Application of transcription factor CdZF-HD1 / 9 in regulating squalene synthesis

Abstract

This invention pertains to modern agriculture, specifically the application of transcription factors CdZF-HD1 / 9 in regulating squalene synthesis. Using the CdSQS promoter as bait, this invention screens a yeast single-hybrid library to identify ZF-HD transcription factors that interact with the CdSQS promoter. ZF‑HD1 and ZF‑HD9 .clone ZF‑HD1 and ZF‑HD9 The nucleotide sequence was further confirmed by dual-luciferase assay and EMSA. ZF‑HD1 and ZF‑HD9 Interacts with CdSQS. ZF‑HD1 and ZF‑HD9 Genetic transformation of Camellia oleifera from Vietnam resulted in increased expression levels of squalene synthase and higher squalene content in the induced hairy roots. This invention provides a scientific basis for finding optimal genetic modification and exogenous regulation methods to increase squalene content in Camellia oleifera, and offers important theoretical and practical support for promoting the development of the Camellia oleifera industry in Hainan.

Description

Technical Field

[0001] This invention belongs to the field of modern agricultural technology, specifically involving the application of transcription factor CdZF-HD1 / 9 in regulating squalene synthesis. Background Technology

[0002] Camellia oleifera is an important woody oilseed tree species in my country. Its seed oil is rich in unsaturated fatty acids and squalene, and is widely used in the food, pharmaceutical, and cosmetic industries. Squalene has various biological functions in vivo, including antioxidant and immunomodulatory effects. Therefore, improving the squalene synthesis capacity in Vietnamese Camellia oleifera has significant economic and application value.

[0003] Squalene is an unsaturated triterpenoid compound, synthesized primarily through the mevalonate (MVA) pathway or the 2C-methyl-d-erythritol-4-phosphate (MEP) pathway. The MVA pathway produces isopentenyl diphosphate (IPP), which, in the MEP pathway, forms IPP and dimethylallyl diphosphate (DMAPP). These two molecules are then catalyzed by FPP synthase (FPS) to produce farnesyl pyrophosphate (FPP), which is subsequently condensed by squalene synthase (SQS) to synthesize squalene. Squalene synthase (SQS) is a key enzyme in squalene synthesis. Squalene synthase genes have been isolated and identified in plants such as ginseng, licorice, tobacco, and Tripterygium wilfordii. In a yeast erg9 mutant strain lacking SQS activity, squalene content was undetectable. Complementary expression of the SQS mutant restored the ability to synthesize squalene, indicating that the SQS enzyme is involved in ginseng squalene production. Previous studies have demonstrated that CdSQS in Camellia oleifera possesses squalene synthase activity through prokaryotic expression. Yeast single-hybrid assays have also shown an interaction between the WRKY transcription factor and SQS (Li et al., 2024). However, the molecular mechanism of squalene synthesis remains incompletely understood, limiting the development and application of technologies that promote squalene content regulation.

[0004] Zinc finger-homeodomain (ZF-HD) transcription factors are a class of plant-specific transcription factors composed of a zinc finger domain (ZF) and a homeodomain (HD). The ZF domain forms a stable structure through zinc ions and cysteine ​​or histidine residues, enabling it to specifically bind to DNA / RNA or promote protein-protein interactions. The HD domain, on the other hand, has DNA-binding function. Working synergistically, ZF-HD proteins can bind to the promoter regions of target genes in the form of homodimers or heterodimers, thereby regulating gene expression. Although ZF-HD transcription factors contain a zinc finger domain, they differ from other types of zinc finger proteins. For example, the C2H2-type zinc finger protein family mainly regulates gene expression by binding to DNA through the zinc finger domain, while the ZF-HD family requires both the zinc finger domain and the homeodomain to exert their regulatory effects. Currently, most reports on the functions of ZF-HD gene family members focus on bioinformatics analysis, plant growth and development, and abiotic stress research. In plant growth and development, AtZHD5 responds to cytokinin induction, promoting leaf cell enlargement and enhanced bud regeneration in Arabidopsis thaliana (Kim et al., 2019). In rice, the OsZHD1 and OsZHD2 genes influence growth by regulating leaf curling and root development (Yoon et al., 2023). Regarding plant abiotic stress, the AtZHD1 gene expression increases under drought, salt, and ABA stress, enhancing drought tolerance in Arabidopsis thaliana. In tomato, the SlZHD13 gene expression is upregulated under drought and salt stress; silencing this gene reduces drought and salt tolerance in tomato plants (Ting et al., 2019). Furthermore, in terms of plant secondary metabolism regulation, the SmZHD12 gene can activate the expression of key genes in the anthocyanin biosynthesis pathway in eggplant, namely SmCHS, SmANS, SmDFR, and SmF3H, and regulate anthocyanin biosynthesis in Arabidopsis thaliana through heterologous expression (Chen et al., 2024). In rapeseed, the BnZF-HD2 transcription factor participates in the regulation of rapeseed seed oil synthesis through phosphorylation (Fu Yifan, 2023).

[0005] However, ZF-HD has not yet been characterized and its functions identified in Vietnamese Camellia oleifera, especially its role in regulating the synthesis of triterpenoids. Summary of the Invention

[0006] The first objective of this invention is to provide a zinc finger-homologous domain transcription factor for camellia oleifera.

[0007] A second aspect of the present invention aims to provide a gene encoding the zinc finger-homologous domain transcription factor of the camellia oleifera described in the first aspect of the present invention.

