CN115819537B - AP2/ERF transcription factor, and coding gene and application thereof - Google Patents
AP2/ERF transcription factor, and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses an AP2/ERF transcription factor, its coding gene and application, the amino acid sequence of the transcription factor is shown in SEQ ID NO.1, the full-length nucleotide sequence of the gene is shown in SEQ ID NO.2, and the nucleotide sequence of the coding region is shown in SEQ ID NO. 3. The invention discovers that AP2/ERF transcription factor DoAP/ERF 89 which participates in the synthesis and regulation of dendrobium candidum volatile terpenes can be combined with a promoter region of DoPAE through a yeast single-hybrid technology and a double-luciferase test, activates the expression of a DoPAE promoter, enhances the activity of the DoPAE promoter, promotes the synthesis of terpenes, plays a key role in regulating and controlling the synthesis of dendrobium candidum terpenes, and provides a theoretical basis for the research of transcription factor DoAP/ERFs.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an AP2/ERF transcription factor, and a coding gene and application thereof.
Background
Dendrobium officinale (Dendrobium officinale Kimura et Migo) is a perennial epiphyte of Dendrobium (Dendrobium Sw.) of Orchidaceae (Orchidacea), which is not only an ornamental plant but also a traditional rare medicinal plant in China. Researches show that the terpenoid is the main component of the volatile substance of the dendrobium candidum, and the main medicinal component dendrobine of the dendrobium candidum is also terpene derivative. Therefore, the research on terpene metabolism is particularly important in the secondary metabolism of dendrobium candidum.
In the whole terpene metabolic pathway, because terpene synthase (TPS) is a direct catalyst for synthesizing terpenes, plants can synthesize and release terpenes with rich types and complex structures, namely, the terpene synthase is a key enzyme in the biosynthesis process of terpenes. Transcriptional activation of synthetic genes by transcription factors is one of the most important regulatory links in plant secondary metabolism, and can effectively control the synthesis and accumulation of secondary metabolites by activating the expression of key functional genes in the synthetic pathway of plant secondary metabolism. At present, transcription factors involved in the regulation of terpene metabolism have been found in plants such as vinca, arabidopsis, maize, citrus sinensis, etc., including AP2, WRKY, zinc fingers, bHLH, etc. In citrus sinensis, citTPS16 catalyzes the accumulation of E-geraniol, while transcription factor CitERF can regulate citrus sinensis fruit E-geraniol synthesis by activating the expression of CitTPS by transcription.
The AP2/ERF transcription factor family is one of the largest transcription factor families in plants, each member of which contains a typical AP2 domain. Many plants have over 100 members of the AP2/ERF transcription factor family. At present, research on AP2/ERF transcription factors related to volatile terpenoid synthesis in dendrobium candidum is still in a starting stage, and the regulation and control effects on key genes in a terpenoid synthesis pathway are not clear.
Disclosure of Invention
The invention mainly aims to provide an AP2/ERF transcription factor capable of regulating and controlling the synthesis of dendrobium candidum terpenes, and a coding gene and application thereof.
In order to achieve the aim, the invention provides an AP2/ERF transcription factor, the amino acid sequence of which is shown as SEQ ID NO. 1. The transcription factor DoAP/ERF 89 is described below.
The invention also provides a gene for encoding the AP2/ERF transcription factor, the full-length nucleotide sequence of the gene is shown as SEQ ID NO.2, and the nucleotide sequence of the encoding region of the gene is shown as SEQ ID NO. 3.
The invention also provides an expression vector containing the gene.
The invention also provides a host bacterium containing the expression vector.
The invention also provides an application of the transcription factor or the gene in regulating and controlling the synthesis of dendrobium candidum terpenes.
Further, the application activates a dendrobium candidum DoPAE gene promoter through the transcription factor or the gene.
The beneficial effects of the invention are as follows:
The invention discovers that AP2/ERF transcription factor DoAP/ERF 89 which participates in the synthesis and regulation of the volatile terpenes of dendrobium candidum can be combined to the promoter region of DoPAE through a yeast single-hybrid technology and a double-luciferase test, and the DoAP/ERF 89 is proved to play a key role in regulating and controlling the terpenes synthesis of dendrobium candidum by activating the expression of DoPAE promoter, enhancing DoPAE promoter activity and promoting the terpenes synthesis. The invention provides a theoretical basis for researching transcription factors DoAP/ERFs and provides thought and basis for further revealing a transcription regulation mechanism for synthesizing dendrobium candidum terpenes.
