, not only provides a direct theoretical basis for enhancing the total flavonoid content of Ginkgo biloba using gene regulation technology, but also lays the foundation for its regulatory mechanism in the synthesis of total flavonoids in Ginkgo biloba. Furthermore, the established Ginkgo biloba-derived transient overexpression and gene silencing technology system overcomes the technical bottleneck of difficult genetic manipulation in Ginkgo biloba, providing an efficient research tool for gene function research in Ginkgo biloba and other difficult-to-transform forest trees. This invention not only fills a key gap in the DNA damage repair pathway of Ginkgo biloba, but also provides important gene resources for improving the UV resistance and total flavonoid content of Ginkgo biloba. Attached Figure Description

[0036] Figure 1 The images show the clone of GbNAC8 (A), bacterial culture detection (B), and sequence alignment (C).

[0037] Figure 2 The images show the phenotypic and chlorophyll fluorescence of Arabidopsis thaliana, Ginkgo biloba and poplar after UV treatment (A), and the maximum photosynthetic efficiency (Fv / Fm) of photosystem II of Arabidopsis thaliana, Ginkgo biloba and poplar under UV irradiation treatment (BD).

[0038] Figure 3 This is a phylogenetic tree analysis of GbNAC8 and NAC8 proteins from other species.

[0039] Figure 4The images show PI staining (A) of wild-type (Control) Arabidopsis thaliana and OE-GbNAC8 transgenic Arabidopsis thaliana after UV irradiation, and NBT staining (B) of wild-type (Control) Arabidopsis thaliana and OE-GbNAC8 transgenic Arabidopsis thaliana after UV irradiation.

[0040] Figure 5 The phenotype and root length statistics of wild-type (Control) Arabidopsis and OE-GbNAC8 transgenic Arabidopsis after treatment with the DNA damage inducer bleomycin (A), and NBT staining of wild-type (Control) Arabidopsis and OE-GbNAC8 transgenic Arabidopsis after bleomycin treatment (B).

[0041] Figure 6 The results show that the GbNAC8-pTRV2 silencing material was verified by fluorescence quantitative PCR (A), and the degree of DNA damage in GbNAC8-TRV2 Ginkgo seedlings and control Ginkgo seedlings after UV irradiation was detected by γ-H2AX immunofluorescence assay (B).

[0042] Figure 7 The results show the quantitative fluorescence detection of Ginkgo callus overexpressing GbNAC8 (A), the detection of total flavonoid content in Ginkgo callus overexpressing GbNAC8 (B), and the detection of total flavonoid content in TRV2-GbNAC8 silent leaves (C). Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0044] Example 1

[0045] Cloning the GbNAC8 gene

[0046] (1) Based on the Ginkgo genome and transcriptome data, the target DNA fragments used in the overexpression vectors (SEQ ID NO.3 and 4) and silencing vectors (SEQ ID NO.7 and 8) were amplified, respectively. The primers are shown below. The target gene was obtained by amplification using Ginkgo leaf cDNA template.

[0047]

[0048] (2) PCR amplification was performed using PrimeSTAR Max (Takara, Japan). The PCR system is as follows:

[0049]

[0050] Gently mix the above mixture, briefly centrifuge at low speed, and then place it in a standard PCR instrument. Set the following program:

[0051]

[0052] Electrophoresis: Remove the gene amplification product from the PCR instrument and spot an appropriate amount of the product onto a 1% agarose gel using an electrophoresis apparatus. After about 20 minutes, remove the gel and observe it using an imaging system to obtain the target fragment. Figure 1 A).

[0053] Example 2

[0054] Construction of GbNAC8 gene overexpression vector PRI-101 and silencing vector pTRV2

[0055] (1) In this experiment, TaKaRa QuickCut restriction enzyme (TaKaRa, Japan) was used to perform enzyme digestion reactions on pRI 101-AN vector, pTRV2 (TaKaRa, Japan) and GbNAC8 gene. The specific reaction system is as follows:

[0056]

[0057] After mixing all solutions in the system, the mixture was briefly centrifuged and incubated in a 37°C water bath for 30 min to stop the enzyme digestion reaction. The enzyme digestion bands were observed by agarose gel electrophoresis. Subsequently, the target gene and vector fragments were digested and recovered for subsequent vector ligation reactions.