[0008] A third aspect of the present invention is to provide a recombinant expression vector.

[0009] The fourth aspect of this invention is to provide a recombinant microorganism.

[0010] The fifth aspect of this invention aims to provide the application of the camellia oleifera zinc finger-homologous domain transcription factor described in the first aspect, the gene described in the second aspect, the recombinant expression vector described in the third aspect, and the recombinant microorganism described in the fourth aspect in the preparation of products that regulate squalene synthesis.

[0011] The sixth aspect of this invention is to provide a method for producing squalene.

[0012] The seventh aspect of this invention aims to provide a method for cultivating camellia oleifera varieties with high squalene expression.

[0013] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0014] The first aspect of this invention provides a camellia oil zinc finger-homologous domain transcription factor ( Camellia drupifera Zinc Finger-Homeodomain (CdZF-HD), the camellia oleifera zinc finger-homeodomain transcription factor is CdZF-HD1 or CdZF-HD9, and its amino acid sequence is shown as a1) or a2):

[0015] a1) CdZF-HD1, the amino acid sequence of which is shown in SEQ ID NO: 10;

[0016] a2) CdZF-HD9, the amino acid sequence of which is shown in SEQ ID NO: 12.

[0017] A second aspect of the present invention provides a gene encoding the zinc finger-homologous domain transcription factor of the camellia oleifera described in the first aspect of the present invention, characterized in that:

[0018] The nucleotide sequence of the gene is shown in b1) or b2):

[0019] b1) CdZF-HD1 The nucleotide sequence is shown in SEQ ID NO: 9;

[0020] b2) CdZF-HD9 The nucleotide sequence is shown in SEQ ID NO: 11.

[0021] A third aspect of the present invention provides a recombinant expression vector encoding the camellia zinc finger-homologous domain transcription factor described in the first aspect of the present invention.

[0022] In some embodiments of the present invention, the recombinant vector includes the gene described in the second aspect of the present invention.

[0023] In some embodiments of the present invention, the recombinant vector includes at least one of the following: Escherichia coli expression vector, yeast expression vector, Bacillus subtilis expression vector, lactic acid bacteria expression vector, Streptomyces expression vector, filamentous fungal expression vector, plant expression vector, or insect expression vector.

[0024] A fourth aspect of the present invention provides a recombinant microorganism comprising the recombinant expression vector described in the third aspect of the present invention.

[0025] In some embodiments of the present invention, the recombinant microorganisms include commonly used engineered strains in the biological field such as *Escherichia coli*, *Bacillus subtilis*, *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Agrobacterium*. *Escherichia coli* is recommended to be used... Escherichia coli DH5α, or other types of Escherichia coli, such as Bacillus subtilis, can be used. Bacillus subtilis 168. Bacillus subtilis WB600 and other Bacillus subtilis types used for protein expression, as well as Saccharomyces cerevisiae, can be used. Saccharomyces ceresiae CEN.PK2-1C Saccharomyces cerevisiae S288C In addition to other types of Saccharomyces cerevisiae that can be used for protein expression, Pichia pastoris can be used... Pichia pastoris GS115 In addition to other Pichia pastoris types that can be used for protein expression, Agrobacterium types such as GV3101 and ATCC15834 can be used as conventional Agrobacterium types.

[0026] Based on common knowledge in the field, the above-mentioned recombinant vectors correspond to recombinant microorganisms.

[0027] A fifth aspect of the present invention provides the use of the camellia oleifera zinc finger-homologous domain transcription factor described in the first aspect, the gene described in the second aspect, the recombinant expression vector described in the third aspect, and the recombinant microorganism described in the fourth aspect in the preparation of products that regulate squalene synthesis.

[0028] In some embodiments of the present invention, the product includes products for preparing squalene and products for increasing the expression level of the squalene synthase gene.

[0029] A sixth aspect of the present invention provides a method for producing squalene, comprising the step of overexpressing the gene described in the second aspect of the present invention in camellia oleifera.

[0030] A seventh aspect of the present invention provides a method for cultivating a Camellia oleifera variety with high squalene expression, comprising the step of overexpressing the gene described in the second aspect of the present invention in Camellia oleifera.

[0031] In some embodiments of the present invention, the camellia oleifera variety with high squalene expression has the following characteristics: the squalene expression level is increased relative to a reference level; the reference level is the level of the wild type.

[0032] In some embodiments of the present invention, the step of overexpressing the gene described in the second aspect of the present invention involves introducing the recombinant vector described in the third aspect of the present invention or the recombinant microorganism described in the fourth aspect of the present invention into Camellia oleifera tissue or Camellia oleifera cells.

[0033] In some embodiments of the present invention, the introduction method includes using at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

[0034] In some embodiments of the present invention, the above-mentioned camellia oil includes, but is not limited to, Vietnamese camellia oil ( Camellia drupifera ).

