Promoter of paulownia fortunei PfD14 gene, recombinant vector and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a paulownia fortunei PfD14 gene promoter, a recombinant vector and application of the paulownia fortunei PfD14 gene promoter. The sequence interval of the promoter comprises that the 3'end is located at the upstream 1bp of the paulownia fortunei SEQ ID NO.1 gene coding sequence ATG, and the 5 'end is located at the upstream 1988bp of the paulownia fortunei SEQ ID NO.1 gene coding sequence ATG; the 5 'UTR sequence is an upstream 1bp-296bp interval sequence of the paulownia fortunei PfD14 gene coding sequence ATG of the paulownia fortunei SEQ ID NO.1. The promoter of the paulownia fortunei PfD14 gene and the core region of the paulownia fortunei PfD14 gene provided by the invention have important significance on research on a paulownia fortunei branch improvement technology and excellent germplasm cultivation.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a promoter, a recombinant vector and application of a Paulownia davidiana PfD14 gene. Background Art

[0002] The PfD14 gene of Paulownia leucophylla encodes the α / β hydrolase DWARF14 (D14) in the Paulownia leucophylla strigolactone signal transduction pathway. Currently, there are problems with the unclear function and regulatory mechanism of this gene and its limited application.

[0003] Strigolactones (SLs) are a new type of plant hormone that plays an important role in inhibiting plant branching and can work together with other hormones to regulate the plant structure. Paulownia alba is an important fast-growing timber in my country. It is often used for street tree greening and land desertification control. It has both economic and ecological benefits and is deeply loved by forest farmers. However, it is susceptible to phytoplasma infection and witches' broom disease during the production process, which leads to uncontrolled growth of axillary buds and causes serious economic losses. In rice, SLs can inhibit the growth of tiller buds, thereby controlling the number of rice tillers. Although it is known that strigolactones are related to plant branching regulation, its specific role in the uncontrolled growth of axillary buds caused by Paulownia witches' broom disease has not yet been clarified.

[0004] D14 is a key gene in the signal transduction process of strigolactones and is an atypical receptor for strigolactones (SLs). D14 can bind to strigolactones and initiate the signal transduction process of strigolactones. At the same time, the ubiquitination and degradation of D14 are responsible for terminating signal perception. The NTD domain of D14 is phosphorylated, which can inhibit the ubiquitination modification and protein degradation of D14, thereby regulating the tillering development of rice. Therefore, by studying the function and mechanism of action of the PfD14 gene in white Paulownia, we can reveal the regulatory mechanism of Paulownia growth and development, improve the disease resistance and growth rate of Paulownia, optimize its wood quality, and promote the development of the Paulownia industry.

[0005] A promoter is a DNA sequence that enables gene transcription. The transcribed RNA is further translated and modified to form functional proteins. Tissue-specific promoters refer to genes that are only expressed in a specific tissue or organ, or only at a specific stage in the growth and development of plants. Such as Arabidopsis leaf-specific promoters, tomato fruit-specific promoters, rice anther-specific promoters, cotton fiber-specific promoters, etc. Specific promoters can not only enable exogenous genes to play a role in the receptor, but also effectively reduce the adverse effects on plants. At present, the continuous and efficient expression of the white-flowered Paulownia PfD14 gene in the receptor plant not only causes a waste of energy in the organism, but also the expression in all tissues may be toxic to the plant itself. Therefore, the use of a specific Paulownia PfD14 gene promoter is a feasible way to address this risk. Based on this, it is necessary to carry out research on the specific Paulownia PfD14 gene promoter. Summary of the invention

[0006] In order to carry out research on the specific Paulownia tomentosa PfD14 gene promoter, the present invention provides the promoter of the Paulownia tomentosa PfD14 gene and its core region and application. To achieve the above purpose, the present invention adopts the following technical scheme.

[0007] The invention provides a promoter of the Paulownia tomentosa PfD14 gene for driving gene-specific expression. The sequence interval of the promoter includes a 3' end located at the 1st bp upstream of the Paulownia tomentosa SEQ ID NO.1 gene coding sequence ATG, and a 5' end located at the 1988bp upstream of the Paulownia tomentosa SEQ ID NO.1 gene coding sequence ATG.

