Dendrobium officinale dehydrin protein DcDHN1 as well as coding gene and application thereof

Abstract

The invention discloses a Dendrobium officinale Kimura et Migo dehydrin protein DcDHN1 as well as a coding gene and application thereof, the amino acid sequence of the dehydrin protein DcDHN1 is as shown in SEQ ID NO.2, and the coding gene is a nucleotide sequence as shown in SEQ ID NO.1; the invention also provides application of the dehydrin protein DcDHN1 and the coding gene thereof in improving cold resistance of dendrobium officinale. According to the invention, the phenotype is observed after overexpression of DcDHN1 in Escherichia coli by using a genetic engineering technology. The DcDHN1 positively regulates the low temperature resistance, and provides important theoretical basis and gene resources for the cultivation of high-quality new varieties of dendrobium officinale.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and abiotic stress resistance technology, specifically relating to a Dendrobium officinale dehydrin protein DcDHN1, its encoding gene, and its applications. Background Technology

[0002] Dendrobium officinale ( Dendrobium catenatum Dendrobium officinale (Tiepi Shihu) is a perennial herb belonging to the genus Dendrobium in the family Orchidaceae. It possesses medicinal properties such as nourishing yin and clearing heat, benefiting the stomach and promoting the production of body fluids. The fresh or dried stems are used medicinally, and it is a traditional, precious, and rare Chinese medicinal herb. Currently, Dendrobium officinale is mainly cultivated artificially. Especially in artificial semi-wild cultivation, low temperature is the main abiotic stress factor restricting its growth and development and hindering its industrial-scale production. Low temperatures can lead to the formation of intracellular ice crystals and cell membrane rupture, thereby inhibiting photosynthetic efficiency and, in severe cases, causing plant death.

[0003] Late embryogenesis abundant protein (LEA) is a group of proteins discovered during the late embryonic development of plant seeds. These proteins are highly hydrophilic and thermostable, belonging to intrinsically disordered proteins (IDPs). Based on the conserved PFAM domains of LEA proteins, plant LEA proteins can be divided into eight subfamilies: LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6 / PvLEA18, Dehydrin, and SMP. Dehydrins (DHNs) typically contain highly conserved K fragments [EKKGIME / DKIKEKLPG], Y fragments [(V / T)D(E / Q)YGNP], and S fragments [LHRSGS4-10(E / d)3]. Based on these three conserved regions, dehydrins can be classified into YnSKn, YnKn, SKn, Kn, and KnS types. Although the functions of dehydrin in many plants have been identified, there is limited research on its role in Dendrobium officinale's response to low-temperature stress, which warrants further investigation. Summary of the Invention

[0004] In view of this, the present invention provides a Dendrobium officinale dehydrin protein DcDHN1, its encoding gene, and its application.

[0005] The amino acid sequence of the Dendrobium officinale dehydrin protein DcDHN1 described in this invention is shown in SEQ ID NO.2; it includes an S-fraction consisting of a serine residue and three conserved lysine-rich K-fractions, belonging to the SK3 type dehydrin.

[0006] The present invention also provides an application of the above-mentioned Dendrobium officinale dehydrin protein DcDHN1 in improving the cold resistance of Dendrobium officinale.

[0007] The present invention also provides a coding gene for the above-mentioned Dendrobium officinale dehydrin protein DcDHN1, wherein the coding gene is a nucleotide sequence as shown in SEQ ID NO.1.

[0008] The aforementioned encoding genes were obtained through cloning in Dendrobium officinale and / or artificial synthesis.

[0009] The present invention also provides a plant overexpression vector containing the encoding gene of the above-mentioned Dendrobium officinale dehydrin protein DcDHN1.

[0010] The above-mentioned plant overexpression vector is pHB- DcDHN1 -YFP.

[0011] The present invention also provides the application of the encoding gene of the above-mentioned Dendrobium officinale dehydrin protein DcDHN1 in improving the cold resistance of Dendrobium officinale.