[0035] The beneficial effects of this invention are:

[0036] This protocol employed a Vietnamese Camellia oleifera cDNA library construction and a yeast single-hybrid screening library to screen for ZF-HD transcription factors CdZF-HD1 and CdZF-HD9, which interact with the CdSQS promoter. Using Vietnamese Camellia oleifera leaf cDNA as a template, [the following was amplified / developed / adapted]... CdZF-HD1 and CdZF-HD9 The coding sequence (CDS) was determined. Further validation was performed using luciferase complementation (LCA) and electrophoretic migration variation analysis (EMSA). CdZF-HD1 and CdZF-HD9 It possesses the ability to specifically bind to DNA. By constructing an overexpression vector and using Agrobacterium-mediated genetic transformation of Vietnamese tea leaves, the molecular level of its activity was explored. CdZF-HD1 and CdZF-HD9 This study investigated the regulatory role of squalene synthesis, further elucidated the synthesis mechanism of squalene in Vietnamese camellia, provided theoretical support for the breeding of new varieties, and the industrial synthesis of squalene in camellia, and offered important theoretical and practical support for promoting the development of the Hainan camellia industry. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1This image shows the construction of a cDNA library from *Camellia oleifera* from Vietnam. Electrophoresis results are as follows: A - mRNA; B - double-stranded cDNA; C - primary library colony identification; D - primary library volume identification; E - nuclear system secondary library colony identification; F - nuclear system secondary library volume identification; G - membrane system secondary library colony identification; H - membrane system secondary library volume identification. The markers used, from top to bottom, are 2000, 1000, 750, 500, 250, and 100 bp.

[0039] Figure 2 The results of the concentration screening for pAbAi-pSQS single-hybrid self-activation are shown.

[0040] Figure 3 The results are for yeast one-hybrid assays. A: Yeast one-hybrid screening library (a is a plate plot of the initial screening portion; b is a secondary screening plot). B: Yeast one-hybrid plate verification.

[0041] Figure 4 Camellia oleifera from Vietnam CdZF-HD1 and CdZF-HD9 Gene PCR gel electrophoresis image, where A is CdZF-HD1 The result is that B is CdZF-HD9 The result.

[0042] Figure 5 for CdZF-HD1, CdZF-HD9, SlZHD17, AtHB-22 and DcHB30 The sequence alignment analysis results.

[0043] Figure 6 for CdZF-HD1 / 9 The results of protein interaction verification with CdSQS are shown in A: Dual-luciferase assay (DLR). B: Electrophoretic migration variation analysis (EMSA).

[0044] Figure 7 The results show the positive identification of hairy roots of Camellia oleifera mediated by Agrobacterium rhizogenes. In the figure, A is a schematic diagram of the vector structure; B is the fluorescence result after transfection.

[0045] Figure 8 The results show the expression levels of related genes in hairy roots after overexpression of CdZF-HD1 and CdZF-HD9. Detailed Implementation

[0046] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0047] The main conclusions of this invention are as follows:

[0048] (1) CdZF-HD1 and CdZF-HD9 interact with the CdSQS promoter to activate CdSQS expression;

[0049] (2) This indicates that CdZF-HD1 / 9 positively regulates squalene synthesis by activating the expression of CdSQS.

[0050] Specific experimental data are shown in the following examples. The nucleotide sequence of the CdSQS gene is shown in SEQ ID NO: 23, and the nucleotide sequence of the CdSQS promoter is shown in SEQ ID NO: 24.

[0051] Example 1: Construction of a Vietnamese Camellia oleifera cDNA library (Uncut type)

[0052] 1. RNA extraction and purification

[0053] RNA extraction was performed according to the instructions of the Rapid Universal Plant RNA Extraction Kit (Beijing Huayueyang Biotechnology Co., Ltd.). The extracted RNA was detected by 1% agarose gel electrophoresis, and the RNA concentration was measured using a nucleic acid analyzer. Quality-compliant RNA samples were stored at -80℃ for later use. mRNA isolation and purification were performed according to the FastTrack MAG mRNA isolation kit instructions (Invitrogen).

[0054] 2. Construction of a Vietnamese Camellia oleifera cDNA library (Uncut type)

[0055] pAbAi - pCdSQS After plasmid transformation, 100 µL of 1 / 10, 1 / 100, 1 / 1000, and 1 / 10000 dilutions were spread onto 100 mm SD / -Leu plates to calculate the transformation efficiency: Transformation Efficiency = cfu × suspension volume (mL) / volume of plated (mL) × amount of DNA (μg). The remaining bacterial culture was spread onto 150 mm SD / -Leu plates, 300 µL per plate, for a total of 100 plates; incubated at 30 ℃ for 3-5 days until single colonies reached a size of 1-2 mm. (The carrier DNA was pre-denatured twice: tubes containing carrier DNA were boiled in boiling water for 5 min, then immediately placed on ice for 2 min, and then repeated once.)

[0056] After culturing the 100 plates, remove them and place them in a 4 ℃ freezer for 3-4 hours. Add 5 mL of frozen culture medium to each plate. Gently scrape the plates with a sterile glass rod to allow cells to enter the liquid. Collect all the liquid into a flask (approximately 500 mL) and mix thoroughly. Calculate the cell density using a hemocytometer. Aliquot the solution into 1.5 mL sterile centrifuge tubes, 1 mL per tube, for a total of 100 tubes; this is the yeast working solution. Aliquot the remaining solution into 50 mL centrifuge tubes; this is the yeast stock solution. Calculate the library titer: Spread 100 µL of 1 / 10, 1 / 100, 1 / 1000, and 1 / 10000 library dilutions onto 100 mm SD / -Leu plates and incubate at 30 ℃ until clones appear (approximately 2-3 days). Count the number of clones on each plate. Note: Titer (cfu / mL) = Number of clones on plate / 100 µL × Dilution factor × 1 × 10 3 µL.

[0057] Select clones and incubate overnight at 30℃ and 250 rpm; centrifuge at 10000 rpm for 2 min and remove the supernatant; resuspend the bacterial culture in 200 µL of 0.2% SDS and incubate at 95℃ for 15 min; centrifuge at 10000 rpm for 2 min and use the supernatant as a PCR template; add the following reagents to the PCR tube in the order shown in Table 1, then place it in the PCR instrument for reaction, followed by electrophoresis detection.

