Paphiopedilum spicerianum trehalose hydrolase gene PaTRE and application thereof

Abstract

The application discloses a Paphiopedilum spicerianum trehalase gene PaTRE and application thereof. The PaTRE gene is cloned from a Paphiopedilum spicerianum protocorm, connected to a plant expression vector, and introduced into tobacco through an Agrobacterium-mediated leaf disc transformation method. Genome PCR detection results show that the PaTRE gene has been introduced into tobacco; real-time fluorescent quantitative PCR detection results show that the PaTRE gene can be expressed in the transgenic tobacco; and trehalase activity detection results show that the trehalase activity in the PaTRE transgenic tobacco leaves is significantly higher than that in the control tobacco. The application proves that the PaTRE gene has the function of improving the trehalase activity of plant tissues, and can be used for plant trehalose metabolism regulation, functional gene research and molecular breeding, and provides a technical basis for related gene resource development.

Description

Technical Field

[0001] This invention belongs to the fields of plant molecular biology and genetic engineering technology, specifically relating to a Paphiopedilum trehalose hydrolase gene PaTRE and its application. Background Technology

[0002] Orchids are a diverse group of angiosperms, with the genus *Paphiopedilum* possessing high ornamental and conservation value due to its unique cup-shaped lip, rich flower colors, and long flowering period. However, *Paphiopedilum* seeds have incomplete embryonic development and lack endosperm, making their germination under natural conditions highly dependent on symbiotic fungi for exogenous nutrition and carbon sources. This results in low natural germination rates and long reproductive cycles, limiting the conservation and large-scale propagation of *Paphiopedilum* germplasm resources.

[0003] Carbon source supply is a crucial factor influencing seed germination and early protocorm development in orchids. Symbiotic fungi can convert external carbon sources into sugars for their own storage or transport. Trehalose, a common non-reducing disaccharide in fungi, is also an important form of carbon source storage and transport. Orchid protocorms can obtain the carbon source and energy required for growth and development by utilizing trehalose from fungi. Trehalose needs to be broken down into glucose by trehalose hydrolases, which then enter the cellular sugar metabolism process. Therefore, trehalose hydrolases are key enzymes in the trehalose metabolic pathway and may be an important molecular node connecting the carbon source supply from symbiotic fungi with the carbon metabolism of orchids themselves.

[0004] Currently, there is limited research on the function of trehalose hydrolase genes in Paphiopedilum species, and there is a lack of verification on the expression and biological function of trehalose hydrolase genes derived from Paphiopedilum in heterologous plants. Therefore, cloning the Paphiopedilum trehalose hydrolase gene and verifying its regulatory role on trehalose hydrolase activity through plant genetic transformation is of great significance for developing plant sugar metabolism regulatory gene resources and studying the trehalose utilization mechanism in orchids. Summary of the Invention

[0005] This invention provides a PaTRE gene, derived from Paphiopedilum baihulanum, and its application. The PaTRE gene was cloned from Paphiopedilum baihulanum, and a plant overexpression vector was constructed. Through genetic transformation, PaTRE was overexpressed in tobacco, significantly increasing the activity of trehalose hydrolase in plant tissues. This provides a new gene resource and technical solution for the regulation of plant sugar metabolism and molecular breeding.

[0006] The first objective of this invention is to provide a Paphiopedilum trehalose hydrolase gene PaTRE, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] A second objective of this invention is to provide a protein encoded by the PaTRE gene of Paphiopedilum trehalose hydrolase, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0008] This invention cloned the PaTRE gene from the protocorm of Paphiopedilum 'White Flag', which has an open reading frame length of 1791 bp and encodes 596 amino acids.

[0009] The third objective of this invention is to provide a recombinant expression vector containing the PaTRE gene of Paphiopedilum trehalose hydrolase described above.

[0010] Preferably, the recombinant expression vector is constructed based on pGreen 35S-GFP.

[0011] A fourth objective of this invention is to provide a recombinant host cell containing the Paphiopedilum trehalose hydrolase gene PaTRE or a recombinant expression vector described above.

[0012] The fifth objective of this invention is to provide the application of the Paphiopedilum trehalase gene PaTRE, the protein encoded by the Paphiopedilum trehalase gene PaTRE, the recombinant expression vector, or the recombinant host cell in improving the activity of trehalase in plant tissues.

[0013] Preferably, the plant is tobacco.

[0014] The sixth objective of this invention is to provide a method for improving the activity of trehalose hydrolase in plant tissues, comprising: introducing the above-mentioned Paphiopedilum trehalose hydrolase gene PaTRE or the above-mentioned recombinant expression vector into plants, thereby overexpressing the gene PaTRE in plants and thus improving the activity of trehalose hydrolase in plant tissues.