[0008] The purpose of the present invention is to carry out research on the specific promoter of the Paulownia tomentosa PfD14 gene, and to provide a promoter of the Paulownia tomentosa PfD14 gene for driving gene-specific expression. The promoter of the Paulownia tomentosa PfD14 gene can be used to drive the efficient and specific expression of the target gene in a recipient plant, which can effectively avoid its continuous and efficient expression in the recipient plant, will not cause waste of energy in the organism, and will not be expressed in all tissues, and thus will not cause toxicity to the recipient plant itself. The promoter of the Paulownia tomentosa PfD14 gene for driving gene-specific expression provided by the present invention can effectively solve the impact caused by the continuous and efficient expression of the Paulownia tomentosa PfD14 gene in the recipient plant.

[0009] Among them, the nucleotide sequence of the Paulownia candidum PfD14 gene is shown as SEQ ID NO.1.

[0010] The promoter of the Paulownia alba PfD14 gene used to drive gene-specific expression is referred to as the promoter of the Paulownia alba PfD14 gene for short.

[0011] The primers for the promoter PPfD14 of the Paulownia alba PfD14 gene include PfD14-pro-F and PfD14-pro-R.

[0012] The nucleotide sequence of PfD14-pro-F is shown in SEQ ID NO.3:

[0013] 5'-AAAAAAAAAAAAGGCATAGTAACTATTTG-3'.

[0014] The nucleotide sequence of PfD14-pro-R is shown in SEQ ID NO.4:

[0015] 5'-TATTTCCTTCCAAATTTACAAAAATTCA-3'.

[0016] The promoter P of the Paulownia leucophylla PfD14 gene PfD14 The preparation method comprises the following steps:

[0017] (1) Take 2 μL of DNA from Paulownia tomentosa for PCR amplification. The reaction system is as follows: KOD one Mix 25 μL, PfD14pro2000-F 2.5 μL, PfD14pro2000-R 2.5 μL, and make up to 50 μL with sterile water.

[0018] (2) After gel recovery, refer to the instructions of Clone JET PCR cloning kit to connect 1 μL of the promoter fragment of the PfD14 gene of Paulownia thunbergii to the PJET1.2 cloning vector. The specific reaction system is as follows: 5 μL of 2×Reaction buffer, 0.5 μL of PJET1.2 cloning vector, 0.5 μL of T4 DNA ligase, and make up to 10 μL with sterile water; the promoter fragment of the PfD14 gene of Paulownia thunbergii was obtained. PfD14 The recombinant vector pJET1.2-PfD14-pro was obtained. PfD14 Promoter.

[0019] (3) A promoter of the PfD14 gene of Paulownia leucophylla PfD14 The method for constructing a recombinant vector comprises: using HandIII / BamHI double restriction enzyme to connect P PfD14 The pJET1.2-PfD14-pro1-7 and pGreenII 0800-luc plasmids were obtained, the promoter fragment and pGreenII 0800-luc fragment cut out by enzyme were recovered by gel, and then ligated and transformed into Escherichia coli to construct the plant expression vector pGreen-Pf14-pro1-7-Luc.

[0020] Preferably, the promoter further comprises a 5'UTR sequence.

[0021] Preferably, the length of the 5'UTR sequence is 296 bp.

[0022] Preferably, the 5'UTR sequence is a sequence in the interval 1 bp to 296 bp upstream of the ATG coding sequence of the Paulownia candidum SEQ ID NO. 1 gene.

[0023] Preferably, the nucleotide sequence of the promoter is as shown in SEQ ID NO.2.

[0024] Preferably, the promoter can be induced by any one or more of light, low temperature, abscisic acid and gibberellin.

[0025] The invention also provides a recombinant vector or a transgenic recombinant bacterium containing the promoter.

[0026] Preferably, in the recombinant vector, the transcription of the Paulownia candidum PfD14 gene is promoted by the promoter.

[0027] Preferably, the recombinant vector is pGreen-Pf14-pro-Luc.