[0058] Table 1. Reaction system for yeast library cloning identification

[0059]

[0060] 3. Experimental Results

[0061] The results are as follows Figure 1 As shown, 48 clones were randomly selected from the cDNA library, and clear insertion sequences were detected. The clone positivity rate in the library was 100%, and the library capacity coverage was 100%, indicating that the constructed yeast cDNA library was of qualified quality.

[0062] Example 2: Screening library of CdSQS promoter, a key gene for squalene synthesis

[0063] 1. Construction of pCdSQS monohybrid bait carrier

[0064] The pAbAi plasmid was double-digested using two restriction endonucleases, Sma I and Xho I. The digestion system is shown in Table 2. After gel electrophoresis, the target band was recovered, and the recovered product was labeled as x-pAbAi.

[0065] Table 2. Double enzyme digestion system of pAbAi vector

[0066]

[0067] by pCdSQS Using plasmids as templates, amplification was performed using primers with homologous arms (Table 3). PCR After the system and reaction are complete, the product is recovered by gel extraction. The product with homologous arms is labeled as follows: pS The object is marked as pS .

[0068] Table 3. List of pCdSQS cloning primers for Camellia oleifera from Vietnam

[0069]

[0070] Then, pS was ligated to x-pAbAi using the homologous recombination kit 2×Ezmax® Universal CloneMix (Tulugang Biotechnology Co., Ltd.). The reaction system is shown in Table 4, resulting in the single-hybrid recombinant vector pAbAi-pSQS. Positive clones were then screened and sequenced using E. coli DH5α. Plasmids were extracted from the correctly sequenced positive clones, named pAbAi-pSQS, and stored at -20℃ for later use. The remaining bacterial culture was then mixed with 20% glycerol and stored at -80℃.

[0071] Table 4 x-pAbAi and pS linkage systems

[0072]

[0073] 2. Construction of bait vector strains

[0074] The pAbAi-pSQS bait recombinant plasmid was linearized using BstBI restriction endonuclease. The restriction enzyme digestion system is shown in Table 5. The products were labeled as x-pAbAi-pSQS.

[0075] Table 5 Single enzyme digestion system of pAbAi-pSQS vector

[0076]

[0077] 1 μg of linearized x-pAbAi-pSQS plasmid was integrated into Y1HGold competent strain, plated on SD / -Ura plates, and cultured for 3-5 days.

[0078] On SD / -Ura plates, select 4-5 single-clone yeast strains and place them in 50 µL of pure water. Identify them using Matchmaker® Insert Check PCR Mix 1, as shown in Table 6. After agarose gel electrophoresis for 30 min, check the size of the PCR bands in the bacterial culture to see if it matches the target band length. Correctly identified clones can be preserved, and the colonies are labeled pCdSQS-Y1H.

[0079] Table 6. Colony PCR reaction system and procedure for the pAbAi-pSQS gene in Camellia oleifera from Vietnam.

[0080]

[0081] 3. Screening of yeast single-hybrid bait strains based on self-activation concentration

[0082] Single colonies of pCdSQS-Y1H were picked from SD / -Ura plates and resuspended in 0.9% NaCl solution to adjust the OD of the yeast culture. 600 The value is 0.002; 100 µL of resuspended yeast culture was spread onto SD / -Ura plates containing different concentrations of AbA with AbA (0, 100, 150, 200, 300, 500, 700, 900 ng / mL). The optimal concentration of AbA is indicated by the minimum or complete absence of yeast plaques.

[0083] 4. Yeast one-hybrid screening library

[0084] The transformed plasmid was a secondary plasmid of a nuclear system yeast library (II cDNA Library Construction Kit (Invitrogen; A11180)). 100 µL of 1 / 10 and 1 / 100 dilutions were spread onto SD / -Leu plates to calculate transformation efficiency; the remaining bacterial culture was spread onto SD / -Leu / AbA(300) plates, totaling approximately 50 plates. The plates were incubated at 30 ℃ for 3-5 days until single clones reached a size of 1-2 mm, completing the initial screening. Positive clones from the initial screening plates were transferred to the same selection medium, SD / -Leu / AbA(300), for secondary screening. Larger positive clones were selected for PCR identification, and the amplification products were detected by agarose gel electrophoresis.

[0085] 5. Extraction, amplification, and identification of positive clone yeast plasmids

[0086] Yeast plasmid extraction for positive clones was performed using a classic yeast plasmid miniprep kit (Beijing Cooler Master Technology Co., Ltd.). Single colonies were picked and cultured for sequencing. Successfully sequenced bacterial cultures were amplified, plasmids were extracted, and stored at -20°C for later use. The remaining bacterial culture was mixed with 20% glycerol and stored at -80°C. The sequencing results were simultaneously compared with data from NCBI and transcriptome databases.

[0087] 6. One-to-one verification

[0088] The pCdSQS-Y1H yeast strain was streaked on SD / Ura solid medium and incubated upside down in a 30°C incubator for 2-3 days. Then, competent cells were prepared, and the identified interaction plasmids were transferred into them. The cells were plated on SD / -Leu and SD / -Leu / AbA (300) plates and incubated in a 30°C incubator for 3-5 days. Single colonies grew to a size of 1-2 mm.

