CN112063632B - Dendrobium officinale transcription factor DobHLH4 and application thereof - Google Patents

Abstract

The invention discloses a dendrobium officinale transcription factor DobHLH4 and application thereof. The transcription factor DobHLH4 can be combined with a G-box (CACGTG) combination site of a terpene synthetase DoTPS10 gene promoter region to activate the expression of the DoTPS10 gene, so that the increase of the linalool content in the dendrobium officinale flowers is promoted. The transcription factor DobHLH4 can be used for genetic engineering breeding for improving the aromatic flowers of the dendrobium officinale, and the aromatic odor of the dendrobium officinale flowers is improved.

Description

Dendrobium officinale transcription factor DobHLH4 and application thereof

Technical Field

The invention belongs to the technical field of plant molecular biotechnology and genetic engineering, and particularly relates to a dendrobium officinale transcription factor DobHLH4 and application thereof.

Background

Dendrobium officinale (Dendrobium officinale Kimura et Migo) is a perennial epiphytic herb of the genus Dendrobium (Dendrobium) of the family Orchidaceae. Dendrobium plants, phalaenopsis, oncidium and cattleya are called as 'four-great ornamental orchids in the world', and have extremely high ornamental value due to attractive flower fragrance, gorgeous flower color and peculiar flower shape (Zhugen, Guo flutter, the molecular biology research progress of important ornamental orchidaceae plants. botany report, 2004,21(4): 471-.

The flower fragrance is one of three elements of color, fragrance and shape of ornamental plants, and is known as the soul of flowers. The flower fragrance not only can improve the ornamental value of orchids, but also is related to the pollination characteristic of plants. Certain floral substances of orchids are the same as pheromones that attract pollinating insects, and may attract insects to help pollinate them, as well as natural enemies of herbivores, to reduce damage to themselves and to increase reproductive reproduction and adaptability of ethnic groups (Schiestl F.P., Peak R.two animals attracting pollination regulators with the same aqueous biology. functional Ecology,2005,19(4):674 and 680; Gervasi D., passage M.A., Sauuve, M., et al. floral science and species differentiation a pair of sexually targeted organisms and Evolution, 6027, 7(15): 60234).

However, most varieties of phalaenopsis and dendrobium which are cultivated at present do not have flowery odour, which is mainly because the traditional breeding focuses on the aspects of flower color, flower type, high yield and resistance, artificial forward breeding and screening are not carried out aiming at flowery odour characteristics, and breeding traits linked with flowery odour are not excavated, so that a large number of flowers lose flowery odour characteristics in the long-term breeding process (Zhanying, Wang Yan, Li Jian Shao, and the like. GC-MS analysis of aroma components of different dendrobium orchids. Guangxi plants, 2011,31(3):422 hai 426; Li Chong, Huang Ming, Huang Sha, and the like. 4 dendrobium plant flower volatile component analysis. Tropical subtropical plant academic report, 2015,23(4):454 and 462;). Understanding the molecular mechanism behind floral scent is the first step of molecular genetic breeding, and with the favor of the consumer market on the scent varieties, the synthetic regulation and control of floral scent substances gradually become a new research hotspot by improving the floral scent and improving the commercial value of ornamental flowers by utilizing the genetic engineering technology. Linalool, An acyclic monoterpene alcohol, is An important material basis for monoterpene floral substances of orchids (Hsiao Y.Y., Pan Z.J., Hsu C.C., et al, research on biology and biotechnology, plant and Cell Physiology,2011,52(9): 1467) 1486; Ramya M., Jang S., An H.R., et al, volatile organic compounds From: From synthesis and function to gene regulation. International Journal of Molecular Sciences,2020,21(3): 1160). Therefore, the identification of the transcription factor involved in the synthesis and regulation of linalool has important biological and industrial significance.

Linalool, a volatile monoterpene aromatic substance, is produced by catalyzing Geranyl diphosphate (GPP) with Terpene synthase (TPS). TPS requires the divalent metal cation Mg²⁺Or Mn²⁺The catalytic environment is neutral, and the C end and the N end are formed by alpha helical regionsDomain structure, N-terminal contains a section of aspartic acid conservation region DD_XXD, arginine RRX is also present₈W conserved region, with DD_XXD formation of R-D carbon-hydrogen bonds plays a role in stabilizing the structure (Chen F., Tholl D., Bohlmann J., et al., the family of depends synthesis in plants: a mid-size family of genes for specific methods of using the same as a high-density two-dimensional family of plant Journal,2011,66(1): 212. alpha.,. Dudareva N., Klemmeen A., Muhlemann J.K., et al. biosynthesis, function and methodology of planar organic compounds.New Phytologist,2013,198(1): 16-32). The expression of the TPS gene is regulated by a transcription factor, and the transcription factor can be mutually combined with a cis-acting element on a promoter of the TPS gene so as to regulate the expression level of the TPS gene. Members involved in the regulation of terpenoid synthesis have been identified mainly in the AP2/ERF, bHLH, MYB, NAC, WRKY and bZIP families (Chuang, Y.C.; Hung, Y.C.; Tsai, W.C.; et al. PbbHLH4 derivatives flow monoterpene biosyntheses in Phalaenopsis organisms. journal of Experimental Botany,2018,69(18): 4363-. Research aiming at regulating and controlling synthesis of monoterpene aromatic substance linalool by transcription factors is rare, and mainly focuses on pattern plant tomatoes, namely SlMYC1 positively regulates synthesis of monoterpene substances in leaf and stem trichomes, but negatively regulates synthesis of sesquiterpenes in stem trichomes, SlWRKY73 and SlMYC1 can activate tomato linalool synthase SlTPS5, meanwhile, SlEOT1 and SlMYC1 can synergistically activate SlTPS5 and regulate and control synthesis of terpenoid substances (Xu J., van Herwijnen Z.O.,

plant cells, 2018,30(12): 2988-. However, the DoTPS10 promoter was not activated by any of the transcription factors in Dendrobium officinale flowers that are homologous to SlEOT1, SlMYC1 and SlWRKY 73. So far, the upstream transcription factor of the biosynthesis of linalool in dendrobium officinale is not clear, and the transcription factor for regulating the biosynthesis of linalool has not been identified.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a transcription factor DobHLH4 participating in synthesis regulation of the volatile monoterpene substance linalool of dendrobium officinale, wherein the transcription factor DobHLH4 can promote biosynthesis of the monoterpene aromatic substance linalool.

The first purpose of the invention is to provide a transcription factor DobHLH4, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention also provides a protein coded by the transcription factor DobHLH4, and the amino acid sequence of the protein is shown in SEQ ID NO. 4.