Drawings
FIG. 1 is a phylogenetic tree analysis of the Dendrobium officinale AP2/ERF protein.
FIG. 2 is a GC-MS analysis of recombinant DoPAES protein in vitro.
FIG. 3 is a DoAP/ERFs and DoPAES co-expression analysis.
FIG. 4 is a diagram showing subcellular localization of DoAP/ERF 89.
FIG. 5 is a yeast single hybridization assay DoAP to verify DoAP/ERF 89 interactions with DoPAES.
FIG. 6 shows the regulatory effect of luciferase complementary imaging analysis transcription factor on promoter.
FIG. 7 is DoAP/ERF 89 transient transformation of Dendrobium candidum protocorm terpenes analysis.
Detailed Description
The invention is further described below with reference to examples:
the various materials and equipment used in the examples below, unless otherwise specified, are commercially available products well known in the art.
Example 1: AP2/ERF transcription factor screening
The possible presence of Cis-acting elements in the DoPAES promoter region was analyzed using the databases Plant Cis-ACTING ELEMENTS (http:// bioinformation. Psb. Ugent. Be/webtools/plantcare/html /) and Plant Cis-acting regulatory DNA ELEMENTS (https:// www.dna.affrc.go.jp/PLACE/action= newplace). CDS (coding region) sequences of the Arabidopsis AP2/ERF are subjected to local library building, and 111 candidate DoAP/ERF genes are identified. And (3) carrying out correlation network analysis and gene coexpression analysis on 111 DoAP/ERFs and DoPAE S, and finally screening out transcription factors DoAP/ERF 89, wherein the amino acid sequence of the transcription factors is shown as SEQ ID NO. 1.
Example 2: nucleotide sequence of coding gene of transcription factor DoAP/ERF 89
Extracting RNA of dendrobium candidum protocorms by using a plant RNA extraction kit; using reverse transcription kit (Whole goldOne-Step gDNA Removal AND CDNA SYNTHESIS SuperMix) was reverse transcribed into cDNA as described, amplified to give the complete CDS (coding region) sequence of the coding gene for DoAP/ERF 89, and full-length primers were designed using PRIMER PREMIER 5.0 software:
DoAP2/ERF89-F1:ATGGTGACTGTGAGAGAGGAGATGC
DoAP2/ERF89-R2:TCACATCATGATTTCTTGATCTCCC
PCR amplification is carried out by taking cDNA as a template, and the reaction system is as follows:
TABLE 1PCR amplification reaction System and reaction conditions
DoAP2/ERF89 is shown as SEQ ID NO.1, the full-length nucleotide sequence of the coding gene is shown as SEQ ID NO.2, and the nucleotide sequence of the coding region is shown as SEQ ID NO. 3.
Example 3: subcellular localization of DoAP/ERF 89
The coding gene DoAP/ERF 89 is transformed into pMD18-T vector, plasmid is extracted, double enzyme digestion is carried out, and the reaction condition is that the constant temperature of 37 ℃ is used for 3 hours. The target fragment was separated by 1% agarose gel electrophoresis, and the target fragment and the vector (pCAMBIA 1300, pDR 196) were subjected to gel recovery using a kit (SanPrep column DNA gel recovery kit, division of bioengineering (Shanghai)). The resulting pCAMBIA1300-DoAP2/ERF89 plasmid was transformed into Agrobacterium GV3101 and colony PCR identified.
The agrobacterium transformation procedure was as follows:
(1) Taking out the stored competent Agrobacterium from-80 ℃ and melting the competent Agrobacterium on ice;
(2) Adding 1 μg plasmid DNA into 100 μl of the mixture, mixing, sequentially standing on ice for 5min, liquid nitrogen for 5min, and ice-bath for 5min at 37deg.C;
(3) Adding 700 mu L of LB liquid medium without antibiotics, and culturing for 2-3 h at 28 ℃ in a shaking way;
(4) Centrifuging at 5000rpm for three minutes to collect bacteria, collecting about 50 μl of supernatant, gently sucking and beating the re-suspended bacteria mass, coating on LB plate containing corresponding antibiotics, and culturing in 28 deg.C incubator for 2-3 days
(5) Single colony is selected for culturing and identification, and 50% glycerol is added to each 300 mu L of the identified correct bacterial liquid, and 700 mu L of the bacterial liquid is stored at-80 ℃.