[0015] Preferably, the plant is tobacco.

[0016] Beneficial effects:

[0017] This invention involves constructing the PaTRE gene into a plant expression vector and introducing it into tobacco using Agrobacterium-mediated transformation to obtain PaTRE transgenic tobacco plants. PCR results showed that the PaTRE gene had been introduced into the tobacco genome; real-time quantitative PCR results showed that the PaTRE gene was expressed in transgenic tobacco; and trehalose hydrolase activity assays showed that the trehalose hydrolase activity in the leaves of PaTRE transgenic tobacco was significantly higher than that in control tobacco. These results indicate that the PaTRE gene has the function of increasing the activity of trehalose hydrolase in plant tissues and can be used for the regulation of plant trehalose metabolism, research on related functional genes, and plant molecular breeding. Attached Figure Description

[0018] Figure 1This is a diagram showing the structure and functional element distribution of the PaTRE plant overexpression recombinant vector in Example 1.

[0019] Figure 2 This is a diagram showing the PCR identification results of the positive clone of the PaTRE plant overexpression recombinant vector in Example 1.

[0020] Figure 3 The image shows the PCR identification results of the PaTRE transgenic tobacco plants in Example 2; where WT-1 and WT-2 are control tobacco plants, AF is a candidate transgenic tobacco plant, and M is the DNA molecular weight standard.

[0021] Figure 4 The figure shows the expression of the PaTRE gene in tobacco and the detection results of trehalose hydrolase activity in Example 3; where A is the real-time fluorescence quantitative PCR detection result of the PaTRE gene, B is the detection result of trehalose hydrolase activity in tobacco leaves, WT is the empty vector control tobacco, and OE is the PaTRE transgenic tobacco.

[0022] Figure 5 The image shows the colorimetric results of trehalose hydrolase activity in the leaves of PatRE transgenic tobacco in Example 3; where WT1-WT3 are empty control tobacco, and AC is PatRE transgenic tobacco. The darker the color, the higher the trehalose hydrolase activity. Detailed Implementation

[0023] To ensure that the objectives, technical solutions, and advantages of this invention are clearer and more complete, the technical solutions in the embodiments of this invention will be described in more detail below with reference to the accompanying drawings. Specific embodiments are further illustrations of this invention and are not intended to limit it. Based on the embodiments of this invention, embodiments obtained by those skilled in the art without inventive technological improvements will be considered to fall within the scope of protection of this invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0024] The plant materials used in this invention include Paphiopedilum white flag bulbs and tobacco ( Nicotiana tabacum NC89 was used, with Paphiopedilum protocorms used for cloning and sequence acquisition of the PaTRE gene, and Tobacco used for overexpression and functional verification of the PaTRE gene. Vectors used included the pClone007 cloning vector and the pGreen 35S-GFP plant overexpression vector. Strains and competent cells used included *Escherichia coli* DH5α (purchased from Beijing Qingke Biotechnology Co., Ltd.) and *Agrobacterium tumefaciens* pSoup-EHA105 (purchased from Shanghai Weidi Biotechnology Co., Ltd.).

[0025] The main reagents used included the pClone007 cloning vector (purchased from Beijing Qingke Biotechnology Co., Ltd.); 2×Phanta Flash Mix high-fidelity PCR enzyme (purchased from Nanjing Novizan Biotechnology Co., Ltd.); a plant RNA extraction kit (purchased from Beijing Huayueyang Biotechnology Co., Ltd.); the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit (AT311) and PerfectStart® Green qPCR SuperMix real-time PCR reagent (AQ601) (purchased from Beijing TransGen Biotech Co., Ltd.); and a trehalase test kit (BK2807B) (purchased from BIOESN). Agarose, DNA molecular weight standards, nucleic acid dyes, gel extraction kits, plasmid extraction kits, homologous recombination reagents, restriction endonucleases, antibiotics, and plant tissue culture reagents used in gene cloning, vector construction, and tobacco genetic transformation were all commercially available products.

[0026] The main instruments used include a clean bench (AIRtech, SWCJ1), a ball mill (Retsch, MM400), a microcentrifuge (Hermle, Z216 MK), a micro spectrophotometer (NanoDrop 2000), a real-time quantitative PCR instrument (Analytik Jena, qTOWER384), an electric thermostatic water bath (Shanghai Yiheng, HWS-12), and a spectrophotometer or microplate reader.

