Brassica rapa BoaWRKY8 gene and application thereof

Abstract

This invention belongs to the field of biotechnology, specifically relating to the BoaWRKY8 gene in Chinese kale and its applications. This invention discloses a gene that regulates resistance to Botrytis cinerea (gray mold) in Chinese kale. BoaWRKY8 The gene has the nucleotide sequence shown in SEQ ID No: 1. This invention also provides the use of the above-mentioned BoaWRKY8 gene from Chinese kale: BoaWRKY8 It positively regulates the expression of genes involved in the synthesis of indoleglucosinolates. BoaWRKY8 Gene overexpression promotes the synthesis of total indole-glucosinolates in Chinese kale. BoaWRKY8 By promoting the synthesis and accumulation of indoleglucosinolates, the plant's resistance to Botrytis cinerea is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the effect of the BoaWRKY8 gene in Chinese kale on increasing indoleglucosinolate content and its resistance to Botrytis cinerea (…). Botrytis cinerea Applications in resistance. Background Technology

[0002] Brassica vegetables are the most widely cultivated, highest-yielding, and most consumed vegetable group in China and even globally, playing a crucial role in ensuring vegetable supplies in certain regions. Chinese kale (Kale) Brassica oleracea var. alboglabra Brassica oleracea, a type of vegetable belonging to the Brassica genus, is widely loved by consumers for its unique flavor and nutritional value. However, during its production process, it is often infected by various pathogens, seriously affecting its yield, quality, and food safety. Discovering and utilizing the inherent disease-resistant genes of Brassica oleracea crops has significant theoretical value and application prospects.

[0003] Botrytis cinerea ( Botrytis cinerea Botrytis cinerea (Gray Mold) is a typical necrotrophic pathogenic fungus belonging to the kingdom Fungi, phylum Ascomycota, class Rhizoctonia, order Hyphomycetes, family Sclerotioraceae, and genus Botrytis. It is widely distributed and has diverse hosts, causing gray mold in various crops and resulting in severe pre- and post-harvest losses. Under suitable conditions, the disease spreads rapidly, leading to rot of leaves, stems, and flowers, forming water-soaked lesions and producing a gray mold layer, severely reducing the commercial value of crops. For cruciferous vegetables such as kale, the damage caused by gray mold is increasingly prominent, becoming one of the important biological stress factors threatening the stable development of their industry.

[0004] Secondary metabolites of cruciferous plants, represented by indole-type glucosinolates, play a crucial role in antifungal activity. These compounds and their degradation products are closely related to the innate immunity and broad-spectrum antifungal activity of plants, and are an important material basis for Brassica plants to resist biotic stress. Chinese kale is rich in glucosinolates.

[0005] BoaWRKY8 is a WRKY transcription factor gene in Chinese kale (Brassica oleracea var. alboglabra, abbreviated Boa), belonging to the plant-specific WRKY family. Existing research indicates that genes in this family play important roles in plant stress responses and disease resistance regulation; for example, WRKY8 can enhance Arabidopsis resistance to viruses by regulating hormone signaling pathways. However, whether WRKY8 participates in regulating the synthesis of plant secondary metabolites (especially indoleglucosinolates), and how it affects antifungal activity through this metabolic pathway, has not yet been reported. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a gene that increases the content of indoleglucosinolates in Chinese kale and its resistance to Botrytis cinerea, and its uses.

[0007] To address the aforementioned technical problems, this invention provides a gene for regulating resistance to Botrytis cinerea in Chinese kale. BoaWRKY8 The gene has the nucleotide sequence shown in SEQ ID No: 1.

[0008] This invention also provides genes BoaWRKY8 The encoded protein has the amino acid sequence shown in SEQ ID No:2 (with a stop codon at the end).

[0009] The present invention also provides plasmids containing the above-mentioned genes and plant expression vectors containing the above-mentioned genes, including gene overexpression vector 35Spro::BoaWRKY8-GFP and gene silencing VIGS vector BoaWRKY8-PTY.