[0058] (2) Following the instructions for TaKaRa T4 DNA Ligase (TaKaRa, Japan), the expression vector recovered after the enzyme digestion reaction was ligated with the target DNA fragment product recovered from the purification gel. The system is as follows:

[0059]

[0060] The solutions in the system were mixed in a microtube and reacted in a metal bath at 16°C for 5-6 h.

[0061] (4) Escherichia coli transformation

[0062] Following the instructions for TaKaRa E.coli DH5α Competent Cells, the ligated product was mixed with competent cells, and after ice bath, heat shock, and recovery, an appropriate amount was spread on LB plates, the plates were inverted, and cultured overnight at 37°C.

[0063] (5) Screening and sequencing analysis of positive clones

[0064] Single colonies were selected from the screening culture plate and inoculated into LB liquid medium, and incubated overnight at 37°C and 200 rpm. The recombinant transformants were then directly detected by PCR using the overnight culture as a template.

[0065] Reaction system:

[0066]

[0067] Reaction procedure:

[0068]

[0069] Clones that tested positive for bacterial culture PCR ( Figure 1 B) The gene was sent to Sangon Biotech (Shanghai) for sequencing and identification. The gene sequence of the overexpression vector PRI-101, as shown in SEQ ID NO. 1, is the full-length CDS sequence of the gene GbNAC8, which will be used for subsequent experiments. The amino acid sequence of the expressed protein is shown in SEQ ID NO. 2 and compared with the Ginkgo genome sequence. Figure 1 C). The pTRV2 gene sequence, which is linked to the viral gene silencing vector, is shown in SEQ ID NO. 5 and is the specific 289bp CDS sequence of the target gene GbNAC8.

[0070] SEQ ID NO. 1

[0071] ATGGTGGCTCAAGAGTGGCTAGGGTTACCTGCTGGGGTGAAATTTGACCCCTCTGATCAAGAACTGTTGGGACATTTAGAATCAAAATTAGGGGTTGGAAATGTCAAACCTCATCCTTTAATTGATGAGTTTATTCCCACGCTTGATAAAGATGATGGGATCTGCTATGTCCATCCTGAAAAGCTGCCAGGTGTCAAACAAGATGGAAGCATAGTTCATTTTTTCCATAGAACAGCTAAGGCCTACACCACAGGCACTCGTAAGCGACGAAAGATCCATTGCGAGGGTGATCAGAGTGGGGGAGATGTTCGCTGGCACAAGACAGGCAAAACAAGGCCTGTTAATGAGAATGGGATTCAGAAGGGTTGGAAAAAGATCATGGTTCTTTATATGAGCACTGGAAAGGGTGAAAAGCCTGAGAAAACAAACTGGGTGATGCATCAGTACCATCTGGGTATTGAGGAGGACGAGAAGGACCGCGAATTTGTGGTTTCAAAAGTATTCTACCAACAGCAATGTAAGCAACTTGACAAACATGAAGATGAAGTACCTCAAGAGACTGGGGAAGCATTAATTCCTAAAGGAGATCCAGTCACTCCTAAAACAAATACTCCTGAACTACCTCGTCCAGGAAAGCGGCATGCTGATCTTGATACCATC