[0089] 7. Spot verification

[0090] Select single clones from the plate and add them to 800 µL of YPDA liquid medium. Incubate overnight at 30 °C, centrifuge at 5000 rpm for 1 min, discard the supernatant, resuspend in 1 mL of sterile ddH2O, centrifuge at 5000 rpm for 1 min, discard the supernatant, and resuspend again in 800 µL of sterile ddH2O. Transfer 100 µL of the resuspended bacterial solution to a new sterile 1.5 mL centrifuge tube, add 700 µL of sterile ddH2O, and dilute at a ratio of 1:10:100:1000. Spot 10 µL of each bacterial solution onto SD / -Leu / AbA (0 and 300) plates and incubate at 30 °C for 3-5 days, observing their growth.

[0091] 8. Experimental Results

[0092] like Figure 2 As shown, pCdSQS-Y1H was plated on SD / -Ura plates containing different concentrations of AbA (0, 100, 150, 200, 300, 500, 700, 900 ng / mL). It was found that pCdSQS-Y1H yeast did not grow in SD / -Ura / AbA containing 300 ng / mL, indicating that CdSQSpro was inhibited at a concentration of 300 ng / mL.

[0093] like Figure 3 As shown, a total of 64 clones grew on the SD / -Leu / AbA (300 ng / mL) screening plate. Figure 3(A). The candidate positive clones on the initial screening medium were transferred again to SD / -Leu / AbA (300 ng / mL) screening medium for further screening. A total of 50 candidate positive clones grew ( Figure 3 (B) Plasmids were extracted from 50 positive yeast cells by shaking and transformed into E. coli for propagation, followed by sequencing. Gene sequences of 50 interacting proteins were successfully obtained. The sequenced gene sequences were compared and annotated with the NCBI database and our transcriptome.

[0094] Six proteins that may interact with pCdSQS were screened, as shown in Table 7.

[0095] Table 7

[0096]

[0097] The results of yeast single-spot plate and one-to-one validation showed that AD-02 (ZF-HD1) and AD-55 (ZF-HD1) had strong interaction, while AD-24, AD-28 and AD-59 had weak interaction.

[0098] Example 3: Vietnamese Camellia Oil CdZF-HD1 and CdZF-HD9 Cloning of gene CDS sequences

[0099] 1. CdZF-HD1 and CdZF-HD9 Cloning of gene CDS sequences

[0100] Using Vietnamese Camellia oleifera cDNA as a template, the target fragment was amplified using PrimerSTAR Max DNA Polymerase.

[0101] Table 8 PCR Primer Sequences

[0102]

[0103] The experimental procedure is as follows: Add each sample to a 0.2 ml Eppendorf tube, and follow the steps in Table 9.

[0104] Table 9 PCR Reaction System

[0105]

[0106] 2) Purification of PCR products

[0107] Under UV light, quickly cut the gel containing the target DNA fragment and transfer the gel into a 2 mL centrifuge tube. Subsequent procedures were performed according to the DNA recovery kit instructions (Tiangen Biotech (Beijing) Co., Ltd.) to recover the target fragment. The concentration and purity of the purified product were tested, and qualified DNA products were stored at -20℃.

[0108] 3) Connection and transformation of target fragments

[0109] The reaction conditions for the connection are shown in Table 10.

[0110] Table 10

[0111]

[0112] 4. Bioinformatics analysis of CdZF-HD1 / 9 from Vietnamese Camellia oleifera

[0113] Predict using NCBI-Conserved Domain CdZF-HD1 and CdZF-HD9 The conserved structural domains were identified; the CdZF-HD1 and CdZF-HD9 protein sequences were aligned using DNAMAN 8.0 software; a phylogenetic tree was constructed using the MEGA11 neighbor-joining method with 1000 replicates; and online analysis was performed using the ExPASy-ProtParam tool. CdZF-HD1 and CdZF- HD9 Physicochemical properties of the encoded proteins; prediction of subcellular localization using the Plant-mPLoc online website; prediction using the SOPMA online tool. CdZF-HD1 and CdZF-HD9 Protein secondary structure; predicted using the SWISS-MODEL online tool. CdZF-HD1 and CdZF-HD9 The tertiary structure of proteins.

[0114] 5. Experimental Results

[0115] The results are as follows Figure 4 As shown, using cDNA from Vietnamese tea leaves as a template, the CdZF-HD1 (Unigene0077051) and CdZF-HD9 (Unigene0075736) genes were amplified using PrimerSTAR Max DNA Polymerase. The coding sequence (CDS) lengths of CdZF-HD1 and CdZF-HD9 were 741 bp and 996 bp, respectively. The sequencing results were consistent with the transcriptome sequence.

[0116] The nucleotide sequence of ZF-HD1 is as follows:

[0117] ATGGAATTTGAAGAGAACGAAGACCAGGAAGAGGAAATCGGGATTCAGGTGCCGCCGGTGTTGAATTACGAGGCGGTGCTGGGAAACTCCGGGAGGCCGAAAATGGGGATGAGTGGTGCCGGAGGAGAGGGGGTGTCTCCGGTTGCGGCGGCCGGGAGGAAAGTCGGCGGGAGCTACAGATACAGAGAGTGCCTGAAGAACCACGCTGTGGGGATCGGCGGCCACGCCGTCGACGGTTGTGGAGAATTTTTACCGGCGGGAGATGAAGGAACCCTAGACGCCCTAAAATGCGCGGCCTGCAATTGCCACCGCAACTTCCACCGCAAAGAGGGCGACGGAGAGGGCTTCCACCACCAACAACAGTTCCATCATCCTCACCACCACCCCCAATTCTCCCCTTACTATCGCACCGCCCACCCGGCCGGGTACCTCCACGTCACGCCGCCGCCGCATCAGCGGCCTCTAGCGCTGCCATCGACTTCTCGGGAAGATGAAGATATGTCGAATCCAAGCAGCAGCGGCGGTGGTGGTAGCGGCGGTGGGTCGAAAAAGCGGTTCAGAACAAAATTTACACAGGAACAGAAGGATAAGATGCTGGTGTTGGCGGAGAACTTGGGGTGGCGCATCCAGAAGCAGGATGAGGCTTCGGTGCAGCAGTTCTGCGCCGAGACTGGTGTGAAAAGGCATGTTCTCAAGGTGTGGATGCACAACAATAAGCATACTCTCGGTAAAAAACCCTAA (SEQ ID NO: 9).

[0118] The amino acid sequence of ZF-HD1 is as follows:

[0119] MEFEENEDQEEEIGIQVPPVLNYEAVLGNSGRPKMGMSGAGGEGVSPVAAAGRKVGGSYRYRECLKNHAVGIGGHAVDGCGEFLPAGDEGTLDALKCAACNCHRNFHRKEGDGEGFHHQQQFHHPHHHPQFSPYYRTAHPAGYLHVTPPPHQRPLALPSTSREDEDMSNPSSSGGGGSGGGSKKRFRTKFTQEQKDKMLVLAENLGWRIQKQDEASVQQFCAETGVKRHVLKVWMHNNKHTLGKKP (SEQ ID NO: 10).

[0120] The nucleotide sequence of ZF-HD9 is as follows:

[0121] ATGGACATAGCCACCGCCACCGCCACCGCCACCGCCACCGCCACAGCCAGTGTTAAAACCCCAGAAGCTGAAACCGAAGCACCGACTCGGATCCAACCCAGCAAACCCTTATCCTTCACCAACGGCGTACTCAAGCGCCACCACCGCCACCACCACCACCACCTGAGTACCCCACCTGTTGTGGTCACTTACAAGGAGTGTCTCAAGAACCACGCGGCCAGCCTCGGAGGCCACGCCGTCGATGGCTGCGGCGAGTTCATGCCTTCTCCCACATCGAGCCCCACCGACCCCACCTCTCTAAAATGCGCCGCTTGTGGCTGCCACCGCAACTTCCACCGTCGTGACCCCGAAGAACCGCTCCCAGCGCCTACCAGCACCGCCGCCCAACACGTCATCGAGTACCAGCCCCACCACCGCCACCACCCACTTCCTCCACCACCAGCACCACACCCACACGGCGGCCACAGCAGCCCAAATTCAGCATCTCCGCCGCCGATCTCTTCCTCCTACTACCCATCCGCACCCCACATGCTCCTCGCCCTCAGCGCAAGCTTGCCGGGCCCACCGCCAGAAAACAACCCATCAATGGCCCCCACTACCGGCACGACAGCTGCGAATTCCAGTGGGAGGAAGCGATTCAGAACAAAATTCACGCAGGATCAGAAGGAGAGGATGCACGAATTGGCAGAGAGAGTTGGGTGGAAGATGCAGAAGAGGGACGAAGAGTTGATCAACGAGTTCTGCAGCGACGTTGGGGTCGACAGAGGTGTGTTCAAAGTCTGGATGCACAACAATAAAAACACTTTTGGGAAGCACAACAACGGTAACGGTAATGGTAATGGTAGCGGAAATAGTCTTGACAACCATGAAAATAACAGCAATAACAACAACCACCACCACCACCACCACCACCAAAATCTGAGCAATCAACACGAAAACAATGATAGTGTAAGTGCACATGTTGTTGCGACTAATGGGTCTTCTTCTTCTTCTTAA(SEQID NO: 11).

[0122] The amino acid sequence of ZF-HD9 is as follows:

[0123] MDIATATATATATATASVKTPEAETEAPTRIQPSKPLSFTNGVLKRHHRHHHHHLSTPPVVVTYKECLKNHAASLGGHAVDGCGEFMPSPTSSPTDPTSLKCAACGCHRNFHRRDPEEPLPAPTSTAAQHVIEYQPHHRHHPLPPPPAPHPHGGHSSPNSASP PPISSSYYPSAPHMLLALSASLPGPPPENNPSMAPTTGTTAANSSGRKRFRTKFTQDQKERMHELAERVGWKMQKRDEELINEFCSDVGVDRGVFKVWMHNNKNTFGKHNNGNGNGNGSGNSLDNHENNSNNNNHHHHHHHQNLSNQHENNDSVSAHVV (SEQ ID NO: 12).

[0124] The results are as follows Figure 5 As shown, CdZF-HD1 and CdZF-HD9 The amino acid sequence of [the substance] is similar to that of Arabidopsis thaliana, which has been reported to have functionally validated [products]. AtHB-22 ),tomato( SlZHD17 ) and carnations ( DcHB30 The sequences of ZF-HD family members were compared, and the results showed that... CdZF-HD1 and CdZF-HD9 and SlZHD17 , AtHB-22 , DcHB30 Having the same ZF and HD conservative structural domains indicates CdZF-HD1 and CdZF-HD9 They may recognize the same binding motifs and play the same or similar roles.