The invention also provides a recombinant vector containing the transcription factor DobHLH 4.

Preferably, the recombinant vector is pCAMBIA3300-DobHLH 4.

The invention also provides an engineering bacterium containing the recombinant vector.

Preferably, the engineering bacteria is agrobacterium GV3101(pSoup-p19) strain containing recombinant vector pCAMBIA3300-DobHLH 4.

The invention also provides application of the transcription factor DobHLH4 in promoting linalool biosynthesis.

Preferably, the application is the application of the transcription factor DobHLH4 in promoting the biosynthesis of the linalool in the dendrobium officinale flower.

The invention also provides application of the transcription factor DobHLH4 in promoting expression of DoTPS10 genes.

In the invention, the transcription factor DobHLH4 can be combined with a promoter region G-box (CACGTG) combination site of a monoterpene substance linalool synthase DoTPS10 gene to activate the expression of the DoTPS10 gene, thereby promoting the biosynthesis of the monoterpene substance linalool. Through verification, in different stages of dendrobium officinale flower opening and exogenous methyl jasmonate (MeJA) treatment, the DobHLH4 gene expression is obviously and positively correlated with the content of DoTPS10 gene and linalool. By adopting a genetic transformation technology to over-express the DobHLH4 gene, the linalool content in the dendrobium officinale flowers can be obviously increased. Therefore, the DobHLH4 can remarkably promote the biosynthesis of linalool in the dendrobium officinale flowers and improve the aromatic smell of the dendrobium officinale flowers.

Therefore, the transcription factor DobHLH4 can be used as an important candidate gene in the development of genetic engineering and germplasm improvement of the dendrobium officinale belonging to the Orchidaceae, is used for genetic engineering breeding for improving aromatic flowers of the dendrobium officinale, and improves the aromatic smell of the dendrobium officinale flowers.

Drawings

FIG. 1 shows the amplification of the Dendrobium officinale transcription factor DobHLH4 gene sequence.

FIG. 2 shows the expression level of the transcription factor DobHLH4 gene of Dendrobium officinale Kimura et Migo at different developmental stages, including bud stage, initial flowering stage, full flowering stage and decay stage, i.e., B, S, F, W; the bar of each group of data represents the standard error (n is more than or equal to 10), the statistical analysis is the comparison of each treatment and the control, and the difference of p less than 0.05 has statistical significance by adopting one-factor analysis of variance.

FIG. 3 shows the expression level of linalool synthase gene DoTPS10 of Dendrobium officinale in different developmental stages, including bud stage, initial flowering stage, full flowering stage and decay stage, i.e., B, S, F, W; the bar of each group of data represents the standard error (n is more than or equal to 10), the statistical analysis is the comparison of each treatment and the control, and the difference of p less than 0.05 has statistical significance by adopting one-factor analysis of variance.

FIG. 4 is a mass spectrum of linalool standard in GC-MS analysis.

FIG. 5 shows the content of linalool in Dendrobium officinale at different stages of development, including bud stage, initial flowering stage, full flowering stage and decay stage, i.e., B, S, F, W; the bar of each group of data represents the standard error (n is more than or equal to 10), the statistical analysis is the comparison of each treatment and the control, and the difference of p less than 0.05 has statistical significance by adopting one-factor analysis of variance.

FIG. 6 shows the plant binary expression vector pCABIA3300 used, with NCBI accession number KP 795973.

FIG. 7 is a schematic diagram of construction of a dendrobium officinale DobHLH4 overexpression vector.

FIG. 8 shows the semi-quantitative PCR detection of Dendrobium officinale flowers overexpressing DobHLH4 gene; lane 1, a Dendrobium officinale flower overexpressing DobHLH 4; lane 2-3, non-over-expression treated flowers of Dendrobium officinale.

FIG. 9 shows the expression level of DoTPS10 gene in Dendrobium officinale flowers overexpressing DobHLH4 gene; the bar of each group of data represents the standard error (n is more than or equal to 10), the statistical analysis is the comparison of each treatment and the control, and the difference of p less than 0.05 has statistical significance by adopting one-factor analysis of variance.

FIG. 10 shows the linalool content in Dendrobium officinale flowers overexpressing DobHLH4 gene; the bar of each group of data represents the standard error (n is more than or equal to 10), the statistical analysis is the comparison of each treatment and the control, and the difference of p less than 0.05 has statistical significance by adopting one-factor analysis of variance.

FIG. 11 shows that the protein encoded by the Dendrobium officinale transcription factor DobHLH4 is localized in the cell nucleus.

FIG. 12 is a diagram of the vectors used in the dual luciferase system assay for the transcriptional activation of DoTPS10 promoter by DobHLH 4.

FIG. 13 shows the transcriptional activation effect of Dendrobium officinale transcription factor DobHLH4 on DoTPS10 p; the bar of each group of data represents the standard error (n is more than or equal to 6), the statistical analysis is the comparison of each treatment and the control, and the difference of p less than 0.05 has statistical significance by adopting one-factor analysis of variance.

FIG. 14 shows the yeast single-hybrid detection of the binding of the transcription factor DobHLH4 to DoTPS10 p.

FIG. 15 shows yeast single hybridization detection of the binding of the transcription factor DobHLH4 to DoTPS10pm (the G-box (CACGTG) site of the DoTPS10 promoter DoTPS10p region is mutated to DoTPS10 pm).

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The following examples, in which specific experimental methods are not specified, can be carried out according to conventional methods. Such as those described in molecular cloning guidelines, sambrook et al, f, osiber et al, fine compiled guidelines for molecular biology, or according to the instructions of the manufacturer of the product used.