[0072] CAAGAAGAACAATTGGACAGTATCAACAATCTTGGCCAGGTTGTAGCTGTTGACGCAGCAAGCCATCAACCTGTCGGCAACTATGAACCAGAATGGGAGGGACCAAATGATCACGCAAATGCAAATTTCGATGACTTCCACATGGCTTCTCAGTTTAGGGATGATTCTGGACAACTTGTAGATGGATTATTTTTTTGTGATGAGAAACTTCAAAGCTCGTCT TCGAAAGAGAACTCTGGAACAGAAAAGCTCATTCCAGGTCTTTCCGAATACGGTCGCATTGGAGTTGAAGAACTCGAGGAAAATAGGGATGGATATGAAGCATCTGCTGCTCTTGCAAACATTGAACTTGATACACCTCCTGATCTAAATGCTATTGAGTTGCAATTTAGCTCACAGGAAAGCGTATCAGCTTGGGTAAGGGACAATCCAGATTGGACATGA

[0073] SEQ ID NO. 2

[0074] MVAQEWLGLPAGVKFDPSDQELLGHLESKLGVGNVKPHPLIDEFIPTLDKDDGICYVHPEKLPGVKQDGSIVHFFHRTAKAYTTGTRKRRKIHCEGDQSGGDVRWHKTGKTRPVNENGIQKGWKKIMVLYMSTGKGEKPEKTNWVMHQYHLGIEEDEKDREFVVSKVFYQQQCKQLDKHEDEV PQETGEALIPKGDPVTPKTNTPELPRPGKRHADLDTIQEEQLDSINNLGQVVAVDAASHQPVGNYEPEWEGPNDHANANFDDFHMASQFRDDSGQLVDGLFFCDEKLQSSSSKENSGTEKLIPGLSEYGRIGVEELEENRDGYEASAALANIELDTPPDLNAIELQFSSQESVSAWVRDNPDWT

[0075] SEQ ID NO. 5

[0076] GGAGATGTTCGCTGGCACAAGACAGGCAAAACAAGGCCTGTTAATGAGAATGGGATTCAGAAGGGTTGGAAAAAGATCATGGTTCTTTATATGAGCACTGGAAAGGGTGAAAAGCCTGAGAAAACAAACTGGGTGATGCATCAG TACCATCTGGGTATTGAGGAGGACGAGAAGGACCGCGAATTTGTGGTTTCAAAAGTATTCTACCAACAGCAATGTAAGCAACTTGACAAACATGAAGATGAAGTACCTCAAGAGACTGGGGAAGCATTAATTCCTAAAGGAGATC

[0077] PCR detection confirmed the successful construction of the GbNAC8 overexpression vector and the virus-induced gene silencing vector pTRV2. They were named p35S::GbNAC8 and pTRV2-GbNAC8, respectively. The overexpression vector was initiated by the CaMV 35S strong promoter with a NOS terminator added at the end. The overexpression vector contained a kanamycin (KanR) prokaryotic resistance selection tag, a hygromycin (HYG) plant resistance selection tag, and GFP and GUS tags initiated by the 35S strong promoter. The pTRV2 silencing vector was similarly initiated by the CaMV 35S strong promoter, containing approximately 300 bp sequences specific to the RNA2 and GbNAC8 genes from the TRV strain ppk20, with a NOS terminator attached to the 3' end. The VIGS silencing vector contained the kanamycin resistance gene and could be used for bacterial screening. Both expression vectors are equipped with RB and LB T-DNA transfer repeat sequences, which can be used for Agrobacterium-mediated plant genetic transformation or transient expression experiments.

[0078] (3) Transformation of Agrobacterium

[0079] Following the instructions for transformation with Shanghai Weidi GV3101 (Agrobacterium), the constructed p35S::GbNAC8 expression vector plasmid or pTRV2-GbNAC8 silencing vector was mixed with competent cells and subjected to the following steps: static incubation on ice for 5 min, flash freezing in liquid nitrogen for 1 min, water bath at 37℃ for 3 min, and rapid ice bath for 2 min. Then, 700 μL of liquid LB medium was added and cultured with shaking for 2 h. After centrifugation at 5000 rpm for 1 min, 100 μL of the supernatant was collected, gently mixed, and spread onto LB agar plates containing kanamycin and rifampin antibiotics. The plates were incubated upside down at 28℃ for 2-3 days. Single clones were picked from the plates, and an appropriate amount of LB liquid medium was added. The plates were incubated at 28℃ and 220 rpm for 48 h. PCR detection of positive clones yielded Agrobacterium containing either the p35S::GbNAC8 vector or the pTRV2-GbNAC8 vector.