[0125] Example 4: Verification of the interaction between CdZF-HD1 / 9 and CdSQS proteins

[0126] 1. Dual-luciferase assay (DLR)

[0127] 1) The constructed transcription factor experimental group plasmids (pEAQ-HT-DEST2-CdZF-HD1 and pEAQ-HT-DEST2-CdZF-HD9), promoter experimental group plasmid (pH2GW7-CdSQS), and two empty vector plasmids (pEAQ-HT-DEST2 and pH2GW7) were transformed into Agrobacterium GV3101-Psoup-P19 competent cells and plated on LB agar plates containing 50 μg / mL Kan. Single colonies grew after incubation at 28°C for 2-3 days.

[0128] 2) Incubation: Pick a single Agrobacterium colony with a diameter of about 1-2 mm from the plate obtained in the previous step and place it into 5 mL of LB liquid medium (containing 50 μg / mL Kan and 25 μg / mL Rif). Incubate at 28℃ and 200 rpm until the logarithmic growth phase of Agrobacterium (OD600 = 0.8 - 1.5).

[0129] 3) Centrifuge at 5000 rpm for 10 min at room temperature to collect the bacterial cells, and suspend the Agrobacterium cells in the staining buffer (containing 10 mM MgCl2, 10 mM MMEs, 150 μM acetylsalicylic acid, pH = 5.6) until OD600 = 0.8.

[0130] 4) Mix the two bacterial cultures at a ratio of transcription factor to promoter of 1:1 and let stand at room temperature for 2 to 3 hours.

[0131] 5) Injecting tobacco: Select plants in good growth condition, use a 1 mL sterile syringe (with the pillow removed) to draw up the bacterial solution and inject it into the back of the tobacco leaf, marking it with a marker.

[0132] 6) Inoculated tobacco plants are cultured in the dark for 1 day, then transferred to light and cultured for 1-2 days before observation.

[0133] 7) Cut off the leaves of Nicotiana benthamiana, spray the lower epidermis with 1 mM fluorescein potassium salt solution, react in the dark for 3-5 minutes, and then observe the fluorescence using a chemiluminescence imaging system.

[0134] 2. Electrophoretic Mobility Analysis (EMSA) Experiment

[0135] 1) Construct recombinant plasmids pMAL-C5X-CdZF-HD1 and pMAL-C5X-CdZF-HD9.

[0136] 2) Recombinant plasmids MBP-CdZF-HD1 and MBP-CdZF-HD9 were expressed in Escherichia coli BL21.

[0137] 3) Purify the fusion protein according to the instructions of the protein purification kit (Sangon Biotech, Shanghai, China).

[0138] 4) Add the reactants in the following order: probe, protein, binding buffer, mix gently, and incubate in a PCR instrument (25℃) for 30 min.

[0139] The probe sequence is as follows:

[0140] Probe SS-1 FP:

[0141] 5'biotin-TAAAAAAAATTAAAAAGTTAAAAAAATTAACTTTTTATTATTGTT (SEQ ID NO: 13);

[0142] Probe SS-1 RP: AACAATAATAAAAAGTTAATTTTTTAACTTTTTAATTTTTTTTA (SEQ ID NO: 14);

[0143] Probe SS-2 FP:

[0144] 5'biotin-ATTATATCTAAATCATAATTATTTTACTTTTTA (SEQ ID NO: 15);

[0145] Probe SS-2 RP: TAAAAGTAAAATAATTATGATTTAGATATAAT (SEQ ID NO: 16).

[0146] 5) Perform SDS-PAGE gel analysis and Western Blot detection.

[0147] 3. Experimental Results

[0148] Dual-luciferase assay (DLR) as follows Figure 6 As shown in A, CdZF-HD1 and CdZF-HD9 All with CdSQSpro interaction, CdZF-HD9 and CdSQSpro The interaction is stronger. To verify this interaction in vitro, electrophoretic migration analysis (EMSA) was performed using recombinant CdZF-HD1 and CdZF-HD9 proteins with biolabeled DNA probes from the SS1 and SS2 regions. The results are as follows: Figure 6 As shown in B, CdZF-HD1 , CdZF-HD9 The appearance of migration bands upon binding to SS1 and SS2 probes respectively indicates that... CdZF-HD1 and CdZF-HD9 It exhibits specific binding activity to CdSQSpro.

[0149] Example 5 CdZF-HD1 / 9 Promote CdSQS expression

[0150] 1. Construction of overexpression vectors

[0151] 1) Enzyme digestion of pCAMBIA 2300 vector

[0152] Based on the restriction enzyme sites (Smal, Xbal) selected when designing the target gene-specific primers containing the vector adapter, the pCAMBIA 2300 vector was double-digested at the corresponding sites.

[0153] Table 11 Enzyme digestion reaction system

[0154]

[0155] 2) Connection and Transformation

[0156] Table 12 Connection Reaction System

[0157]

[0158] For the successfully sequenced bacterial culture, plasmids were extracted according to the instructions of the General Plasmid Mini Kit (FOREGENE Chengdu Fujie Biotechnology Co., Ltd.). Transformation of Agrobacterium GV3101 was performed according to the product instructions of Shanghai Weidi Agrobacterium GV3101-pSoup-P19 competent cells.