[0027] Example 1: Cloning of the PaTRE gene and construction of a plant overexpression vector

[0028] 1. Cloning of the PaTRE gene

[0029] Total RNA was extracted from the protocorms of *Paphiopedilum baixiang* and reverse transcribed to synthesize cDNA. Specific primers PaTRE-F (SEQ ID NO.3) / PaTRE-R (SEQ ID NO.4) were designed based on the PaTRE gene sequence in the *Paphiopedilum baixiang* transcriptome to amplify the open reading frame of the PaTRE gene using cDNA as a template. The total volume of the PCR reaction system was 50 μL, including 25 μL of 2×Phanta Flash Mix, 2 μL of forward primer, 2 μL of reverse primer, 2 μL of cDNA, and 19 μL of ddH2O. The amplification program was 35 cycles: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 60℃ annealing for 5 s, and 72℃ extension for 20 s, followed by a 1 min extension at 72℃. The sample was then stored at 4℃.

[0030] After PCR products were detected by 1% agarose gel electrophoresis, the target band was excised and recovered from the gel. The recovered product was ligated into the pClone007 cloning vector, transformed into E. coli DH5α competent cells, and after antibiotic selection and colony PCR identification, positive clones were sent for sequencing.

[0031] After sequencing confirmation, the complete open reading frame of Patre was obtained, and its nucleotide sequence is shown in SEQ ID NO.1:

[0032] The amino acid sequence encoded by the PaTRE gene is shown in SEQ ID NO. 2: MAVINSLLPKVPSFFLLFLLLPLLSLPMCASAEAAAACTGDPTQSSTPLVTFLQRLQSEALKTLGPKSFDPKLYVDLPLKRDLADAESAFATLPRVDGVIPNSSLATFLDGYFGEAGGDLVYFEPRDFAPEPAGFLPAVENPEVRAWALQIHSLWKNLSRKVSDEVRQRPERHTLLPLPEPVIIPGSRFREVYYWDSYWIIRGLLVSKMYDTAKSIATNLLWLVDNYGHAFNGARVYYANRSQPPLLSSMVMEIFLKTKDLEFVRKSLPTLIKEHNFWNSGIHKVMIRDLHGHKHSLSRHFAMWNRPRPESGTIDEESASKLINASDKEKFYRQVASAAETGWDFSSRWMRNSSDITTLAATSIIPVDLNAYLYKVEVDLAFFANEVGDVPAHKKFLKASKARLSAFRSIFWNEEKNQWLDYWLTTSKSNCEDVHQFEARNQNGKTFISNFIPLWTLRHISGEDGTSVERILKSFKNSDLLNPAGISTSLLNSGQQWDFPNGWAPLQHMIIEGLANSGSKSALALAEDISVRWIRTNYAAYKTTGAMHEKYDVEGCGMVGGGGEYNPQTGFGWSNGVLLALLEEFGWPIDRKIECQ。

[0033] 2. Construction of PaTRE plant overexpression vector

[0034] Primers with homologous arms, PaTRE-OE-F (SEQ ID NO.5) / PaTRE-OE-R (SEQ ID NO.6), were designed based on the PaTRE gene open reading frame and the pGreen 35S-GFP vector sequence. The PaTRE target fragment was amplified using a correctly sequenced clone plasmid as a template. The pGreen 35S-GFP vector was double-digested with EcoRI and XbaI in a 50 μL reaction mixture, including 5 μL of 10×QuickCut Buffer, 2 μL of EcoRI, 2 μL of XbaI, and 2 μg of plasmid DNA. ddH2O was added to a final volume of 50 μL, and the reaction was incubated at 37℃ for 30 min. The digestion products were detected by 1% agarose gel electrophoresis, and the linearized vector was recovered. The PaTRE target fragment and the linearized vector were ligated using homologous recombination. The total reaction volume was 10 μL, including 5 μL of 2×HIFI Seamless Cloning Mix, 60 ng of linearized vector, 200 ng of PaTRE target fragment, and ddH2O added to a final volume of 10 μL. The reaction was carried out at 50℃ for 15 min. The recombinant product was transformed into *E. coli* DH5α competent cells and cultured overnight at 37℃ inverted position. Six single clones were randomly selected for colony PCR identification and sequencing. The constructed vector drives PaTRE gene expression via the CaMV 35S promoter. The vector structure is shown below. Figure 1 As shown; colony PCR results showed that 2 out of 6 candidate single clones amplified the target band of the expected size (the target fragment was the 1791 bp PaTRE gene). Sequencing results showed that the PaTRE insertion sequence and orientation were correct, and the pGreen 35S-GFP-PaTRE plant overexpression vector was successfully obtained. The results are shown in the figure. Figure 2 As shown.