The Dendrobium officinale (Dendrobium officinale Kimura et Migo) used in the examples was planted in a Dendrobium officinale planting greenhouse (N23 degrees 10 ', E113 degrees 21'; Guangzhou, China) in a south China plant Garden of Chinese academy of sciences; wild type Arabidopsis thaliana (Arabidopsis thaliana) is derived from Columbia ecotype (Columbia, col-0); polysaccharide polyphenol plant RNThe A extraction kit is purchased from Beijing Huayue Biotechnology Limited (catalog number: 0416-50); the reverse transcriptase M-MLV kit was purchased from Promega corporation (cat # M1701); SMARTer^TMRACE cDNA Amplification Kit was purchased from Clontech (cat # 634923); in-

HD Cloning Kit was purchased from Takara (cat # 639648); pMD18-T Vector was purchased from Takara (cat # D101A); SYBR Premix Ex Taq^TMKit is available from Takara corporation (cat # DRR 420A); TaKaRa LA

Purchased from Takara corporation (cat # RR 52A); LB and MS culture media are commonly used in the field, and the formula thereof refers to molecular cloning experimental guidelines of J. SammBruke et al. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 isolation and cloning of the Dendrobium officinale DobHLH4 Gene

(1) Extraction of total RNA of dendrobium officinale flowers and synthesis of cDNA first chain

Taking 100mg of freshly harvested dendrobium officinale flowers, and grinding the flowers into powder under liquid nitrogen. The total RNA of the dendrobium officinale is extracted by adopting a polysaccharide polyphenol plant RNA extraction kit (Beijing Huayuyo Biotechnology limited company, the product number is 0416-50). Using NanoDrop^TMTotal RNA content and purity were determined using a 2000c ultramicrospectrophotometer (Thermo Scientific, Wisconsin, USA) and a 1.0% agarose gel electrophoresis apparatus (Biorad, Calif., USA). Mu.g of purified total RNA was taken, first strand cDNA synthesis was performed according to the instructions of the reverse transcriptase M-MLV kit (Promega corporation, cat. No. M1701), and the reaction product was diluted to the desired concentration and stored in a freezer at-80 ℃.

(2) Amplification and sequence analysis of dendrobium officinale DobHLH4 gene

According to the Dendrobium officinale genome database (Zhang G., Xu Q., Bian C., et al., the Dendrobium catenatum Lindl genome sequence providences insights into polysaccharidesynthase, floral definition and adaptive evaluation scientific Reports,2016,6,19029), using PCR technology, using Dendrobium officinale cDNA as template, using Primer Premier 5.0 software (Premier Biosoft, Calif.) to design the corresponding upstream Primer SEQ ID NO.2 and downstream Primer SEQ ID NO.3, using 2 × Hieff

PCR amplification is carried out on PCR Master Mix (YEASEN company, cat # 10136ES03) to obtain the dendrobium officinale DobHLH4 gene (the sequence is shown as SEQ ID NO. 1). PCR reaction (25. mu.L): reverse transcription template 1.0. mu.L, 2 XHieff

PCR Master Mix 12.5. mu.L, forward primer 1.25. mu.L, reverse primer 1.25. mu.L, ddH₂O (autoclaved) 9. mu.L. PCR reaction procedure: pre-denaturation at 98 ℃ for 3min, followed by 35 cycles of reaction (98 ℃ for 10s,60 ℃ for 20s,72 ℃ for 1min), and final extension at 72 ℃ for 5 min. The PCR product was detected with a 1.0% agarose Gel electrophoresis apparatus (Biorad, Calif.) and the desired fragment (FIG. 1) was recovered using HiPure Gel Pure Micro Kit (Kyowa Meiji technologies Co., Ltd., Guangzhou, cat # D2110-02), ligated to pMD18-T Vector (Takara, cat # D101A), and the ligated product was transformed into E.coli DH 5 alpha (Kyowa Biotech Co., Ltd., cat # DL1001) by heat shock method and spread on a plate containing 100. mu.g mL^-1Ampicillin (Ampicillin) on LB plates at 37 ℃ overnight. Selecting single colony as template, using the upstream and downstream sequences as primers, performing colony PCR verification, and sending the positive clone to Beijing Optimalaceae New Biotechnology Limited (Guangzhou division) for bacterial liquid sequencing. The nucleotide sequence of the obtained dendrobium officinale transcription factor DobHLH4 is shown in SEQ ID No. 1.

The transcription factor DobHLH4 has the nucleotide length of 1596bp (shown in figure 1), codes 531 amino acids (shown as SEQ ID NO. 4), has the protein molecular weight of about 56.54kD, comprises 66 acidic amino acids and 50 basic amino acids, has the theoretical isoelectric point of 5.00, the fat-soluble coefficient of 77.19 and the protein hydrophobicity coefficient of-0.305. The protein comprises 32.20% of alpha-helix, 14.12% of beta-sheet, 9.04% of beta-turn and 44.63% of random coil. NCBI's conserved domain database analysis shows that basic helix-loop-helix (bHLH) domain exists in the protein sequence of DobHLH4 and is located at position 343-.

Example 2 analysis of Gene expression Pattern of Dendrobium officinale transcription factor DobHLH4

Collecting different development stages (bud stage, initial flowering stage, full flowering stage and decay stage) of dendrobium officinale flowers, and performing RNA extraction and reverse transcription reaction thereof according to the method in the example 1- (1). Fluorescent quantitative PCR reaction respectively designs an upstream primer (SEQ ID NO.5) and a downstream primer (SEQ ID NO.6) according to DobHLH4 gene, and SYBR Premix Ex Taq is adopted^TMKit (Takara Co., Ltd., cat # DRR420A) was subjected to fluorescent quantitative PCR amplification. The reaction program was 95 ℃ denaturation for 2min, followed by 40 cycles of reaction (95 ℃ for 15s, 60 ℃ for 1 min). The reference gene is DoEF-1 alpha gene of dendrobium officinale (NCBI accession number is JF 825419). Is reacted in

480Instrument real-time fluorescent quantitative PCR (Roche diagnostics, Mannheim, Germany) was run to obtain data, run 2^-ΔΔCTThe method calculates relative expression data of each sample. The used DoEF-1 alpha upstream primer is shown as SEQ ID NO.7, and the used DoEF-1 alpha downstream primer is shown as SEQ ID NO. 8. Linalool synthase gene DoTPS10(NCBI accession number is MT875215, and the sequence of the NCBI accession number is shown as SEQ ID NO. 26), the used upstream primer is shown as SEQ ID NO.24, and the used downstream primer is shown as SEQ ID NO. 25.

The result shows that the expression level of the DobHLH4 gene is lower in the bud stage of the dendrobium officinale, and the expression level reaches the highest in the initial flowering stage and then gradually decreases along with the gradual development of dendrobium officinale flowers (figure 2). The expression pattern of the linalool synthase gene DoTPS10 in different development stages of dendrobium officinale flowers is consistent with that of the DobHLH4 gene, the change trend of increasing first and then decreasing is shown, and the expression amount reaches the peak value in the initial flowering phase (figure 3). In addition, the expression level of DobHLH4 in different development stages of dendrobium officinale flowers and the expression level of DoTPS10 gene show obvious positive correlation (Pearson correlation coefficient R)²＝0.98，p<0.01), therefore, DobHLH4 is closely related to the biosynthesis of linalool in Dendrobium officinale flowers.