[0010] The present invention also provides a host cell, which is an Escherichia coli cell or an Agrobacterium cell.

[0011] This invention also provides the use of the above-mentioned BoaWRKY8 gene in Chinese kale: BoaWRKY8 It positively regulates the expression of genes involved in the synthesis of indoleglucosinolates.

[0012] An improvement to the use of the Chinese kale BoaWRKY8 gene of the present invention: BoaWRKY8 Gene overexpression promotes the synthesis of total indole glucosinolates in Chinese kale.

[0013] As a further improvement to the use of the Chinese kale BoaWRKY8 gene of the present invention: the indoleglucosinolate synthesis gene includes BoaCYP79B2.1, BoaCYP79B2.2, BoaCYP83B1, BoaCYP81F2.1.

[0014] As a further improvement to the use of the Chinese kale BoaWRKY8 gene of the present invention: BoaWRKY8 The content of indoleglucosinolates can be increased by promoting the expression of the indoleglucosinolate synthesis gene.

[0015] As a further improvement to the use of the Chinese kale BoaWRKY8 gene of the present invention: BoaWRKY8 By promoting the synthesis and accumulation of indoleglucosinolates, the plant's resistance to Botrytis cinerea is enhanced.

[0016] That is, the BoaWRKY8 gene was used to construct transgenic kale, which can improve resistance to Botrytis cinerea.

[0017] The transgenic Chinese kale obtained using the method of this invention significantly increases the content of indole glucosinolates and resistance to Botrytis cinerea in the plant. By measuring the content of indole glucosinolates, it was found that…BoaWRKY8 The gene plays a positive regulatory role in the accumulation of indole-like glucosinolates in Chinese kale. Through botrytis cinerea inoculation experiments, it was found that compared to wild-type WT, BoaWRKY8 The resistance of the gene overexpression material increased significantly, while BoaWRKY The resistance of the 8-gene silencing material was significantly reduced. This discovery lays an important technical and theoretical foundation for using genetic engineering to breed new Brassica vegetable varieties resistant to gray mold.

[0018] It should be noted that: BoaWRKY8 The gene is a key transcription factor associated with resistance in Chinese kale, belonging to class II WRKY transcription factors. However, whether and how this transcription factor directly regulates the biosynthetic pathway of indole-like glucosinolates, a key defense substance, in Brassica plants, thereby conferring resistance to Botrytis cinerea, was unknown prior to the date of this invention application.

[0019] Currently known genes involved in the positive regulation of indoleglucosinolates include the MYB family (MYB51, MYB34, MYB122), the MYC family (MYC2, MYC3, MYC4), and the WRKY family (WRKY33, WRKY20). However, these genes belong to different families or different subclasses of the same family as BoaWRKY8. Therefore, the aforementioned prior art cannot provide the present invention with the " BoaWRKY8 The technical inspiration from "the link between genes and total indole glucosinolates" is that genes are associated with total indole glucosinolates.

[0020] The research of this invention shows that the BoaWRKY8 transcription factor, a Botrytis cinerea resistance gene derived from Chinese kale, plays an important role in combating Botrytis cinerea by regulating the synthesis of indole glucosinolates.

[0021] In summary, this invention relates to the BoaWRKY8 gene in Chinese kale and its effects on increasing indoleglucosinolate content and inhibiting Botrytis cinerea (…). Botrytis cinerea Application in resistance. This invention is the first to construct a resistance-resistant variety of Chinese kale. BoaWRKY8 VIGS gene-silenced plants and transgenic kale materials overexpressing 35Spro::BoaWRKY8-GFP were developed and their functions were studied. The content of indole glucosinolates was determined, and botrytis cinerea inoculation experiments were conducted. BoaWRKY8 Gene regulation of indoleglucosinolates plays a positive regulatory role in the resistance of Chinese kale to gray mold. Attached Figure Description

[0022] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Figure 1 yes BoaWRKY8The content of total indole glucosinolates (IGS) in the leaves of wild-type Chinese kale (left panel) and gene-silenced lines (right panel); In the left image: WT represents wild-type Chinese kale, and OE-1 and OE-3 represent two BoaWRKY8 gene overexpression lines; In the right figure: PTY-EV represents the control line obtained from the empty PTY plasmid, and PTY-15 and PTY-19 represent the two gene-silenced lines with the lowest BoaWRKY8 expression levels obtained from the BoaWRKY8-PTY plasmid.