[0080] Example 3

[0081] Analysis of UV tolerance in Arabidopsis thaliana, Ginkgo biloba, and Populus tomentosa seedlings

[0082] Use a UV lamp (specification 105 μW / cm) 2 Pre-treated Arabidopsis thaliana, Ginkgo biloba, and wild-type poplar seedlings were simultaneously subjected to UV-B irradiation in a climate chamber. Their growth was observed over 24 hours, and samples were taken for chlorophyll fluorometry imaging. Results showed that after 6 hours of treatment, the aboveground parts of Arabidopsis thaliana plants began to exhibit obvious damage phenotypes, and the maximum photosynthetic efficiency (Fv / Fm) of photosystem II in both Arabidopsis thaliana and poplar decreased significantly. After 24 hours of treatment, the leaves of Arabidopsis thaliana and poplar showed obvious damage, with a significant decrease in Fv / Fm. In contrast, Ginkgo biloba plants did not show obvious stress phenotypes within 24 hours, with only a slight decrease in Fv / Fm, indicating that Ginkgo biloba exhibits stronger UV tolerance than Arabidopsis thaliana and poplar. Figure 2 ).

[0083] Example 4

[0084] Evolutionary analysis of GbNAC8 protein

[0085] NAC8 protein sequences from 34 other species were downloaded from Phytozome and Ensembl Plants. Conserved domains of the NAC8 protein were identified and analyzed using the NCBI Conserved Domain Database. The conserved domains of screened NAC8 were extracted using the TBtools Conserved Domain Sequences Extraction for Phylogenetic Tree plugin, and a phylogenetic tree was constructed using MEGA7. Furthermore, motif analysis was performed on the screened NAC8 sequences from multiple species using the MEME database. Phylogenetic analysis showed that the GbNAC8 gene protein is closely related to the NAC8 genes of species such as Norway spruce, *Gnaphalium affine*, *Bryum simonii*, and *Lysimachia nummularia*. The NAC8 genes in angiosperms are more distantly related to the GbNAC8 gene. Figure 3 This suggests that the GbNAC8 gene in Ginkgo biloba may differ functionally from that in other angiosperms.

[0086] Example 5

[0087] Genetic transformation of GbNAC8-PRI-101

[0088] 1. Arabidopsis transformation

[0089] (1) Agrobacterium containing the p35S::GbNAC8 vector obtained in Example 2 was plated on LB agar plates. After culturing, single Agrobacterium clones were picked from the LB agar plates and inoculated into 100 mL of LB liquid medium. The culture was carried out at 28°C for 24 h until the OD600 was 0.5-0.6.

[0090] (2) Place the bacterial culture into a centrifuge tube, centrifuge at 25°C, 4000 rpm for 10 min, and then remove the supernatant.

[0091] (3) Add resuspension solution (5 g sucrose dissolved in 100 mL ddH2O and 50 μL silwet L-77) to the centrifuge tube to resuspend the bottom cells, and let stand at room temperature for 2 h;

[0092] (4) Select healthy Arabidopsis thaliana plants in full bloom and immerse the entire inflorescence in the above-prepared Agrobacterium suspension multiple times for 30 seconds each time.

[0093] (5) After the inoculation is completed, the plants are placed in the dark in the incubator for 24 h, and then transferred to normal light conditions at 22℃ until the seeds mature. Mature seeds are collected for later screening of positive seedlings.

[0094] 2. Gene verification of transgenic materials

[0095] (1) Positive screening: Harvested mature seeds were sterilized in a clean bench (15% sodium hypochlorite for 1 min, 70% alcohol for 1 min, twice, followed by washing with sterile water 3 times). The sterilized seeds were evenly sown on MS solid medium plates containing kanamycin. After vernalization at 4℃ for 2 days, the selective medium was placed in a 22℃ environment under normal light conditions until the seeds germinated. The T1 seedlings that grew well on the selective medium were then transplanted into the soil for further growth.