Example 3 analysis of content of volatile monoterpene substance linalool in Dendrobium officinale flowers

The volatile aroma released from the dendrobium officinale flowers is carried out by a Solid-phase micro-extraction (SPME) method. Dendrobium officinale flowers at different development stages (bud stage, initial flowering stage, full flowering stage and decay stage) are placed in a closed headspace bottle, an SPME extraction head (50/30 μm DVB/CAR/PDMS, Stable Flex, Supelco, USA, Cat: 57329-U) is inserted above the headspace bottle, and the flowers are collected at 25 ℃ to release fragrance for 1 h. The measurement analysis was carried out by means of a gas chromatography-mass spectrometer (GC-MS) (QP2010SE, Shimadzu corporation, Japan) equipped with a SUPELCOWAXTM10 column (30 m. times.0.25 mm. times.0.25. mu.m, Supelco corporation, U.S.A.: 59305-U). The injection port temperature was 230 ℃, using a 10: 1 split mode. Helium was used as a carrier gas at a flow rate of 1mL min^-1. GC temperature was maintained for 3min at 60 ℃ and 5 ℃ for min^-1Heating to 150 deg.C, and heating at 10 deg.C for 10min^-1The temperature is raised to 250 ℃ and kept at 250 ℃ for 5 min. The mass spectrum detection adopts a full scanning mode, and the scanning range is 40 to 600 m/z. Mass spectrometry library NIST-8(NIST/EPA/NIH, USA) and linalool standard (Sigma-Aldrich, USA, Cat: L2602, figure 4) were used for substance identification.

As a result, the content of the volatile monoterpene linalool in the flowers of Dendrobium officinale shows fluctuating changes (increasing and then decreasing) in different development stages, and the linalool content in the early flowering phase is the highest (figure 5). This is in significant positive correlation with the expression level of the transcription factor DobHLH4 gene (Pearson correlation coefficient R)²＝0.90，p<0.01) and simultaneously shows obvious positive correlation with the expression quantity of the linalool synthase gene DoTPS10 (Pearson correlation coefficient R)²＝0.82，p<0.01)。

Example 4 obtaining and identifying Dendrobium officinale transcription factor DobHLH4 Arabidopsis thaliana

(1) Construction of Dendrobium officinale transcription factor DobHLH4 overexpression vector

Based on the sequences on the pCAMBIA3300 vector (FIG. 6) and the transcription factor DobHLH4, in BamH I and BamH I, respectivelyDesigning an upstream primer (SEQ ID NO.9) and a downstream primer (SEQ ID NO.10) at the Nco I enzyme cutting site, carrying out high-fidelity amplification by taking dendrobium officinale cDNA as a template to obtain a DobHLH4 sequence, purifying an amplification product, and recovering a target fragment. The expression vector pCAMBIA3300 was double-digested with BamH I and Nco I. By using In-

The HD Cloning Kit (Takara, Cat. No.: 639648) constructs plant expression vectors, and the detailed procedures are described in the specification. The pCAMBIA3300 vector contains CaMV35S promoter, the DobHLH4 retains ATG, removes stop codon, and is connected to 35S promoter to construct DobHLH4 overexpression vector, and the plant recombinant expression vector is named pCAMBIA3300-DobHLH4 (FIG. 7).

(2) The recombinant plasmid pCAMBIA3300-DobHLH4 was transformed into Agrobacterium GV3101(pSoup-p19)

The recombinant plasmid pCAMBIA3300-DobHLH4 was transformed into Agrobacterium GV3101(pSoup-p19) by heat shock method as follows: mu.g of recombinant plasmid pCAMBIA3300-DobHLH4 and 100. mu.L of competent cells of Agrobacterium GV3101(pSoup-p19) were mixed well, placed on ice for 5min, snap frozen with liquid nitrogen for 5min, quickly transferred to a 37 ℃ water bath and kept warm for 5min, and then placed on ice for 5 min. Adding 700 μ L LB culture medium without antibiotic, and culturing at 28 deg.C in shaker at 100rpm for 2-3 h. The culture was spread on 25mL LB plates (50 mg. multidot.mL in the medium)^-1Kanamycin). The plate was placed upside down in an incubator at 28 ℃ until colonies grew out (about 2 days). The single clone is selected for colony PCR identification, and primers are SEQ ID NO.9 and SEQ ID NO. 10. Agrobacterium GV3101(pSoup-p19) which had been transformed with the recombinant plasmid pCAMBIA3300-DobHLH4 was selected as a positive clone.

(3) Dendrobium officinale flower transient overexpression DobHLH4

After activation, the Agrobacterium containing pCAMBIA3300-DobHLH4 was isolated in 100mL LB liquid medium (50 mg. multidot.mL)^-1Kanamycin), 28 ℃, 180rpm shaking culture overnight (about 16h), to OD₆₀₀0.6 to 0.8. The cells were centrifuged at 5000 Xg for 5min at room temperature to collect the bacterial suspension. 100mL of permeation buffer (0.2mM acetosyringone, 10mM MgCl)₂，10mM MES, pH 5.7), resuspending the precipitated Agrobacterium, centrifuging at 5000 Xg for 5min at room temperature, discarding the supernatant, and adjusting the OD with the permeation buffer₆₀₀The product is approximately equal to 0.6, and is kept stand and activated for 2 hours at the room temperature of 25 ℃ in the dark. And (3) selecting the dendrobium officinale in bud period (namely, buds are formed but not blossomed), and sucking the activated bacteria liquid by using a 1mL syringe and injecting the activated bacteria liquid into the buds until the bacteria liquid just seeps out. Soaking the buds in the bacterial solution, vacuumizing, maintaining for 2min, and air drying. Culturing in a tissue culture room at 23 +/-2 ℃ for 24 hours in the dark, culturing for 2 days under the condition of illumination, and screening positive plants. The semi-quantitative PCR amplification is carried out according to the conventional PCR amplification, and the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 3min, followed by 30 cycles of reaction (98 ℃ for 10s,60 ℃ for 20s,72 ℃ for 1min), and final extension at 72 ℃ for 5 min. The PCR products were detected by electrophoresis on a 1% agarose gel. Expression of the transcription factor DobHLH4 was detectable in all 3 selected dendrobium officinale flowers, whereas overexpression of Lane 1 significantly increased the transcription level of DobHLH4 (fig. 8).