[0024] Figure 2 yes BoaWRKY8 Gene overexpression and gene silencing lines and the structural gene for indole glucosinolate synthesis in wild-type kale leaves BoaCYP79B2.1, BoaCYP79B2.2, BoaCYP83B1, BoaCYP81F2.1 The amount of expression.

[0025] Figure 3 yes BoaWRKY8 Gene overexpression and silencing lines versus wild-type Chinese kale resistance to Botrytis cinerea. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] I. Obtaining Chinese kale BoaWRKY8 Full-length gene sequence: The WRKY8 CDS sequence (XM_013764171.1) of *Brassica napus* TO1000, a species related to Chinese kale, was used as the... BoaWRKY8 The reference sequence for CDS sequence (SEQ ID No: 1) amplification was used. Primers for whole-genome amplification were designed using the NCBI primer design tool. cDNA from Chinese kale "JL27" leaves was used as a template (cDNA extraction was performed using standard techniques, such as CN104561025A). PrimerSTAR high-fidelity enzyme PCR amplification was performed. BoaWRKY8 Excerpt.

[0028] The primer sequences are: F: 5'-TCTAGACCCGGGATGTCTAATGAAACCAAAGAT-3'; R: 5'-AGTGGATCCTCAAGGTTCTTGCTTGAAG-3'.

[0029] The PCR amplification and ligation system was based on CN 112029775 A, namely: PCR amplification reaction system: 25 μl of 2x PrimerSTAR buffer, 5 μl of dNTP Mixture, 1 μl of PrimerSTAR DNA polymerase, 6 μl of ddH2O, 1 μl of cDNA, and 1 μl each of forward and reverse primers, totaling 40 μl. The PCR reaction program was: 94℃ pre-denaturation for 90 seconds; 94℃ denaturation for 30 seconds, 57℃ annealing for 45 seconds, 72℃ extension for 1 minute and 30 seconds, for 35 cycles; and a final extension at 72℃ for 5 minutes. The obtained PCR products were identified by 1% agarose gel electrophoresis, and the amplified bands were then analyzed using an Axygen DNA gel.

[0030] The reagent kit was recovered and purified, and the successfully amplified [products / products] were [absorbed / purified]. BoaWRKY8 The recombinant plasmid was constructed into the pEasy®-T&B Simple Cloning vector (TransGen), and then sent to Qingke Technology Co., Ltd. for sequencing confirmation.

[0031] The obtained genes BoaWRKY8 The nucleotide sequence is shown in SEQ ID No: 1; the amino acid sequence of the protein encoded by this gene is shown in SEQ ID No: 2.

[0032] II. Chinese kale BoaWRKY8 Construction of gene overexpression vectors: BoaWRKY8 The overexpression vector was constructed using pCambia1300-GFP (CAMBIA) as the final vector, resulting in the 35Spro:BoaWRKY8 vector with the CaMV35S recombinant overexpression promoter. The construction process can be found in CN 112029775 A, "Boa kabochalic acid BoWRKY33 gene and its applications".