[0012] Beneficial effects: This invention clones a Dendrobium officinale dehydrin protein DcDHN1, which is located in the cell membrane and nuclear membrane, and significantly responds to low temperature in both roots and leaves, thereby improving the low temperature tolerance of Escherichia coli. This invention provides a basis for its effective application and is of great significance for improving the cold resistance of Dendrobium officinale, especially for realizing genetic engineering breeding. Attached Figure Description

[0013] Figure 1 for DcDHN1 Image of PCR amplification results of the gene; Figure 2 This is a diagram showing the amino acid sequence alignment analysis of homologous proteins of the dehydrin protein DcDHN1; Figure 3 for DcDHN1 Expression patterns of genes in leaves and stems under low temperature treatment; where (a) is the expression pattern in leaves; and (b) is the expression pattern in stems. Figure 4 Subcellular localization of dehydrin protein DcDHN1 in tobacco epidermal cells; Figure 5 for DcDHN1 Growth diagrams of gene-transformed Escherichia coli under low temperature conditions; where (a) is the growth phenotype under normal conditions; and (b) is the growth phenotype under low temperature conditions. Figure 6 for DcDHN1 Graphs showing the changes in OD values ​​of gene-transformed Escherichia coli under low temperature conditions; where (a) shows the changes in OD values ​​under low temperature conditions; and (b) shows the changes in OD values ​​under normal conditions. Detailed Implementation

[0014] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0015] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition), or as recommended in the reagent instructions.

[0016] Example 1: DcDHN1 Gene cloning and sequence analysis Plant material preparation: Dendrobium officinale seedlings, 12-15cm tall. After washing away any residual culture medium from the uniformly growing Dendrobium officinale seedlings, spread them evenly on a tray lined with double layers of filter paper. Allow the surface moisture to dry naturally before transplanting them into cultivation pots containing peat moss substrate (25℃, 65% humidity, 16 h light / 8 h dark, light intensity approximately 40 μmol / m²). -2 s -1 After two weeks of cultivation, 0.2 g of Dendrobium officinale leaves were weighed, quickly wrapped tightly in aluminum foil, and frozen in liquid nitrogen for 15 minutes. They were then transferred to an ultra-low temperature freezer at -80℃ for storage.

[0017] Total RNA extraction was performed using the SteadyPure Universal RNA Extraction Kit (Accurate Biology, AG21019, China). cDNA was synthesized via reverse transcription using the Evo M-MLV Reverse Transcription Kit (Accurate Biology, AG21019, China).

[0018] Primer design and synthesis: Based on the genomic data of *Dendrobium officinale*, primers for the target gene sequence were designed and synthesized by Youkang Biotechnology Co., Ltd. Using cDNA from *Dendrobium officinale* leaves as a template, PCR amplification was performed according to the following reaction system and conditions: 12.5 μL of LATaq DNA Polymerase (purchased from TaKaRa), and 10 μmol / L primer F- DcDHN1 (5'-ATGGCTGAAGAGTTCAAGGATC -3') and primer R- DcDHN1 1 μL each of (5'-TCAATGACTACCAGGACTCTTCT-3'), 2 μL of cDNA, and ddH2O to a final volume of 25 μL. Reaction conditions: pre-denaturation 98℃ for 3 min; 98℃ for 10 s; 55℃ for 15 s; 72℃ for 1 min; 30 cycles; extension at 72℃ for 5 min. DcDHN1 PCR amplification results of gene sequences as follows Figure 1As shown. After separation by gel electrophoresis, the amplified fragment was recovered and ligated with the cloning vector pMD18-T (TaKaRa). The resulting product was then transformed into *E. coli* DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.). After positive clone identification, the cells were sent to Youkang Biotechnology for sequencing analysis. DcDHN1 The full-length open reading frame sequence of the gene is 753 bp, and the detailed sequence results are shown in SEQ ID NO.1.