[0096] (2) Positive Identification: When Arabidopsis T1 plants have grown to 5-6 true leaves, DNA verification is performed using the TPS method. TPS solution preparation: 50 mL Tris-HCl (1M, pH=8.0), 10 mL EDTA (0.5 M, pH=8.0), and 37.27 g KCl, diluted to 500 mL with ddH2O. The obtained Arabidopsis T1 leaf samples were rapidly ground into powder in liquid nitrogen and placed in a 2 mL centrifuge tube. 300 μL of the prepared TPS solution was added, and the tube was incubated in a 65℃ water bath for 30 min, inverting the tube several times every 10 min. After the water bath, the tube was centrifuged at 13000 rpm for 10 min, the supernatant was collected, and an equal volume of isoacetone was added. The mixture was then centrifuged again at 13000 rpm for 10 min, the supernatant was discarded, the precipitate was dried, and 20 μL of ddH2O was added. The extracted DNA was used as a template for PCR amplification of the target gene, and the results were detected by agarose gel electrophoresis. Finally, the positive lines were further screened until homozygous T3 generation transgenic line seeds (OE-GbNAC8) were obtained (for later functional verification).

[0097] 3. DNA damage treatment of transgenic Arabidopsis thaliana

[0098] (1) Wild-type and OE-GbNAC8 Arabidopsis thaliana seeds were sterilized and sown on MS solid medium. After 7 days of growth, they were subjected to ultraviolet irradiation treatment (105 μW / cm²). 2 12h. Samples were taken for PI and NBT staining. The number of dead cells in the roots of OE-GbNAC8 plants was significantly less than that in wild-type plants, meaning that the degree of cell damage in the roots of OE-GbNAC8 plants was significantly lower than that in wild-type plants. Figure 4 A). NBT staining results showed that OE-GbNAC8 plants accumulated less reactive oxygen species in their leaves compared to the wild type. Figure 4 B).

[0099] (2) Wild-type and OE-GbNAC8 Arabidopsis seeds were sterilized and sown on MS solid medium containing bleomycin (25 μg / mL). Phenotypic observation and NBT staining were performed after 7 days of growth. Results showed that under bleomycin treatment, the root length of OE-GbNAC8 transgenic plants was significantly longer than that of wild-type plants. These results indicate that overexpression of GbNAC8 plants can significantly alleviate bleomycin-induced DNA damage. Figure 5 A). Furthermore, NBT staining revealed that, compared to the control, GbNAC8 transgenic plants accumulated less reactive oxygen species in their leaves. Figure 5 B).

[0100] 4. Ginkgo callus transformation

[0101] (1) Agrobacterium containing the p35S::GbNAC8 vector obtained in Example 2 was plated on LB agar plates. After culturing, single Agrobacterium clones were picked from the LB agar plates and inoculated into 100 mL of LB liquid medium. The culture was carried out at 28°C for 24 h until the OD600 was 0.5-0.6.

[0102] (2) Place the bacterial culture into a sterilized centrifuge tube, centrifuge at 25°C, 4000 rpm for 10 min, and then remove the supernatant.

[0103] (3) Add sterile resuspension (100 mL MS liquid medium) to the centrifuge tube to resuspend the bottom cells and place at room temperature for 2 h;

[0104] (4) After disinfecting the ginkgo leaves, cut out the wound and select the tender tissue growing at the wound. Place the complete callus tissue block into the resuspension solution and let it stand for 30 minutes. Then, gently remove it with tweezers, place it on filter paper, absorb the surface bacterial solution, place it on the callus solid culture medium, and culture it in the dark for 2 days. After that, take it out, wrap it with aluminum foil, label it, and store it in a -80℃ refrigerator for subsequent experiments.