[0035] Example 2: Genetic transformation and positive identification of the PaTRE gene in tobacco

[0036] 1. Transformation of Agrobacterium tumefaciens with recombinant vectors

[0037] Thaw Agrobacterium tumefaciens EHA105 competent cells on ice, add 500 ng pGreen 35S-GFP-PaTRE recombinant plasmid (Example 1), gently mix, incubate at 37°C for 5 min, then place on ice for 5 min. Add 700 μL of antibiotic-free LB liquid medium, incubate at 28°C with shaking for 2–3 h, centrifuge at 6000 rpm for 1 min, resuspend the cells in approximately 200 μL of supernatant, spread on LB solid medium containing 50 μg / mL kanamycin, and incubate upside down at 28°C for 2–3 days.

[0038] Positive single colonies were picked and inoculated into LB liquid medium containing kanamycin and cultured overnight at 28°C with shaking at 200 rpm. 50 μL of the bacterial culture was then transferred to 50 mL of LB liquid medium containing kanamycin and cultured until OD500. 600 The bacterial cells were collected by centrifugation at 5000 rpm for 5 min with a pH of 0.6–0.8. After washing with MS liquid medium, the cells were resuspended in MS liquid medium containing 100 μM acetylsyl syringone and allowed to stand in the dark for 2–3 h to obtain the tobacco infection solution.

[0039] 2. Genetic transformation of tobacco leaf discs

[0040] Sterile tobacco NC89 seedlings were used as recipient materials. Sterilized tobacco seeds were treated at 4℃ for 2–3 days, then inoculated onto 1 / 2 MS solid medium and cultured at 25–27℃, light intensity of 2300–2500 Lux, and a photoperiod of 12 h / d. Healthy leaves were selected, the midrib removed, and cut into leaf discs of 0.2–0.5 cm². The leaf discs were immersed in Agrobacterium infection solution for 10–15 min, then removed, and the surface bacterial solution was blotted off with sterile filter paper. The leaf discs were then transferred to a co-culture medium and cultured in the dark for 3 days. The co-culture medium was based on MS medium with the following components added (per liter): 30 g sucrose, 6 g agar, 2 mg 6-BA, and 0.2 mg NAA.

[0041] After co-cultivation, the leaf discs were washed 3-4 times with sterile water, and after drying the surface moisture, they were transferred to a selection medium containing Basta and cephalosporin antibiotics for resistance screening and adventitious shoot induction. The selection medium was based on MS medium with the following components added (per liter): 30g sucrose, 6g agar, 2mg 6-BA, 0.2mg NAA, 2mg Basta, and 400mg penicillin cephalosporin. When the resistant adventitious shoots grew to more than 5 cm, they were cut off and transferred to a rooting medium based on MS medium with the following components added (per liter): 30g sucrose, 6g agar, 2mg Basta, and 200mg penicillin cephalosporin. After the root system was fully developed, the seedlings were hardened off for 7 days, the root medium was washed off, and the seedlings were transplanted to obtain candidate PatRE transgenic tobacco plants.

[0042] 3. Positive identification of genetically modified tobacco

[0043] Genomic DNA was extracted from leaves of candidate transgenic tobacco and empty-vector control tobacco, and PCR amplification was performed using the PaTRE gene-specific primers PaTRE-qF (SEQ ID NO.7) / PaTRE-qR (SEQ ID NO.8). The total volume of the PCR reaction system was 50 μL, including 25 μL of 2×Phanta Flash Mix, 2 μL of forward primer, 2 μL of reverse primer, 2 μL of genomic DNA, and 19 μL of ddH2O. The amplification program was 35 cycles: 98℃ pre-denaturation for 30 s, 98℃ denaturation for 10 s, 60℃ annealing for 5 s, and 72℃ extension for 20 s, followed by a 1 min extension at 72℃.

[0044] PCR products were detected by 1% agarose gel electrophoresis. Results are as follows: Figure 3 As shown, candidate transgenic tobacco lines A to F all amplified the PaTRE target band of the expected size, while the empty vector control tobaccos WT-1 and WT-2 did not amplify the corresponding band, indicating that the PaTRE gene has been successfully integrated into the tobacco genome.