Further detecting the expression of DoTPS10 gene and the content of monoterpene aromatic substance linalool in the dendrobium officinale flowers overexpressing the transcription factor DobHLH4, the result shows that after the DobHLH4 gene is overexpressed in the dendrobium officinale flowers, the expression level of the linalool synthase gene DoTPS10 is increased by 6.5 times compared with the control (figure 9), and the content of monoterpene aromatic substance linalool is increased by 67.7% compared with the control (figure 10). Therefore, the transcription factor DobHLH4 can activate the expression of the linalool synthase gene DoTPS10 through transcription in the dendrobium officinale flowers, and further increase the accumulation of the monoterpene aromatic substance linalool.

Example 5 Dendrobium officinale transcription factor DobHLH4 localized in the nucleus

(1) YFP-DobHLH4 vector construction

The primer pair SEQ ID NO.11 and SEQ ID NO.12 are combined, and the DobHLH4 with the stop codon removed is obtained by utilizing PCR technology amplification, and the PCR system and the reaction procedure are consistent with those in example 1- (2). After the subcellular localization vector pSAT6-NYFP-N1(NCBI accession number: AY818378) is cut at the Nco I site, In-

HD Cloning Kit (Takara, cat # 639648)The cell localization vector and the transcription factor DobHLH4 are specifically operated according to the specification, and positive clones are identified by sequencing.

(2) PEG-mediated transient expression of DobHLH4 in arabidopsis leaf protoplast

Preparing protoplast enzymatic hydrolysate Enzyme solution (1.5% Cellulose R10, 0.3% Macerozyme, 0.1M MES, 20mM KCl, 0.4M Mannitol, pH 5.7), water-bathing at 55 deg.C for 10min, pouring into a culture dish, naturally cooling to room temperature, adding 100 μ L of 1M CaCl per 10mL Enzyme solution₂And 100 μ L of 10% BSA. Selecting the leaf of Arabidopsis thaliana with good growth state and 3-4 weeks of leaf age, tearing off the lower epidermis by using a transparent adhesive tape, putting the leaf into a culture dish containing the Enzyme solution, and placing the leaf in a constant temperature incubator at 50rpm for 3 hours under the illumination condition of 22 ℃. The Enzyme solution containing protoplasts was transferred to a 15mL round bottom centrifuge tube with a 1mL tip removed, centrifuged at 100 Xg for 3min at 4 ℃ and the supernatant slowly aspirated, repeated 2-3 times. With 4-5mL W5 solution (154Mm NaCl, 125mM CaCl)₂5mM KCl, 0.03% MES, 5mM glucose, pH 5.7) was added slowly along the tube wall, shaken gently, the precipitated protoplasts were washed, centrifuged at 100 Xg for 3min at 4 ℃ and the supernatant slowly aspirated. Adding 2mL W5 solution, standing on ice for 30min, centrifuging at 4 deg.C for 3min at 100 Xg, discarding supernatant, adding 2mL MMG solution (15mM MgCl)₂0.1% MES, 0.4Mannitol, pH 5.7), gently resuspended, and kept on ice until use. 10. mu.g of DobHLH4 plasmid, 200. mu.L of protoplast, and PEG Solution (40% PEG4000, 0.2Mannitol, 0.1M CaCl) were sequentially added to a 1.5mL centrifuge tube₂) 210. mu.L, and 840. mu.L of W5 solution, and mixing by turning upside down. Centrifuge at 100 Xg for 2min at 4 ℃ and discard the supernatant and repeat once. Adding 200 mu L W5 solution, mixing immediately, wrapping with tinfoil, and keeping flat for 16h at 22 ℃ in the dark. The slide glass was transferred to a slide glass, and the yellow fluorescent protein was observed by using a Leica TCS SP8 STED3 Xmicroscope (Leica, Inc., Germany) laser confocal scanning microscope.

The results show that the protein encoded by the dendrobium officinale transcription factor DobHLH4 is localized in the cell nucleus (FIG. 11).

Example 6 tobacco Dual-luciferase validation that DobHLH4 transcriptionally activates the promoter of DoTPS10

(1) Construction of pGreen II 62-SK-DobHLH4 recombinant vector

The primer pair SEQ ID NO.13 and SEQ ID NO.14 are combined, and the DobHLH4 with the stop codon removed is obtained by utilizing PCR technology amplification, and the PCR system and the reaction procedure are consistent with those in example 1- (2). The Effect vector pGreen II 62-SK is subjected to double enzyme digestion at the sites of BamH I and Hind III by adopting In-

The HD Cloning Kit (Takara, Cat: 639648) was ligated to pGreen II 62-SK and the transcription factor DobHLH4 to obtain pGreen II 62-SK-DobHLH4 recombinant vector (FIG. 12), the detailed operation method is described in the specification, and the positive clone was identified by sequencing.

(2) Construction of pGreen II 0800-DoTPS10p recombinant vector

According to the dendrobium officinale genome data, a promoter sequence (DoTPS10p) of a DoTPS10 gene is excavated, an upstream primer SEQ ID NO.15 and a downstream primer SEQ ID NO.16 are designed, and a promoter sequence DoTPS10p of the DoTPS10 is cloned by taking dendrobium officinale DNA as a template and is shown as SEQ ID NO. 17. Further, DoTPS10p was analyzed using promoter in-line software plantaCARE (http:// bioinformatics. psb. element. be/wbtools/plantaCARE/html /) and found that a potential transcription factor bHLH binding site G-box (CACGTG) was present at positions-478 to-483.

The primer pair SEQ ID NO.18 and SEQ ID NO.19 are combined, the DoTPS10p sequence containing BamH I and Hind III double restriction sites is obtained by PCR amplification, and the PCR system and the reaction procedure are the same as those in the embodiment 1- (2). The Reporter vector pGreen II 0800-LUC is subjected to double enzyme digestion at BamH I and Hind III sites by adopting In-

The HD Cloning Kit (Takara, Cat: 639648) ligated pGreen II 0800 with the promoter DoTPS10p of the target gene to obtain pGreen II 0800-DoTPS10p recombinant vector (FIG. 12), the detailed operation method is described in the specification, and the positive clones were identified by sequencing.

(3) Transformation of the recombinant plasmid into Agrobacterium GV3101(pSoup-p19)

The recombinant plasmids pGreen II 62-SK-DobHLH4 and pGreen II 0800-DoTPS10p were transformed into Agrobacterium GV3101(pSoup-p19) by heat shock method, the specific method was the same as example 4- (2), and positive clones were identified by sequencing.