[0028] The transgenic recombinant bacteria include Escherichia coli DH5A and / or Agrobacterium tumefaciens GV3101.

[0029] The present invention also provides the application of the promoter in plant breeding. The specific application is the application of the promoter in tobacco and Arabidopsis breeding, and the promoter is used to promote the efficient and specific expression of the target gene in the transgenic tobacco leaves and Arabidopsis roots. Among them, the target gene includes the GUS gene

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention provides a promoter of the PfD14 gene of white Paulownia for driving gene-specific expression. In order to carry out research on the specific promoter of the PfD14 gene of Paulownia, the present invention provides a promoter of the PfD14 gene of white Paulownia. The promoter of the PfD14 gene of white Paulownia can be used to drive the efficient and specific expression of the target gene in the recipient plant, which can effectively avoid its continuous and efficient expression in the recipient plant, will not cause waste of energy in the organism, and will not be expressed in all tissues, and thus will not cause toxicity to the recipient plant itself. The promoter of the PfD14 gene of white Paulownia for driving gene-specific expression provided by the present invention can effectively solve the impact caused by the continuous and efficient expression of the PfD14 gene of white Paulownia in the recipient plant.

[0032] 2. The white Paulownia odorifera P provided by the present inventionPfD14 The promoter can accurately locate the regulated gene, drive the expression of the target gene in the transgenic plant, improve the expression efficiency of the exogenous gene, and can be applied to plant genetic engineering research.

[0033] 3. Paulownia odorifera P. PfD14 The method for preparing the promoter uses a specific P PfD14 Paulownia edulis P PfD14 The promoter is simple to operate and the results are stable and reliable.

[0034] 4. The recombinant vector containing a plant efficient expression driver of the present invention contains the promoter nucleotide sequence, has an appropriate vector size, and is easy to transform in plants; by transforming Nicotiana benthamiana with the vector, a tobacco transgenic plant with efficient expression in plants can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The promoter clone of the PfD14 gene in the present invention; wherein M is 2000DNA maker; Figure 1 A in the figure is the promoter sequence amplification band; Figure 1 B in the figure is the colony PCR identification of the PJET1.2-PfD14 recombinant vector.

[0036] Figure 2 The promoter fragments of different lengths of the PfD14 gene in the present invention are amplified; wherein, Figure 2 A in the figure is a schematic diagram of the promoter fragment of the PfD14 gene, and numbers 1-7 represent promoter fragments P1-P7; Figure 2 B in the figure represents PCR amplification of the promoter fragment, numbers 1-7 represent P1-P7 respectively, and M represents 2000DNAmaker.

[0037] Figure 3 is the identification result of the core region of the promoter of the PfD14 gene in the present invention; wherein, P1 ～ P7 represents fragments of different lengths.

[0038] Figure 4 For the present invention Figure 4 The white Paulownia P PfD14 The results of histochemical staining of transgenic plants driven by promoter GUS; Figure 4 A in it stands for flower (colorless); Figure 4 B in it is the stem (colorless); Figure 4 C in the figure is rosette leaf (colorless); Figure 4 The D in the figure is the root (blue). DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0040] Example 1

[0041] 1. Cloning of the PfD14 gene promoter

[0042] First, the promoter sequence of the PfD14 gene (i.e., the DNA sequence 2000 bp upstream of ATG) was extracted from the genome data of Paulownia truncatum, and primers were designed based on Primer-primer 5.0. Then, cloning was performed using the Paulownia truncatum DNA as a template.

[0043] Among them, the PfD14 gene is the Paulownia candidum PfD14 gene, and its nucleotide sequence is shown in SEQ ID NO.1:

[0044]

[0045] The nucleotide sequence of the promoter of the Paulownia candidum PfD14 gene is shown in SEQ ID NO.2:

[0046]

[0047] The primers for the promoter PPfD14 of the Paulownia alba PfD14 gene include PfD14-pro-F and PfD14-pro-R.

[0048] The nucleotide sequence of PfD14-pro-F is shown in SEQ ID NO.3:

[0049] 5'-AAAAAAAAAAAAGGCATAGTAACTATTTG-3'.