[0033] Specifically, the BoaWRKY8 fragment amplified in the first step and the vector pCambia1300-GFP were... Sma I and BamHThe fragment was digested with enzyme I (37℃, 1 hour), and then ligated into the digested pCambia1300-GFP using T4 DNA ligase (Takara, Japan) at 16℃ for 14 hours to obtain the recombinant plasmid 35Spro:BoaWRKY8. After the reaction, the pCambia1300-BoaWRKY8-GFP plasmid was transformed into E. coli DH5α competent cells. Selection was performed using kanamycin-resistant plates, and colony PCR was used to select the pCambia1300-GFP recombinant plasmid carrying the target fragment (SEQ ID NO: 1), followed by sequencing identification. Sequencing results were analyzed using SnapGene software. Thus, the pCambia1300-BoaWRKY8-GFP vector with the CaMV35S recombinant overexpression promoter was obtained.

[0034] III. Construction of the BoaWRKY8 gene silencing vector in Chinese kale: Refer to CN 112029775 A "Kale" BoWRKY33 Genes and their applications.

[0035] Specifically as follows: BoaWRKY8 Search for a 40bp oligonucleotide sequence in SEQ ID NO: 1 that begins with NTAG, NTAA, or NTGA at the 5' end. Add its inverse complementary sequence to this sequence to form an 80bp oligonucleotide target fragment. The 80bp fragment is: 5'-GTAGTTAAAACCAAGAAGAAAGAGGAGAAGAAGCAAAGAGCTCTTT-GCTTCTTCTCCTCTTTCTTCTTGGTTTTTAACTAC-3'.

[0036] The target fragment was ligated with the PTY vector via enzyme digestion. SnaB The PTY plasmid was digested overnight at 37°C using restriction endonucleases, and a linear fragment of approximately 10045 bp was recovered by agarose gel electrophoresis. This fragment was then treated with alkaline phosphatase and purified. Simultaneously, the 80 bp target fragment was terminally phosphorylated using phosphokinase and purified. Subsequently, the treated vector fragment and the target fragment were ligated using T4 ligase, and the ligation product was transformed into *E. coli* DH5α competent cells. Screening was performed using ampicillin-resistant plates. Positive clones were picked and verified by colony PCR and sequencing. The verified positive clone (expected PCR fragment approximately 560 bp) was named the recombinant vector BoaWRKY8-PTY.

[0037] Four, BoaWRKY8 Construction of kale with overexpression and VIGS gene silencing The pCambia1300-BoaWRKY8-GFP vector plasmid was transformed into Agrobacterium GV3101 strain, and the plantlets were infected with cotyledons of Chinese kale. Callus induction, resistance-induced differentiation, and rooting culture were then performed to obtain tissue culture seedlings. Positive transgenic plants were verified using PCR and RT-PCR. BoaWRKY8 Seeds of the T2 generation of the line with the highest fold increase in expression were sown on a medium containing hygromycin (12 mg / L) for germination, thus obtaining... BoaWRKY8 Transgenic material BoaWRKY8-OE (OE-1, OE-3); BoaWRKY8 The construction of VIGS gene-silenced kale can be referenced in CN 112029775 A "Kale" BoWRKY33 Genes and their applications.

[0038] Chinese kale plants were cultured at 25℃, 60% relative humidity, and a 16-hour light / 8-hour dark light cycle. Leaf infection was performed at the three-leaf stage. Before infection, the plants were subjected to 16-24 hours of dark treatment and thoroughly watered. Approximately 3 μg of BoaWRKY8-PTY plasmid was used per leaf in the experimental group, while the control group used an equal amount of empty PTY plasmid. During the procedure, a small amount of green silicon carbide was sprinkled on the leaves, followed by the addition of the plasmid solution and gentle rubbing, then rinsing and drying. After inoculation, the plants were incubated in the dark for 12 hours, then returned to normal conditions. Two weeks later, RNA was extracted from the terminal new leaves. BoaWRKY8 Expression level analysis was performed using VIGS silent lines with gene expression levels reduced by more than 50%. BoaWRKY8 -PTY (PTY-15, PTY-19) are used for subsequent research.

[0039] five, BoaWRKY8 Analysis of indoleginoside content in kale overexpression and VIGS gene silencing For WT, BoaWRKY8 -OE, PTY-EV, BoaWRKY8 - PTY kale leaves were sampled for glucosinolate extraction and content analysis. The extraction and analysis process followed CN 115976057 A "Kale BoIAA30 Gene and Its Application".