(4) Agrobacterium infection tobacco leaf and LUC/REN fluorescence detection

Preparation of permeation buffer (0.2mM acetosyringone, 10mM MgCl)₂10mM MES, pH 5.7). GV3101(pSoup-p19) Agrobacterium containing pGreen II 62-SK-DobHLH4, pGreen II 0800-DoTPS10p vector was suspended with the above-mentioned permeate and OD was adjusted separately₆₀₀Standing at 28 deg.C under dark condition for 2h to activate. Selecting well-growing tobacco leaves, and injecting the tobacco leaves into a 1mL injector according to the ratio of a promoter to a transcription factor 1: 9, mixing and then jointly transforming the tobacco leaves of the Nicotiana benthamiana, wherein each leaf is a repeat, more than 6 repeats are needed to be made, marking is carried out, and comparison is set (the unloaded SK is used as the comparison). After culturing for 3 days in a tissue culture room at 23 ℃ in a 16h light/8 h dark culture room, using Dual-

The ratio of two luciferases (LUC and REN) in the tobacco leaf is detected by a Reporter Assay System kit (Promega corporation, cargo number: E1910), and the specific operation method is described in the specification.

The results of the dual-luciferase test on the tobacco leaves show that DobHLH4 can significantly activate the activity of DoTPS10 promoter DoTPS10p, and compared with the control no-load, the transcription activation multiple reaches 4.59 times (FIG. 13).

Example 7 Yeast Monohybrid Y1H validation of DobHLH4 binding to DoTPS10p

(1) Construction of JG4-5-DobHLH4 recombinant vector

The primer pair SEQ ID NO.20 and SEQ ID NO.21 are combined, and the DobHLH4 with the stop codon removed is obtained by utilizing PCR technology amplification, and the PCR system and the reaction procedure are consistent with those in example 1- (2). The JG4-5 vector is subjected to double enzyme digestion at EcoR I and Xho I sites, and In-

HD Cloning Kit (Takara, cat # 639648) ligated JG4-5 and the transcription factor DobHLH4, obtainedThe JG4-5-DobHLH4 recombinant vector is obtained, the specific operation method is described in the specification in detail, and the positive clone is identified by sequencing.

(2) Construction of pLACzi-TPS10p recombinant vector

The primer pair SEQ ID NO.22 and SEQ ID NO.23 are combined, the DoTPS10p sequence containing EcoR I and Xho I double enzyme cutting sites is obtained by utilizing PCR technology amplification, and the PCR system and the reaction procedure are consistent with the embodiment 1- (2). The pLACzi vector is subjected to double enzyme digestion at EcoR I and Xho I sites by adopting In-

The HD Cloning Kit (Takara, Cat: 639648) was ligated to pLACzi and the promoter DoTPS10p of the target gene to obtain pLACzi-TPS10p recombinant vector, the detailed operation method is described in the specification, and the positive clone was identified by sequencing.

(3) EGY48 yeast transformation and combination verification

100 mu.L of ice-thawed EGY48 competent cells (Shanghai Weidi Biotechnology Co., Ltd., product number: YC1030) were taken, and 5. mu.g of JG4-5-DobHLH4 recombinant plasmid, 5. mu.g of pLACzi-TPS10p recombinant plasmid, 10. mu.L of Carrier DNA, and 500. mu.L of PEG/LiAc were added in this order, mixed, and subjected to 30-minute water bath at 30 ℃ for 6-8 times. Transferring to 42 deg.C water bath for 15min (mixing for 6-8 times). Centrifuge at 5000 Xg for 1min, discard the supernatant, 400. mu.L ddH₂Resuspend O, centrifuge at 5000 Xg for 1min, discard the supernatant. 50 μ L ddH₂O heavy suspension, plate into SD-Ade/-Trp/-Ura plate, 29 degrees C inverted culture 2 d.

The monoclonal obtained above was spotted on SD-Ade/-Trp/-Ura/X-gal, cultured in an inverted state at 29 ℃ for 2 days, and observed for bluing (bluing in the presence of interaction between the two).

The results show that the concentration of Adenine (Ade) is 100ng mL^-1After co-transformation of pLacZi with JG4-5-DobHLH4 on the SD-Ade/-Trp/-Ura plate, the EGY48 yeast did not grow, while after co-transformation of pLacZi-DoTPS10p (plasmid containing DoTPS10 promoter) and JG4-5-DobHLH4, the EGY48 yeast can grow normally. Meanwhile, the monoclonal transfer to SD-Ade/-Trp/-Ura/X-gal plates appeared blue, indicating that DobHLH4 can bind to the DoTPS10 promoter (FIG. 14).

Furthermore, pLACzi-TPS10pm recombinant vector was reconstructed after mutating G-box (CACGTG) site of region-478 to-483 in TPS10p sequence, the method was identical to that of example 7- (2) above, and the binding of pLACzi-TPS10pm to JG4-5-DobHLH4 after the mutation of DoTPS10 promoter was analyzed. The results show that, on SD-Ade/-Trp/-Ura plates, after co-transforming pLacZi-DoTPS10p with JG4-5-DobHLH4, the EGY48 yeast can grow normally, while after mutating the G-box (CACGTG) site of TPS10p to TPS10pm, co-transforming pLacZi-DoTPS10pm with JG4-5-DobHLH4, EGY48 yeast does not grow (FIG. 15), therefore, DobHLH4 is combined with DoTPS10p through the G-box (CACGTG) site.

Sequence listing

<110> south China plant garden of Chinese academy of sciences

<120> dendrobium officinale transcription factor DobHLH4 and application thereof

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1596

<212> DNA

<213> Dendrobium officinale (Dendrobium officinale Kimura et Migo)