[0159] 3) Genetic transformation of Vietnamese tea stalks

[0160] A culture of Agrobacterium rhizogenes containing the target gene was streaked onto LB agar containing kanamycin (50 mg / L) and incubated at 28°C for 2-3 days. Single colonies of Agrobacterium were picked and transferred to 50 mL of LB liquid medium containing kanamycin (50 mg / L) and incubated at 28°C and 200 rpm until OD600 = 0.8-1.2. The bacteria were collected by centrifugation at 5000 rpm for 10 minutes. Subsequently, the bacteria were resuspended in an inoculum solution (10 mM MES, 10 mM MgCl2, 200 μM As) until OD600 = 0.8 and allowed to stand for 3 hours. One-year-old Vietnamese tea leaves were harvested as leaf explants for genetic transformation. The wounds of the leaf explants were immersed in the inoculum solution and placed together in a vacuum pump for 30 min-1 h. Finally, the Agrobacterium-infused explants were incubated in sterilized vermiculite and cultured in a growth chamber.

[0161] 4) Real-time quantitative PCR

[0162] Table 13 Primer Sequences for RT-qPCR

[0163]

[0164] 5) Detection of squalene content

[0165] Weigh 15.00 ± 0.50 mg of tissue sample and place it in a 1.5 mL centrifuge tube. Accurately add 200 μL of pre-cooled (4°C) n-hexane to the centrifuge tube, add two zirconium oxide beads (3 mm in diameter), and oscillate at 30 Hz for 2 minutes to ensure thorough homogenization. Use a vortex mixer (IKA Vortex Genius 3) at 2800 rpm for 3 minutes to thoroughly mix the sample with the extraction solvent. Place the centrifuge tube in an ice-water bath sonicator (300 W, 40 kHz) and sonicate for 15 minutes. After sonication, allow to stand at room temperature for 10 minutes. Centrifuge using a low-temperature centrifuge (Eppendorf 5424 R) at 4°C, 12000 rpm for 15 minutes. Take 150 μL of the upper n-hexane phase and transfer it to a 1.5 mL glass centrifuge tube. Use a vacuum centrifuge concentrator (Thermo Scientific Savant SPD131DDA) with the following parameters set: temperature 40℃, vacuum degree ≤10 mbar, and concentration time approximately 3 h. Add 100 μL of acetonitrile (chromatographic grade, filtered through a 0.22 μm filter membrane) to the concentrated sample and vortex (2500 rpm) for 1 minute to fully dissolve the residue. Then analyze the sample using the centrifuge.

[0166] 2. Experimental Results

[0167] pCAMBIA 2300-GFP-CdZF-HD1 and pCAMBIA 2300-GFP-CdZF-HD9 were transformed into *Camellia oleifera* stalk tissue using an *Agrobacterium rhizogenes* (ATCC15834)-mediated transformation method. The results are as follows: Figure 7 As shown, transgenic hairy roots with GFP fluorescence were produced in the stalks of tea oil plants carrying different genes, indicating that... CdZF-HD1 and CdZF-HD9 It is expressed in the stem of the oil tea plant.

[0168] The expression levels of related genes in hairy roots are as follows: Figure 8 As shown, compared with the control vector pCAMBIA 2300-GFP, the expression levels of CdZF-HD1, CdZF-HD9, and CdSQS in the transgenic hairy roots were all significantly upregulated, indicating that... CdZF-HD1 and CdZF- HD9 Genes enhance the transcriptional level of CdSQS, promotingCdSQS The expression.

[0169] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. Camellia oleifera zinc finger-homeodomain transcription factor, characterized in that: the Camellia oleifera zinc finger-homeodomain transcription factor is CdZF-HD1 or CdZF-HD9, and the amino acid sequence is shown as a1) or a2): a1) CdZF-HD1, the amino acid sequence is shown as SEQ ID NO: 10; a2) CdZF-HD9, the amino acid sequence is shown as SEQ ID NO:

12. 2.A gene encoding the Camellia oleifera zinc finger-homeodomain transcription factor of claim 1, characterized in that: the nucleotide sequence of the gene is shown as b1) or b2): b1) CdZF-HD1, the nucleotide sequence is shown as SEQ ID NO: 9; b2) CdZF-HD9, the nucleotide sequence is shown as SEQ ID NO:

11. 3.A recombinant expression vector, characterized in that: the recombinant expression vector encodes the Camellia oleifera zinc finger-homeodomain transcription factor of claim 1. 4.A recombinant microorganism, characterized in that: the recombinant microorganism comprises the recombinant expression vector of claim 3. 5.The recombinant microorganism of claim 4, characterized in that: the recombinant microorganism comprises Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Agrobacterium. 6.The Camellia oleifera zinc finger-homeodomain transcription factor of claim 1, the gene of claim 2, the recombinant expression vector of claim 3, or the recombinant microorganism of claim 4 or 5, are used in the preparation of a product for up-regulating squalene synthesis; the product is Camellia oleifera; the Camellia oleifera over-expresses CdZF-HD1 or CdZF-HD9. 7.A method for producing squalene, characterized in that: the method comprises the step of over-expressing the gene of claim 2 in Camellia oleifera. 8.A method for cultivating a Camellia oleifera variety with high expression of squalene, characterized in that: the method comprises the step of over-expressing the gene of claim 2 in Camellia oleifera; the Camellia oleifera variety with high expression of squalene comprises the following characteristics: the expression level of squalene is improved relative to a reference level; the reference level is the level of a wild type. 9.The method of claim 7 or 8, characterized in that: the step of over-expressing the gene of claim 2 is introducing the recombinant vector of claim 3 or the recombinant microorganism of any one of claims 4-5 into Camellia oleifera tissue or Camellia oleifera cells. 10.The method of claim 9, characterized in that: the introduction is by at least one of a Ti plasmid, a Ri plasmid, a plant virus vector, microinjection, electroporation.

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