[0045] Example 3: Expression analysis of PaTRE transgenic tobacco and detection of trehalose hydrolase activity

[0046] 1. Detection of PaTRE gene expression level

[0047] Leaves from the same leaf position of PaTRE-positive transgenic tobacco (OE) and empty-vector control tobacco (WT) were collected, and total RNA was extracted and reverse transcribed to synthesize cDNA. The cDNA concentration was uniformly adjusted to 50 ng / μL. Gene expression levels were detected using PaTRE gene-specific primers qPaTRE-F (SEQ ID NO. 9) / qPaTRE-R (SEQ ID NO. 10). Real-time quantitative PCR was performed using ACTIN7 as an internal reference gene, with primers ACTIN7-F (SEQ ID NO. 11) / ACTIN7-R (SEQ ID NO. 12). The total reaction volume was 20 μL, including 1 μL of cDNA, 1 μL of forward primer, 1 μL of reverse primer, 10 μL of 2×PerfectStart Green qPCR SuperMix, and 7 μL of RNase-free water. The amplification program consisted of 94℃ pre-denaturation for 30 s, followed by 94℃ denaturation for 5 s, 57℃ annealing for 15 s, and 72℃ extension for 10 s, for a total of 40 cycles. Melting curve analysis was then performed, with three biological replicates for each sample. The relative expression level of the PaTRE gene was calculated using the 2^-ΔΔCt method.

[0048] The results are as follows Figure 4As shown in Figure A, the relative expression level of the PaTRE gene in the PaTRE transgenic tobacco was significantly higher than that in the empty vector control tobacco, indicating that the PaTRE gene can be stably expressed in tobacco.

[0049] 2. Detection of Trehalose Hydrolase Activity

[0050] Weigh 0.1 g of fresh leaves from PatRE transgenic tobacco and empty control tobacco, respectively, add 1 mL of pre-cooled extraction solution, grind thoroughly under ice bath conditions, centrifuge at 8000 ×g for 10 min at 4℃, and collect the supernatant as crude enzyme solution. The samples were tested using a trehalase test kit (BK2807B): 90 μL of crude enzyme solution and 150 μL of reagent-1 were added to the test tube, and 90 μL of crude enzyme solution and 150 μL of distilled water were added to the control tube. After mixing, the mixture was reacted in a 45℃ water bath for 15 min, and then the reaction was terminated by incubating in a 95℃ water bath for 5 min. If precipitation occurred, centrifuge at 9000 ×g for 10 min at 4℃, and collect the supernatant for color development.

[0051] Prepare the working solution by mixing reagents 2 and 3 at a volume ratio of 1:1. Take 100 μL of the reaction solution from both the test tube and the control tube, add 900 μL of the working solution, and let stand at room temperature for 15 min. Measure the absorbance at 505 nm. Calculate the absorbance difference as ΔA = A_test tube - A_control tube, and calculate the trehalose hydrolase activity as THL activity = 366 × (ΔA + 0.0042) ÷ W, where W is the fresh weight of the sample, and the enzyme activity unit is nmol·min^2. -1 ·g^ -1 FW. Color development results are as follows Figure 5 As shown, the color development of PatRE transgenic tobacco lines A–C was significantly deeper than that of the unloaded control tobacco lines WT1–WT3; the quantitative results are as follows. Figure 4 As shown in B, the trehalose hydrolase activity of the PaTRE transgenic tobacco is 3700–4100 nmol·min⁻¹. -1 ·g^- 1 FW, empty control tobacco concentration: 50–150 nmol·min^ -1 ·g^ -1 FW indicates that overexpression of the PatRE gene in tobacco can significantly increase the activity of trehalose hydrolase in plant tissues.

[0052] Table 1

Claims

1. A PaTRE gene for trehalose hydrolase from Paphiopedilum blazei, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

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

2.

3. A recombinant expression vector, characterized in that, It contains the PaTRE gene, a trehalose hydrolase from Paphiopedilum baiji as described in claim 1.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector was constructed based on pGreen35S-GFP.

5. A recombinant host cell, characterized in that, The recombinant expression vector containing the PaTRE gene of Paphiopedilum trehalose hydrolase as described in claim 1 or any one of claims 3-4.

6. The application of the PaTRE trehalose hydrolase gene of Paphiopedilum blazei according to claim 1, the protein according to claim 2, the recombinant expression vector according to any one of claims 3-4, or the recombinant host cell according to claim 5 in improving the activity of trehalose hydrolase in plant tissues.

7. The application according to claim 6, characterized in that, The plant in question is tobacco.

8. A method for improving the activity of trehalose hydrolase in plant tissues, characterized in that, The process includes the following steps: introducing the PaTRE trehalose hydrolase gene from Paphiopedilum baiji as described in claim 1 or the recombinant expression vector as described in any one of claims 3-4 into a plant, thereby overexpressing the PaTRE gene in the plant and increasing the trehalose hydrolase activity in plant tissues.

9. The method according to claim 8, characterized in that, The plant in question is tobacco.

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