<400> 1

atgctgtcga ggcttagtgg cgcggtttgg agggagggag aaggggcgga ggaggaagag 60

gacaccgctt cttggacaag agcggccact tgcggcggtg ggatggggga aggcagggac 120

gacatggcgc ttccgtgcct caagtccatt ctcgaggacg actggtacgt agcagcaacc 180

tcgggtagcg gcgggaacgt cggtgggagc tcagtaaacc agggttttgc tggaatgaac 240

ccaccggatt cttctctcct cctcccgccg caggtcgatt cgtcctgctc cccttcctcc 300

gtgtttgcac tcgacccttc cgctcaccag gccttctttc cccacaaacc cgctctttcc 360

tcttttctat ccgctgtgtg ctccaacccg tatgacgccg gcttcgattt tgtctcggat 420

aacccgacct tccttcctcc ggtgaatcct aacgccgcca cctccggctt tggcggtgga 480

atgttgggct tccctaacgt gcagattaat agccaagatt tattgcctat ggccggaagc 540

tcctctttgc taaaccccat ggccttcgat tcttttgaaa attcgccctt tttgagtagg 600

ggaaaggttt taaggcctct ggagatcttc ccaccagtag gcgctcagcc cactcttttc 660

cagaaacggg ctgcggcggc cctgcgccaa aattcaagca gtatgggagg tgataaaggc 720

atgtgtttgg ggcttttggg catggaagct accggtcagt tgaggagctc ttgggatggc 780

gagcttgaga agaggaggag aataaacgac gacgatgagc ttgatgatgc cagcattgat 840

ggctctggct taaattatga ttctgaggat atctgcgaag ttgccgaaac tgtaaaggtc 900

gatgatgaga agaatgatgg cataggagca gcatccggcg gcggcggagg agttggcaat 960

gtttccatta ctaatagcag tctaactggt ggtggagagc tcaaaggccg gagaaagggt 1020

cttcctgcaa agaatttgat ggccgagagg aggaggagga agaagctcaa tgataggctt 1080

tacatgctaa gatctgttgt tccaaagatc agcaagatgg atagagcttc cattcttggt 1140

gatgcaattg agtacttgaa ggagctttta cagagaatca atgaccttca taatgaactt 1200

gagtccacga attcttcgat gcccacctct agcacatcga gctttcaccc tttaacgccg 1260

acaccgccaa ctcttccgtg tcgaatcaag gaggagcttt gcccaagctc attaccgagc 1320

ccgaatggtc aaccagcaag ggtagaagta aggctaaggg aaggcagagc tgtaaacata 1380

cacatgtttt gcgcccgtcg accgggttta ctcctctcaa caatgagagc tcttgatggc 1440

cttggccttg acatccagca agcagtcata agctgcttca atggctttgc cctcgatgtt 1500

tttcgggccg agcaatcaaa ggatggacct ggtgttctgc cagaagaaat caaagaagtt 1560

ctcttgcatt ctgcaggctt ccaaaatgca ctgtga 1596

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agaggaaacg atgctgtcga g 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tcacagtgca ttttggaagc c 21

<210> 4

<211> 531

<212> PRT

<213> Dendrobium officinale (Dendrobium officinale Kimura et Migo)

<400> 4

Met Leu Ser Arg Leu Ser Gly Ala Val Trp Arg Glu Gly Glu Gly Ala

1 5 10 15

Glu Glu Glu Glu Asp Thr Ala Ser Trp Thr Arg Ala Ala Thr Cys Gly

20 25 30

Gly Gly Met Gly Glu Gly Arg Asp Asp Met Ala Leu Pro Cys Leu Lys

35 40 45

Ser Ile Leu Glu Asp Asp Trp Tyr Val Ala Ala Thr Ser Gly Ser Gly

50 55 60

Gly Asn Val Gly Gly Ser Ser Val Asn Gln Gly Phe Ala Gly Met Asn

65 70 75 80

Pro Pro Asp Ser Ser Leu Leu Leu Pro Pro Gln Val Asp Ser Ser Cys

85 90 95

Ser Pro Ser Ser Val Phe Ala Leu Asp Pro Ser Ala His Gln Ala Phe

100 105 110

Phe Pro His Lys Pro Ala Leu Ser Ser Phe Leu Ser Ala Val Cys Ser

115 120 125

Asn Pro Tyr Asp Ala Gly Phe Asp Phe Val Ser Asp Asn Pro Thr Phe

130 135 140

Leu Pro Pro Val Asn Pro Asn Ala Ala Thr Ser Gly Phe Gly Gly Gly

145 150 155 160

Met Leu Gly Phe Pro Asn Val Gln Ile Asn Ser Gln Asp Leu Leu Pro

165 170 175

Met Ala Gly Ser Ser Ser Leu Leu Asn Pro Met Ala Phe Asp Ser Phe

180 185 190

Glu Asn Ser Pro Phe Leu Ser Arg Gly Lys Val Leu Arg Pro Leu Glu

195 200 205

Ile Phe Pro Pro Val Gly Ala Gln Pro Thr Leu Phe Gln Lys Arg Ala

210 215 220

Ala Ala Ala Leu Arg Gln Asn Ser Ser Ser Met Gly Gly Asp Lys Gly

225 230 235 240

Met Cys Leu Gly Leu Leu Gly Met Glu Ala Thr Gly Gln Leu Arg Ser

245 250 255

Ser Trp Asp Gly Glu Leu Glu Lys Arg Arg Arg Ile Asn Asp Asp Asp

260 265 270

Glu Leu Asp Asp Ala Ser Ile Asp Gly Ser Gly Leu Asn Tyr Asp Ser

275 280 285

Glu Asp Ile Cys Glu Val Ala Glu Thr Val Lys Val Asp Asp Glu Lys

290 295 300

Asn Asp Gly Ile Gly Ala Ala Ser Gly Gly Gly Gly Gly Val Gly Asn

305 310 315 320

Val Ser Ile Thr Asn Ser Ser Leu Thr Gly Gly Gly Glu Leu Lys Gly

325 330 335

Arg Arg Lys Gly Leu Pro Ala Lys Asn Leu Met Ala Glu Arg Arg Arg

340 345 350

Arg Lys Lys Leu Asn Asp Arg Leu Tyr Met Leu Arg Ser Val Val Pro

355 360 365

Lys Ile Ser Lys Met Asp Arg Ala Ser Ile Leu Gly Asp Ala Ile Glu

370 375 380

Tyr Leu Lys Glu Leu Leu Gln Arg Ile Asn Asp Leu His Asn Glu Leu

385 390 395 400

Glu Ser Thr Asn Ser Ser Met Pro Thr Ser Ser Thr Ser Ser Phe His

405 410 415

Pro Leu Thr Pro Thr Pro Pro Thr Leu Pro Cys Arg Ile Lys Glu Glu

420 425 430

Leu Cys Pro Ser Ser Leu Pro Ser Pro Asn Gly Gln Pro Ala Arg Val

435 440 445

Glu Val Arg Leu Arg Glu Gly Arg Ala Val Asn Ile His Met Phe Cys

450 455 460

Ala Arg Arg Pro Gly Leu Leu Leu Ser Thr Met Arg Ala Leu Asp Gly

465 470 475 480

Leu Gly Leu Asp Ile Gln Gln Ala Val Ile Ser Cys Phe Asn Gly Phe

485 490 495

Ala Leu Asp Val Phe Arg Ala Glu Gln Ser Lys Asp Gly Pro Gly Val

500 505 510

Leu Pro Glu Glu Ile Lys Glu Val Leu Leu His Ser Ala Gly Phe Gln

515 520 525

Asn Ala Leu

530

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aaccagcaag ggtagaagta ag 22

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gctctcattg ttgagaggag taa 23