[0019] Example 2: Amino acid sequence alignment analysis of dehydrin protein DcDHN1 homologous proteins pass DcDHN1 Open reading frame sequence deduction revealed that the dehydrin protein DcDHN1 is a protein containing 250 amino acid residues, as shown in SEQ ID NO.2. Amino acid sequence alignment analysis was performed using DNAMAN software (the alignment included *Apostasia shenzhenica*, *Phalaenopsisequestris*, *Phoenix dactylifera*, and *Vanilla planifolia*; the alignment results are shown below). Figure 2 .Depend on Figure 2 It is known that the dehydrin protein DcDHN1 contains an S-segment consisting of a serine residue and three conserved lysine-rich K-segments, belonging to the typical SK3 type dehydrin.

[0020] Example 3: DcDHN1 Gene expression patterns in leaves and stems under low temperature treatment Dendrobium officinale grown in a greenhouse was subjected to low-temperature stress treatment in a 4℃ incubator (photocycle: 16 h light / 8 h dark). Samples were collected at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after stress treatment. The samples were flash-frozen in liquid nitrogen and then stored in an ultra-low temperature freezer at -80℃ for subsequent relative expression analysis. To ensure experimental reproducibility and data stability, three biological replicates were set up for each treatment.

[0021] The methods for extracting total RNA from different tissues and synthesizing cDNA were the same as in Example 1. DcDHN Design specific primers qF-1 for the coding sequence of a gene. DcDHN1 :5'-GGTTGTTTGACTTCGTCCA-3',qR- DcDHN1 :5'-GCCGTCCTCTATGTGAATCT-3', DcActin2The internal reference gene was used, and the upstream and downstream primer sequences were: qF: 5'-TTGTGTTGGATTCTGGTGATGGTGT-3', qR: 5'-TTTCCCGTTCTGCTGTTGTTGTGAA-3'. Quantitative detection was performed using a Bio-Rad real-time quantitative PCR instrument. The PCR reaction system consisted of: 10 μL 2× SYBR Green Pro Taq HSPremix (purchased from Aikerui Biotechnology), 0.4 μL each of upstream primer qF and downstream primer qR, 2 μL cDNA, and water to a total volume of 20 μL. The reaction program was 94℃ for 30 s; 95℃ for 5 s; 58℃ for 15 s; 72℃ for 15 s; 40 cycles. Each treatment was performed in triplicate (biological replicates) and in triplicate (technical replicates). Two... −ΔΔCT The data were analyzed using SPSS 26 for statistical analysis and GraphPad Prism 10.4.1 for plotting. DcDHN1 Gene expression patterns, such as Figure 3 As shown, Figure 3 for DcDHN1 Schematic diagrams of gene expression patterns in leaves and stems under low-temperature treatment; where (a) shows the expression pattern in leaves; and (b) shows the expression pattern in stems. It can be seen that... DcDHN1 The upregulation of expression in response to low temperature suggests that it may regulate plant resistance to low temperature stress.

[0022] Example 4: Subcellular localization of dehydrin protein DcDHN1 in tobacco epidermal cells Construction of plant expression vectors: using plants containing DcDHN1 Using recombinant plasmids of gene sequences as templates, BamH I, Spe I restriction endonuclease was used to construct the target gene into the pHB-YFP vector via homologous recombination, thus obtaining the plant overexpression vector pHB- DcDHN1 -YFP (abbreviated as) DcDHN1 -YFP); the ligation product was transformed into E. coli competent cells, and after verifying the positive clone by bacterial PCR and confirming that the sequencing was correct, the recombinant plasmid was extracted and transformed into Agrobacterium GV3101 competent cells.