[0105] 5. Detection of gene expression and determination of total flavonoid content in transgenic materials

[0106] (1) Real-time quantitative PCR was used to detect the expression of exogenous genes at the RNA level. Primer Premier 5.0 software was used to design fluorescent quantitative primers for GbNAC8. The forward primer was 5'-AACCACTGACTAGCAGGCTTTCTTG-3', and the reverse primer was 5'-GCCGCATTGGAAGTGATGAATTGG'. The internal control (GbGAPDH) forward primer was 5'-ATCACGGGAGTCTTCAC-3', and the reverse primer was 5'-GACCTTCAACAATGCCAAAC'. The results showed that the expression level of GbNAC8 in the transgenic Ginkgo callus tissue obtained in step 4 was significantly increased (…). Figure 7 A).

[0107] (2) Determination of flavonoid content in transgenic materials

[0108] The total flavonoid content of Ginkgo biloba callus tissues from control (non-transgenic, with identical culture conditions) and transgenic Ginkgo biloba was determined using a plant flavonoid extraction kit (Suzhou Keming Biotechnology Co., Ltd., China). The results showed that the total flavonoid content in callus tissues overexpressing GbNAC8 was significantly increased. Figure 7 B). The above results indicate that, in addition to enhancing the plant's resistance to ultraviolet radiation, GbNAC8 also has the function of regulating flavonoid synthesis, and that overexpressing the GbNAC8 gene increases the total flavonoid content in Ginkgo biloba.

[0109] Example 6

[0110] Genetic transformation of the pTRV2-GbNAC8 gene

[0111] 1. Ginkgo leaf transformation

[0112] (1) Agrobacterium containing the pTRV2-GbNAC8 vector obtained in Example 2 was plated on LB agar plates. After incubation, single Agrobacterium colonies were picked from the LB agar plates and inoculated into 100 mL of LB liquid medium, and incubated at 28°C for 24 h until OD. 600 It is 0.5;

[0113] (2) Place the bacterial culture into a centrifuge tube, centrifuge at 25°C, 4000 rpm for 10 min, and then remove the supernatant.

[0114] (3) Add resuspension solution (100 mL MS liquid medium containing 100 μM acetylsyl syringone) to the centrifuge tube to resuspend the bottom cells and incubate at room temperature for 2 h;

[0115] (4) Use a sterile syringe (with removable needle) to draw up the bacterial solution and inject it into the back of the leaf until the bacterial solution submerges the entire leaf;

[0116] (5) The infected ginkgo leaves were cultured in the dark at 25°C for 1 day, and then under light for 2 days. One portion was treated with ultraviolet irradiation and then flash-frozen in liquid nitrogen and stored in an ultra-low temperature freezer for subsequent immunofluorescence detection. The other portion was directly flash-frozen in liquid nitrogen for subsequent determination of total flavonoid content.

[0117] 2. Gene expression detection in transgenic materials

[0118] (1) Real-time quantitative PCR was used to detect the expression of exogenous genes at the RNA level. Primer Premier 5.0 software was used to design fluorescent quantitative primers for GbNAC8. The forward primer was 5'-AACCACTGACTAGCAGGCTTTCTTG-3', and the reverse primer was 5'-GCCGCATTGGAAGTGATGAATTGG'. The internal control (GbGAPDH) forward primer was 5'-ATCACGGGAGTCTTCAC-3', and the reverse primer was 5'-GACCTTCAACAATGCCAAAC'. The results showed that the expression level of GbNAC8 in the transgenic Ginkgo biloba leaves obtained in step 1 was significantly downregulated. Figure 6 A).

[0119] 3. Immunofluorescence detection of transgenic materials

[0120] (1) Weigh out the ultraviolet light (specification 105 μW / cm). 2 0.1 g of control and GbNAC8-pTRV2 silent plant samples after 48h irradiation were flash-frozen in liquid nitrogen, 200 μL of 1×PBS was added, and the wounds were quickly cut with a blade. The samples were then placed on ice for 10 min and the suspended cells were collected.