[0050] The nucleotide sequence of PfD14-pro-R is shown in SEQ ID NO.4:

[0051] 5'-TATTTCCTTCCAAATTTACAAAAATTCA-3'.

[0052] The specific steps are as follows.

[0053] (1) Extraction of DNA from Paulownia odorifera

[0054] ① Take 0.2 g of young leaf tissue of Paulownia davidiana and put it into a centrifuge tube containing steel balls, quickly put it into liquid nitrogen for quick freezing, and then quickly put the sample into a pre-cooled cryo-grinder and grind it into powder.

[0055] ② Add 800 μL SLS DNA extraction solution to the centrifuge tube and shake vigorously for 5 minutes.

[0056] ③ Add 800 μL of a mixed solution of Tris-equilibrium phenol-chloroform-isoamyl alcohol with a volume ratio of 25:24:1, mix for 5 minutes, and centrifuge at 12000 rpm at room temperature for 10 minutes.

[0057] ④ Carefully aspirate 500 μL of supernatant and transfer to a 1.5 mL centrifuge tube, then add an equal volume of pre-cooled isoamyl alcohol. Centrifuge at 12000 rpm, room temperature for 10 minutes.

[0058] ⑤ Add 500 μL of 75% ethanol to rinse the DNA precipitate, centrifuge at 12000 rpm for 3 min, and repeat this step once.

[0059] ⑥ Add 500 μL of 100% anhydrous ethanol and let stand at room temperature for 3 minutes.

[0060] ⑦ Centrifuge at 12000rpm for 2min, discard the supernatant, let stand for 7min, and dry the residual anhydrous ethanol in the tube.

[0061] ⑧Add 50 μL ddH2O to dissolve the DNA precipitate.

[0062] ⑨Use NanoDrop2000 to determine the purity and concentration of the extracted DNA.

[0063] (2) PCR system is shown in Table 1:

[0064] Table 1 PCR system

[0065] Components Addition amount KODoneMix 25μL PfD14pro2000-F 2.5μL PfD14pro2000-R 2.5μL DNA 2μL <![CDATA[ddH2O]]> 18μL Total volume 50μL

[0066] (3) Gel recovery of target fragment

[0067] The above PCR products were separated by 1% agarose gel electrophoresis to identify the target bands. After the correct position was identified, the bands were recovered using the TIANGEN agarose gel DNA recovery kit. The specific steps are as follows:

[0068] Add 500μL of balancing solution to the adsorption column to rinse the adsorption membrane, centrifuge at 12000rpm for 1min, and discard the waste liquid; put the gel block containing the target band into a 2mL centrifuge tube, add an equal volume of sol solution, and put it into a 50℃ water bath for sol treatment until the gel block is completely dissolved; transfer the dissolved gel solution to the adsorption column with a collection tube, let it stand at room temperature for 2min, and centrifuge at 12000rpm for 30s; discard the filtrate and add 600μL of rinsing solution PW, let it stand at room temperature for 5min, centrifuge at 12000rpm for 30s, and repeat this step once after discarding the waste liquid; put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2min; take out the adsorption column and put it into a new 1.5mL centrifuge tube, leave it open at room temperature for 10min to allow the rinsing solution to evaporate fully; add 30μL ddH2O, stand at room temperature for 2 minutes, centrifuge at 12000rpm for 2 minutes, collect the DNA solution; use NanoDrop2000 to measure the concentration of the DNA solution.

[0069] (4) Ligation reaction

[0070] According to the instructions of Clone JET PCR Cloning Kit, 1 μL of PfD14 promoter 2000 fragment was connected to PJET1.2 cloning vector. The specific reaction system is as follows: 22°C for 30 min.