[0040] The specific steps are as follows: High-performance liquid chromatography (HPLC) was used to determine the glucosinolate content. Three-week-old Chinese kale leaves were freeze-dried and ground into powder. 0.02 g of the powder was weighed and extracted with 2 ml of 90% methanol solution, then allowed to stand at room temperature for 1 hour. After centrifugation, the supernatant was collected and eluted with 1 ml of ddH2O to obtain desulfurized glucosinolate samples. The samples were filtered through a 0.45 μm filter before analysis. The analysis was performed using a Shimadzu HPLC system (Shimadzu, Japan) equipped with an SPD-M20A detector and a Hypersil C18 column (5 μm, 4.6 mm × 250 mm Elite Analytical Instruments Co., Ltd., Dalian, China). The mobile phase was chromatographic grade acetonitrile and ddH2O. The analytical procedure for glucosinolate content was as follows: total flow rate 1.0 mL / min; detection wavelength 226 nm; detection program: 0-5 min, 98.5% acetonitrile; 5-20 min, gradient elution from 98.5% acetonitrile to 80% acetonitrile; 20-30 min, 80% acetonitrile. Each injection was 20 μL. The glucosinolate content in the sample was calculated using O-Nitrophenyl-β-D-galactopyranoside (ONPG) as an internal standard.

[0041] The results are as follows Figure 1 As stated above. Figure 1 The results showed that the total indoleglucosinolate content was significantly increased in BoWRKY8-OE and significantly decreased in BoaWRKY8-PTY. According to... Figure 1 It can be concluded that BoWRKY8 overexpression promotes the accumulation of indoleglucosinolates.

[0042] six, BoaWRKY8 Study on the expression levels of indoleginoside gene in Chinese kale overexpression and VIGS gene silencing Leaves of WT, BoaWRKY8-OE, PTY-EV, and BoaWRKY8-PTY kale were sampled, RNA was extracted, and reverse transcribed into cDNA. Gene expression levels were detected using real-time quantitative PCR. RNA extraction and expression analysis followed CN112029775 A, "Kale". BoWRKY33 Genes and their applications.

[0043] Specifically, total RNA was extracted from Chinese kale leaves using the Trizol method. After grinding the sample in liquid nitrogen, Trizol extraction buffer was added, followed by chloroform extraction, isopropanol precipitation, and washing with 75% ethanol to obtain RNA precipitate. The precipitate was dissolved in DEPC water and its concentration was determined using Nanodrop. Subsequently, the RNA was reverse transcribed into cDNA using Prime Script RT Master Mix (Takara, Japan). Real-time quantitative PCR was performed using SYBR Green Master Mix (Takara, Japan) on an Applied Biosystems Step One system (Applied Biosystems, USA). Boaβ-actin As an internal reference gene, 2 -ΔΔCT The method calculates the relative expression level of genes.

[0044] Indole-glucosinolate synthesis gene BoaCYP79B2.1 The corresponding primers are: F: 5'-CTCCGTGAAGCTTTCCGTCT-3', R: 5'-AACTTTTGGGTTACGGCCCA-3'; Indole-glucosinolate synthesis gene BoaCYP79B2.2 The corresponding primers are: F: 5'-CATCGGAATGGTCCCAACGA-3', R: 5'-TGTTTCCTAGCCTCACGCAG-3'; Indole-glucosinolate synthesis gene BoaCYP83B1 The corresponding primers are: F: 5'-GTGGACTTCAAGGGGCAAGA-3', R: 5'-ACTCCAAGATGCATAGCGGG-3'; Indole-glucosinolate synthesis gene BoaCYP81F2.1 The corresponding primers are: F: 5'-AATAGTGGTGCAAGCCATCC-3', R: 5'-GCATTGTGTTGCTCTCTCCA-3'; Internal reference gene Boaβ-actin The corresponding primers are: F:5'-CCAGAGGTCTTGTTCCAGCCATC-3', R:5'-GTTCCACCACTGAGCACAATGTTAC-3'; The results are as follows Figure 2 As stated above. Figure 2 This indicates that the indoleglucosinolate synthesis gene BoaCYP79B2.1, BoaCYP79B2.2, BoaCYP83B1, BoaCYP81F2.1 The expression level inBoaWRKY8 All of them increased significantly in -OE, BoaWRKY8 -PTY decreased significantly. According to Figure 2 It can be seen that BoaWRKY8 can promote the accumulation of indoleglucosinolates by promoting the expression of the indoleglucosinolate synthesis gene.