[0121] (2) After fixing with fixative for 15 min, centrifuge at 800 g for 5 min to remove the fixative. Wash once with washing buffer, then resuspend the cell solution in a small amount of washing buffer and add it to a glass slide to make a smear. After drying thoroughly, wash three times with washing buffer for 5 min each time;

[0122] (3) Use an immunohistochemistry pen to draw circles on the slide and let it dry;

[0123] (4) After adding an appropriate amount of immunostaining blocking solution, incubate in a humidified chamber for 20 min;

[0124] (5) After removing the immunostaining blocking solution, add an appropriate amount of γ-H2AX rabbit monoclonal antibody and incubate overnight at 4°C;

[0125] (6) After removing the γ-H2AX rabbit monoclonal antibody, wash three times with washing buffer for 10 min each time. After the last wash, try to remove as much washing buffer as possible. Add an appropriate amount of anti-rabbit 488 and incubate at room temperature in a humidified chamber for 1 h.

[0126] (7) After removing the anti-rabbit 488, wash with washing solution 3 times, 10 min each time. After the last wash, try to remove as much washing solution as possible.

[0127] (8) Add an appropriate amount of nuclear staining solution (DAPI) and incubate at room temperature for 5 min. After staining, remove the nuclear staining solution and wash three times with washing solution for 5 min each time. After the last wash, try to remove as much washing solution as possible.

[0128] (9) Finally, add an appropriate amount of anti-fluorescence quenching mounting solution, cover with a coverslip, and observe and photograph under a laser confocal microscope. The results showed that the DNA damage degree of GbNAC8-pTRV2 silenced plants was significantly higher than that of the control. Figure 6 B). All the above results indicate that GbNAC8 plays an important role in the repair of DNA damage in Ginkgo biloba.

[0129] 4. Total flavonoid detection in genetically modified materials

[0130] The total flavonoid content of control and transgenic Ginkgo biloba leaves was determined using a plant flavonoid extraction kit (Suzhou Keming Biotechnology Co., Ltd., China). It was found that the total flavonoid content of transiently silenced GbNAC8 leaves was significantly reduced. Figure 7 C). The above results indicate that GbNAC8 not only enhances the UV resistance of Ginkgo biloba, but also regulates flavonoid synthesis.

Claims

1. A gene GbNAC8 that promotes DNA damage repair and flavonoid synthesis in Ginkgo biloba, characterized in that, The nucleotide sequence of the gene GbNAC8 is shown in SEQ ID NO.

1.

2. A protein encoded by the gene GbNAC8 as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A biomaterial containing the gene GbNAC8 as described in claim 1, characterized in that, The biological materials include recombinant expression vectors, recombinant plasmids, transgenic plant tissues, transgenic callus tissues or transgenic plants, and host bacteria.

4. The application of the gene GbNAC8 of claim 1, the protein of claim 2, or the biomaterial of claim 3 in improving the DNA damage repair capacity of plants.

5. The application according to claim 4, characterized in that, Application of the biomaterials in improving the plant's tolerance to DNA damage inducers and its resistance to DNA damage from ultraviolet radiation.

6. The application of the gene GbNAC8 of claim 1, the protein of claim 2, or the biomaterial of claim 3 in reducing the accumulation of reactive oxygen species in plants.

7. The application according to any one of claims 4-6, characterized in that, The biological materials are Arabidopsis thaliana or Ginkgo biloba.

8. The application of the gene GbNAC8 of claim 1, the protein of claim 2, or the biomaterial of claim 3 in increasing the total flavonoid content of Ginkgo biloba.

9. The application according to claim 8, characterized in that, Overexpression of the gene GbNAC8 increases the total flavonoid content of Ginkgo biloba, while silencing the gene GbNAC8 decreases the total flavonoid content of Ginkgo biloba.

10. The application of the gene GbNAC8 of claim 1, the protein of claim 2, or the biomaterial of claim 3 in cultivating Ginkgo biloba varieties resistant to ultraviolet radiation and / or with high flavonoid content.

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