Agrobacterium containing the gene of interest was placed in LB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin for expansion culture. And when the bacterial liquid OD 600 =0.6-0.8, centrifuging for 10min at 4 ℃ at 4500r/min, and collecting bacteria. The cells were suspended in an infection buffer (1/2 MS,1% sucrose, 0.5mmol/L MgCl 2, 200. Mu. Mol/L acetosyringone, 10mmol/L ethanesulfonic acid and 0.01% Silwett-77) and allowed to stand at 28℃for about 3 hours in the absence of light.
(1) The fusion expression vector DoPAES-GFP was transferred into protoplasts obtained from Arabidopsis leaves using the modified polyethylene glycol method, with pCAMBIA1304-GFP empty vector as a control.
TABLE 2 cleavage reaction System and reaction conditions
TABLE 3 connection of reaction systems and reaction conditions
(2) About 1cm 2 of fresh onion flake lower epidermis is selected, immersed in agrobacterium suspension, dark cultured for 30min, and then subjected to suction of excess liquid with filter paper, and placed on a co-culture medium (1/2 MS,1% sucrose, 0.03% hydrolyzed casein, 0.28% proline, 10. Mu. Mol/L2, 4-D, 2. Mu. Mol/L6-BA, 200. Mu. Mol/L acetosyringone and 0.8% agar), and dark cultured at 22℃for 20h to 24h.
The transient expression arabidopsis protoplast and the lower epidermis of the onion flake are placed under a laser confocal microscope for scanning shooting, and the distribution of GFP green fluorescence is observed. The excitation wavelengths of GFP and chlorophyll were 488nm and 640nm, respectively. Subcellular localization is shown in FIG. 4, where DoAP2/ERF89 is localized to the nucleus.
Example 4: single hybridization test of yeast with interaction of coding gene of transcription factor DoAP/ERF 89 and DoPAES gene
(1) Preparation of Yeast competence (used as is, but not after cryopreservation):
taking out yeast strain Y1HGold from refrigerator at-80deg.C, sucking 10-20 μl, coating on YPDA solid culture medium, and culturing in incubator at 28deg.C for 2d-4d until the diameter of bacterial plaque is about 2 mm; 2-3 clones are respectively selected from YPDA solid culture plates and are respectively put into 20mL of YPDA liquid culture medium, the temperature is kept constant at 28 ℃, and the culture is carried out overnight at 180r/min until the OD 600 is more than 1.5; about 10mL of bacterial liquid is inoculated in 100mL of fresh YPDA liquid culture medium, the temperature is kept constant at 28 ℃,180r/min is cultured until OD 600 = 0.4-0.6, and 1 000 Xg of the bacterial liquid is centrifugated for 5 minutes at room temperature to collect bacteria; removing the supernatant, re-suspending cells with 10mL of sterile water, centrifuging for 5min at 6 000r/min, and removing the supernatant; the pellet was resuspended in 1.5mL 1 XLiAc to give competent cells.
(2) Bait vector transformation:
pAbAi empty vector plasmids to be transformed and recombinant vectors pAbAi-DoPAESpro are transformed into yeast competent cells according to Table 6, and after transformation, the cells are spread on SD/-Ura solid medium for culture, positive clones are screened for subsequent experiments, and linearization is required before transformation by using restriction enzyme BstB I.
TABLE 4 cleavage reaction System and reaction conditions
TABLE 5 connection of reaction systems and reaction conditions
Background level expression of Y1H [ pBait-AbAi ] strain:
The Y1H [ pBait-AbAi ] strain was resuspended with 0.9% NaCl to OD 600 =0.002, 100. Mu.L was spread on SD/-Ura deficient medium containing different concentrations (0 ng/mL,200ng/mL,400ng/mL,600ng/mL,800 ng/mL) of aureobasidin (Aureobasidin A, abA) and incubated upside down at 28℃for 2-3d in the absence of light. If there is a white single colony on the plate, the pBait-AbAi strain has self-activation, otherwise, the AbA concentration can be proved to inhibit the self-activation, and the lowest AbA concentration for inhibiting the growth of the pBait-AbAi strain is determined.