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcttgagaag gagcccaagt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccaacagcca cagtttgtcg 20

<210> 9

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cggtacccgg ggatccatgc tgtcgaggct tagtggc 37

<210> 10

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cccttgctca ccatggcagt gcattttgga agcctgc 37

<210> 11

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atttacgaac gatagccatg ctgtcgaggc ttagtggc 38

<210> 12

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agatctgagt ccggaccatg gtcagtgcat tttggaagcc t 41

<210> 13

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tagaactagt ggatccatgc tgtcgaggct tagtggc 37

<210> 14

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cggtatcgat aagcttcagt gcattttgga agcctgc 37

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tgtgctttcg acctcttgat g 21

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgtttggagc ggctttctta 20

<210> 17

<211> 680

<212> DNA

<213> Dendrobium officinale (Dendrobium officinale Kimura et Migo)

<400> 17

tgtgctttcg acctcttgat gatgatttca tttcaagatg ttctttgtgt taatagtcta 60

acccttaatg ttaacgatgt ttactgaaca agccaaaatg aaaaatgaga gagagtgcaa 120

gtgaaaggtt tgtaagagat ggcaaaagag gaaagcacga ggggggatga tagaaaacat 180

aaagggaaag agaaaaccac gtgcgagtag tacacatgat aaaaatgagt ttttaggtta 240

gtttgatcaa gcgactcatg tagattatga tcttacaaag aagatttatt atactatggt 300

aagatatagt taacgagatt aaggttttat ttggaaaagt tttctttttt attcctaaag 360

cagcttcgta gtaaaaaaat aaattttata ctaagaagct gatcatttaa gaagcaaaaa 420

gaaagccgtc ccaaggcttt gtttgggaca acttttttac tgatttgtaa agctgctttt 480

taatacaaat atatctagtt caataaattt ttgtactata aatttatttt aggaagcaat 540

aagaaagctg tcccaaatga agcttaaggc ttcgtttggg acgacttttt attgcttctt 600

aaagcaattt tttaatgcaa aaataaattt tttgtactaa aaagctgctt taagaagcaa 660

taagaaagcc gctccaaacg 680

<210> 18

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cggtatcgat aagctttgtg ctttcgacct cttgatg 37

<210> 19

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tagaactagt ggatcccgtt tggagcggct ttcttattg 39

<210> 20

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tgcctctccc gaattcatgc tgtcgaggct tagtggc 37

<210> 21

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tccaaagctt ctcgagtcac agtgcatttt ggaagcc 37

<210> 22

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tgaaaagctt gaattctgtg ctttcgacct cttgatg 37

<210> 23

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gagcacatgc ctcgagcgtt tggagcggct ttcttattg 39

<210> 24

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gctgcctctc aacagctaat a 21

<210> 25

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ctggaagcga ttggatgtag t 21

<210> 26

<211> 1797

<212> DNA

<213> Dendrobium officinale (Dendrobium officinale Kimura et Migo)

<400> 26

atggctttgt attttcgccc tcccatgctt tcgtacagta agaagcagcc agtgctcttt 60

agcttttctt ccacgccaac atttctaatt cttcaaggaa gtattacaag attccatccc 120

ataagctgcc tctcaacagc taatacaaaa acttctcctg ctcgtcgcac agcaaatttt 180

cagccaacca tttgggatga taactacatc caatcgcttc cagttcattt catggaggag 240

aagtataaac accaaagaga taaattgaag gaggaagtca ggtgtttgat tggccaacaa 300

catggacttg ttaaacagct tgaacttgtg gatgccctac gacagttagg aattgattat 360

cattttgatt cagagatcat gaatttgttg agttgtatta gttcatcaac ggaaaatata 420

cataacttga taaagaataa caatctttat ggctctgcat tgctctttag gcttctaagg 480

gagcatggca tcaataatgc tgcaatacta agagttgata ctctgataag ctgcttcaaa 540

aaagtgagag aaagcttcaa tcccaatcat caatatgatg ttaaagaaat gctcagtttg 600

tatgaggctt cctaccttgc tatggaagga gaggaagaac tggatgaggc aggaaagttt 660

gctatggagc acttaagacg tcttgataga tctttactaa gtccacaact tattgaagaa 720

atagatcatg ccttggagct acccttgcat tggaggatgc caaggctgca tagtaggtgg 780

ttcattgatg cttatgggaa acaagaaaat gttaatccca tattgcttga gttggccaaa 840

ctagatttca acatagtaca gagcatttat atgacagagc ttaaagaaat ctctaattgg 900

tggagaaatc ttggtgttct ttgtgatgag cttgatttta taagagacag gctagtggag 960

aaccatttat gggctttagg atttacattt caacctaaat tttggagaag caggaaagca 1020

atcaccaaga ttaattgttt ggtaacaaca attgatgatg tttatgatgt ttatggcact 1080

ctagatgagc tagagatctt tacaaatgcc gttgaagatt ggaaaatgga tgcagctcaa 1140

caacttccaa attgtatgaa gatatgcttg atggcacttt tcaatacaat gaatgaagtt 1200

gcgtactcat ttttaaagga aaaagaattg gatattcttc catgccttaa aagagtgtgg 1260

gtcgatttat gcaaagcata cttaattgaa gcaaggtggt atcataatag atacacccca 1320

acattggatg agtacttgga gaatgcttgg gtaacaatat ccggaaattg tggactatca 1380

gcatcatatt gtctaagtga tgacttgaat gttgaagctc ttgatagtat aaaattttat 1440

ccacccatcg ttcaacattc atgtatgatt ttccgactct acaatgattt gggaactgac 1500

atggcagaga tccaaagagg agacgtttta aaatcaattc aatgctatat gaatgagaaa 1560

aatgtttcag actcagctgc tcgagactat ataagatgtt taataagaaa ttattggaaa 1620

aaattgaatg gagaatatat tacattttca acatctatag aatctttcag aaaagcttta 1680

gtagatgttc cacgaacagc acaaagcttc tatcactatg gagatggata tggagagccc 1740

ggtggtaaaa ccaaggatca aattttctta atgatgattg aaccaattcc actttaa 1797

Claims (2)

1. The application of the transcription factor DobHLH4 in promoting synthesis of linalool in dendrobium officinale flowers is disclosed, wherein the nucleotide sequence of the transcription factor DobHLH4 is shown in SEQ ID No. 1.

2. The application of a transcription factor DobHLH4 in promoting the expression of a DoTPS10 gene is disclosed, wherein the nucleotide sequence of the transcription factor DobHLH4 is shown as SEQ ID NO. 1.

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