[0023] Instantaneous conversion of tobacco leaves: Select leaves containing... DcDHN1 Agrobacterium monoclonal strains of -YFP and pHB-YFP (empty vector) were added to a solution containing 50 mg L... -1 Kan and 100 mg L -1 In Rif's LB liquid medium, cultured at 28°C on a shaker at 180-200 rpm until the bacterial culture OD... 600The value reached approximately 1.2; the bacterial cells were collected by centrifugation at 5000 rpm for 15 min, the precipitate was resuspended in MS liquid medium, and the OD value of the bacterial culture was adjusted. 600 The concentration was adjusted to 0.6, and acetylsyl syringone (AS) and MES buffer (final concentrations of 0.2 mM and 10 mM, respectively) were added. The mixture was gently inverted and incubated in the dark at room temperature for at least 3 hours. The bacterial culture was injected into the underside of tobacco leaves using a 1 mL disposable syringe, and the plants were cultured in the dark for 48 hours. Subsequently, a 1 cm² section of the injected leaf was cut, and subcellular localization was observed using a laser confocal microscope. The results are as follows: Figure 4 As shown, by Figure 4 It is known that the dehydrin protein DcDHN1 is located in the cell membrane and nuclear membrane.

[0024] Example 5: Overexpression DcDHN1 Effects of low temperature stress on Escherichia coli Construction of prokaryotic expression vectors: using homologous recombination. Construction of prokaryotic expression vectors pET32a-DcDHN1 was transformed into E. coli, and the results were verified by colony PCR and sequencing. The positive plasmid was then transformed into competent Rosetta (DE3) cells of E. coli.

[0025] The initial concentrations of the pET32a-DcDHN1 recombinant bacteria and the pET32a control bacteria (empty vector) were uniformly adjusted to OD=0.8, and 50 mg L was used. -1 Amp's liquid LB medium was prepared at 10... 0 10 -1 10 -2 10 -3 10 -4 10 -5 Dilute the bacterial suspension. Spot the diluted bacterial suspension onto a plate at a concentration of 50 mg / L. -1 The plates containing Amp solid LB medium were placed in incubators at 37°C (normal conditions) and 18°C ​​(low temperature) for 12 hours, respectively, and then observed. The results are as follows. Figure 5 As shown. By Figure 5 It can be seen that under normal conditions, empty load and expression DcDHN1 The bacteria have similar growth states and express themselves at low temperatures. DcDHN1 The bacterial growth was significantly better than that of the empty vector strain. Figure 5 In the middle, the scale bar is 0.5 cm.

[0026] After inducing pET32a-DcDHN1 recombinant bacteria and pET32a control bacteria (empty vector) with 0.2 mM IPTG for 6 h, the OD was adjusted. 600 1. Take 500 μL of bacterial culture and add it to 15 mL of liquid LB medium (containing 50 mg L). -1In Amp and chloramphenicol). The bacteria were cultured at 37℃ (normal conditions), 18℃ (low temperature), and 220 rpm, and the OD of Escherichia coli was measured at 0h, 1h, 3h, 5h, 7h, and 9h. 600 Values. The experiment was repeated three times, with three parallels set each time, and bar charts were plotted. Figure 6 .Depend on Figure 6 It can be seen that under normal conditions, empty load and expression DcDHN1 No significant difference in OD values ​​between bacterial cultures; expression at low temperature DcDHN1 The bacterial OD value was significantly higher than that of the empty vector strain, indicating that DcDHN1 It has the ability to withstand low temperatures.

[0027] Unless otherwise specified, all technologies mentioned above refer to existing technologies.

[0028] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A Dendrobium candidum dehydrin protein DcDHN1, characterized in that, The amino acid sequence of the dehydrin protein DcDHN1 is shown as SEQ ID NO.

2.

2. Application of the Dendrobium candidum dehydrin protein DcDHN1 in claim 1 to improve the cold resistance of Dendrobium candidum.

3. A coding gene encoding the Dendrobium officinale dehydrin protein DcDHN1 according to claim 1, characterized in that, The coding gene is a nucleotide sequence shown as SEQ ID NO.

1. 4.The Dendrobium candidum desiccation-responsive protein DcDHN1-encoding gene of claim 3, characterized in that, The coding gene is obtained by cloning and / or artificial synthesis in Dendrobium candidum.

5. A plant overexpression vector, characterized by, 6. Application of the coding gene of the Dendrobium candidum dehydrin protein DcDHN1 in claim 3 or 4 to improve the cold resistance of Dendrobium candidum. ​

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