Preparation of the competent yeasts carrying pBait-AbAi was carried out as above, and the transformation products were spread on SD/-Leu solid medium and cultured upside down at 28℃for 3-5d. Positive clones were screened for gradient dilution and plated on SD/-Leu solid medium with 600 ng/mLAbA.
TABLE 6 Yeast competent transformation System
As shown in FIG. 5, doAP2/ERF89 can effectively bind to DoPAES promoter region and regulate DoPAES expression.
Example 5: transient transformation gene DoAP/ERF 89
By analyzing pGreenII 0029 62-SK and pGreenII 0800-LUC vectors, a proper Xhol-Xmal restriction site is selected, and a homologous recombinase is used for constructing recombinant plasmids. The DoAP/ERF 89 encoding gene was ligated into pGreenII 0029 62-SK vector, the promoter DoPAES was inserted into pGreenII 0800-LUC vector, and the two recombinant plasmids were transformed into Agrobacterium tumefaciens GV3101, respectively. Recombinant plasmid pGreenII-0800-LUC and empty pGreenII 0029 62-SK were co-transferred as controls, recombinant plasmid pGreenII-0800-LUC and recombinant plasmid pGreenII 0029 62-SK were co-transferred into tobacco, and firefly luciferase and Renilla luciferase activities after infiltration for 72h were analyzed using a dual luciferase report analysis system (E710, promega) and a Modulus luminometer (GloMax 96, promega). The experimental results are shown in FIG. 6, in which the LUC driven by the DoAP2/ERF89 and DoPAES promoters are co-expressed in N.tabacum leaves, doAP/ERF 89 significantly up-regulates DoPAES expression.
Example 6: detection of volatile terpenoid components of dendrobium candidum protocorms
The volatile terpenoid of the dendrobium candidum protocorm is detected by headspace-solid phase microextraction (HS-SPME) -gas chromatography (Agilent 7890B)/mass spectrum (Agilent 7000B) combination (GC-MS). Solid phase microextraction fibers composed of 100 μm polydimethylsiloxane (SUPELCO) were placed in a headspace bottle containing the sample and adsorbed at room temperature for 1 hour. The volatile terpenoids collected were analyzed on a GC-MS Triple Quad apparatus equipped with HP-5MS (30 m. Times.0.25 mm. Times.0.25 μm) molten silicon capillary column. Chromatographic conditions: helium (1 mL/min) is used as carrier gas, the temperature of the sample inlet is 250 ℃, the sample injection is not split, the initial temperature is 60 ℃, the sample is kept for 6min, and the temperature is raised to 300 ℃ at 5 ℃/min and is kept for 5min. Mass spectrometry conditions: the temperature of the EI ion source is 230 ℃, the electron energy is 70ev, the temperature of the four-stage rod is 150 ℃, the temperature of the adapter is 280 ℃, and the mass scanning range is 50-400 m/z. The detected volatile compounds were retrieved by NIST (National Institute of STANDARDS AND Technology) 2011 standard library and the resulting compounds were aligned in combination with published literature to determine the volatile terpenes detected.
To the sample, 200. Mu.g/kg.h of ethyl decanoate was added as an internal standard. Quantification was performed based on the peak area of the internal standard. The volatile content was expressed in μg/kg.FW.h.
The test results are shown in FIG. 2 and FIG. 7, and the in vitro enzyme activity of FIG. 2 proves that DoPAES can catalyze the synthesis of beta-patchoulene. FIG. 7 shows that DoAP/ERF 89 can activate DoPAES promoter expression, and enhance DoPAES promoter activity, thereby promoting synthesis of beta-patchoulene in Dendrobium officinale.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (2)
- The application of the AP2/ERF transcription factor in the regulation and control of the synthesis of dendrobium candidum terpenes is characterized in that the amino acid sequence of the transcription factor is shown as SEQ ID NO.1, and the application activates a dendrobium candidum DoPAE gene promoter through the transcription factor.
- 2. The application of the gene for encoding the AP2/ERF transcription factor in the regulation and control of the dendrobium candidum terpenoid synthesis is characterized in that the full-length nucleotide sequence of the gene is shown as SEQ ID NO.2, the nucleotide sequence of the coding region of the gene is shown as SEQ ID NO.3, and the application activates the dendrobium candidum DoPAE gene promoter through the gene.
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