[0071] Wherein, the connection reaction system is shown in Table 2:

[0072] Table 2 Ligation reaction system

[0073] Components Addition amount 2×Reactionbuffer 5μL DNA recovery products 1μL pJET1.2 cloning vector 0.5μL T4 DNA ligase 0.5μL <![CDATA[ddH2O]]> 3μL Total volume 10μL

[0074] (5) Conversion reaction

[0075] The above ligation product was added to 30 μL DH5α competent cells, and placed in an ice bath for 30 min; heat-shocked in a water bath at 42°C for 90 s; quickly placed in an ice bath for 2 min; 700 μL of antibiotic-free LB liquid culture medium was added, and cultured for 1 h at 37°C and 220 rpm; centrifuged at 5000 rpm for 5 min, 600 μL of supernatant was aspirated and discarded, and the remaining bacterial liquid was resuspended and coated on a plate containing 100 mg·L -1 Amp + The cells were cultured on LB solid medium at 37°C overnight.

[0076] Among them, overnight culture refers to a culture time ≥ 12h.

[0077] (6) Colony PCR Identification

[0078] Positive clones were identified on the transformation plate by colony PCR. The reaction system is shown in Table 3:

[0079] Table 3 PCR reaction system

[0080]

[0081]

[0082] Reaction procedure: 94℃, 3min; 94℃, 20s, 68℃, 20s, 72℃, 1min, 34 cycles; 72℃, 5min. The amplified products were separated by 1% agarose gel electrophoresis, and the positive clones with the correct band at the target position were added to 5mL containing 100mg·L - 1 Amp + The clones were cultured in LB liquid medium at 37°C with shaking for 16 h and then sent to Qingke Biotechnology Co., Ltd. for sequencing. The clones with correct sequencing were saved and named PJET1.2-PfD14pro2000.

[0083] The results of cloning the PfD14 gene promoter are as follows Figure 1 shown.

[0084] 2. Identification of the core functional region of the PfD14 gene promoter

[0085] 1. Construction of pGreenII 0800-Luc expression vector

[0086] (1) PCR amplification

[0087] According to the obtained PfD14 gene promoter sequence, 7 pairs of homologous recombination primers were designed. Using pJET1.2-PfD14-pro plasmid as template, promoter fragments of different lengths were amplified respectively ( Figure 2 B).

[0088] Among them, the PfD14 gene promoter sequence amplification band is as follows Figure 1 As shown in A; PJET1.2-PfD14 recombinant vector colony PCR identification as shown in Figure 2 As shown in B.

[0089] The PCR reaction systems of PfD14 promoter fragments of different lengths are shown in Table 4:

[0090] Table 4 PfD14 promoter fragment PCR reaction system

[0091] Components Addition amount KODoneMix 25μL PfD14proPn-F 2.5μL PfD14proPn-R 2.5μL pJET1.2-PfD14pro2000 plasmid 2μL <![CDATA[ddH2O]]> 18μL Total volume 50μL

[0092] (2) Preparation of linearized vector

[0093] According to the multiple cloning site on the pGreenII 0800-luc vector, the vector was double-digested with HandIII and BamHI.

[0094] Among them, the reaction system for preparing the linearized carrier is shown in Table 5:

[0095] Table 5 Reaction system for linearized vector preparation

[0096] Components volume Component Volume B H 2μL HandⅢ 2μL 10*FastDigestGreenbuffer 5μL pGreenII0800-luc vector 5μL <![CDATA[ddH20]]> 35μL

[0097] (3) Gel recovery and purification of target fragment and linearized vector

[0098] The above PCR and enzyme digestion products were separated by 1% agarose gel electrophoresis to separate the target bands, and after the correct position was identified, the bands were recovered using the TIANGEN agarose gel DNA recovery kit. The specific steps were the same as above.

[0099] (4) Homologous recombination

[0100] The promoter fragments of different lengths and the pGreenII 0800-luc fragment were ligated using the Novozymes CE II one step cloning kit.

[0101] Wherein, the connection reaction system is shown in Table 6:

[0102] Table 6 Ligation reaction system

[0103] Components volume Component Volume Linearized pGreenII0800-luc vector 0.8μL PfD14proPn purified fragment 1μL 5xCEIIbuffer 2μL ExnaseII 1μL <![CDATA[ddH2O]]> 5.2μL

[0104] (5) Transformation system

[0105] The ligation product was transformed into E. coli DH5α competent cells, and the transformation steps were the same as above.