[0045] Note: BoaWRKY8 is a regulatory gene for indoleglucosinolate synthesis. BoaCYP79B2.1, BoaCYP79B2.2, BoaCYP83B1, BoaCYP81F2.1 These are structural genes of the indoleglucosinolate pathway, and they all play a positive regulatory role in the accumulation of indoleglucosinolates.

[0046] seven, BoaWRKY8 Analysis of resistance of Chinese kale to Botrytis cinerea by overexpression and VIGS gene silencing Botrytis cinerea inoculation experiments were conducted on leaves of WT, BoaWRKY8-OE, PTY-EV, and BoaWRKY8-PTY Chinese kale.

[0047] Culture and propagation of Botrytis cinerea: Inoculate the spore suspension of Botrytis cinerea onto 2×V8 solid medium (36% V8 juice, 0.2% CaCO3, 2% agar powder) and incubate upside down at 25-28℃ in the dark. It can be used for inoculation experiments after about 10 days.

[0048] Botrytis cinerea inoculation experiment: Spores were scraped off with a sterile scalpel and suspended in spore suspension buffer (4% maltose, 3% peptone). The spore suspension was filtered through gauze and stirred with a magnetic stirrer to ensure even distribution of spores. The spore concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 5 Plaque area / mL. Using a pipette, apply 100 μL to the center of the leaf. Observe the plaque area after 4 to 5 days. Photograph the plaque area uniformly, and then measure and count the area using ImageJ software.

[0049] The results are as follows Figure 3 As stated above. Figure 3 This indicates that plants overexpressing BoaWRKY8-OE showed milder disease symptoms, while BoaWRKY8 Gene silencing lines BoaWRKY8 -PTY Chinese kale plants are severely affected by the disease.

[0050] It should be noted that an increase in the total indole glucosinolate content in plants not only provides the plants with advantages in stress resistance and defense, but also enhances the anti-cancer, anti-inflammatory, and antioxidant benefits of consuming the plant.

[0051] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The BoaWRKY8 gene of Chinese kale, characterized by: BoaWRKY8 The nucleotide sequence of the gene is shown in SEQ ID No:

1.

2. The use of the BoaWRKY8 gene in Chinese kale as described in claim 1, characterized in that: BoaWRKY8 It positively regulates the expression of genes involved in the synthesis of indoleglucosinolates.

3. The use of the BoaWRKY8 gene in Chinese kale according to claim 2, characterized in that: BoaWRKY8 Gene overexpression promotes the synthesis of total indole glucosinolates in Chinese kale.

4. The use of the BoaWRKY8 gene in Chinese kale according to claim 2 or 3, characterized in that: Indole-glucosinolate synthesis genes include BoaCYP79B2.1, BoaCYP79B2.2, BoaCYP83B1, BoaCYP81F2.

1.

5. The use of the BoaWRKY8 gene in Chinese kale according to claim 4, characterized in that: BoaWRKY8 The content of indoleglucosinolates can be increased by promoting the expression of the indoleglucosinolate synthesis gene.

6. The use of the Chinese kale BoaWRKY8 gene according to any one of claims 2 to 5, characterized in that: BoaWRKY8 By promoting the synthesis and accumulation of indoleglucosinolates, the plant's resistance to Botrytis cinerea is enhanced.

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