[0106] (6) Colony identification

[0107] Positive clones were selected for colony PCR. The PCR system is shown in Table 7.

[0108] Table 7 PCR system

[0109] Components Addition amount 2×TaqPCRstarmix 5μL PGreen-F 0.5μL PGreen-R 0.5μL Bacterial liquid 4μL Total volume 10μL

[0110] The clones at the correct position were selected and sent for testing. After the sequencing results were compared by NCBI-blast, the bacterial solution with correct sequencing was retained to obtain expression vector plasmids with promoter fragments of different lengths fused to the Luc reporter gene and marked as pGreen-Pf14-pro-P1-Luc to pGreen-PfD14-pro-P7-Luc.

[0111] The amplification results of promoter fragments of different lengths of PfD14 gene are shown in Figure 2 shown.

[0112] 2. Tobacco instant transformation

[0113] The correctly sequenced pGreen-Pf14-pro-Pn-Luc plasmid and the pGreenII 0800-luc empty vector plasmid were respectively transformed into Agrobacterium competent GV3101-pSoup (P19).

[0114] The operation steps refer to the instructions: add 2 μL of recombinant plasmid and 20 μL of competent cells to a sterilized 1.5 mL centrifuge tube, ice bath for 30 min; quick freeze in liquid nitrogen for 5 min; bath in 37°C water for 5 min, and then quickly bath in ice for 5 min; add 140 μL of antibiotic-free YEP liquid culture medium, shake at 28°C, and resume culture for 3 h; take 80 μL of bacteria and evenly spread it on a plate containing 100 mg·L -1 kana+ and 50mg·L -1 The cells were incubated on Rif+ YEP solid medium at 28°C for 3 days, and positive single colonies were picked for colony PCR and agarose electrophoresis identification. The correct clones were selected for preservation and the colony identification was the same as above.

[0115] Among them, Agrobacterium competent cell GV3101-pSoup (P19) was purchased from Weidi Biotechnology.

[0116] Pick the correct single clone and add it to 1 mL of YEP liquid medium containing the above three resistances, and culture it overnight at 28℃ and 200 rpm; inoculate 1 mL of overnight cultured bacteria into 5 mL of YEP liquid medium containing the above three resistances and continue to culture until OD 600 =0.9; centrifuge at 5000rpm for 10min to collect the cells, and resuspend them with resuspension solution to OD 600 =0.6, and injected into the lower epidermis of tobacco leaves after being placed at room temperature for 2.5 h; 3 days after injection, the Luc signal was observed to analyze the strength of the activation function of different fragments.

[0117] The results are as follows Figure 3 As shown in the figure, it can be seen that the three longer segments P1, 2 and 3 can all activate the expression of the Luc reporter gene, and the signal is strong, while the activation ability of the P6 and P7 segments is weaker, and the activation activity of the P7 segment is less than that of the P6 segment, indicating that the core promoter segment of the PfD14 gene may be located in the P3 segment, that is, 0 to 839 bp. At the same time, the activation ability of the P5 segment is significantly greater than that of the P4 segment, indicating that the P5 segment is the core promoter region of PfD14, that is, between 290 bp and 839 bp.

[0118] From the above experimental results, it can be seen that in the leaves of Nicotiana benthamiana, the white Paulownia PfD14 promoter has strong transcriptional activation activity, and the 290bp to 839bp region of the promoter is its core activation region. The promoter cloned by the promoter cloning method provided by the present invention can obtain the white Paulownia PfD14 promoter with strong transcriptional activation activity. In addition, the present invention also includes a recombinant vector containing a plant high-efficiency expression promoter, which is easy to transform and detect in Nicotiana benthamiana. By using the vector to transform Nicotiana benthamiana, a highly expressed tobacco transgenic plant can be obtained.

[0119] Among them, the promoter of the PfD14 gene of Paulownia leucophylla PfD14 Also known as Paulownia davidiana PfD14 promoter, P PfD14 Promoter, Paulownia truncatula P PfD14 Promoter and P PfD14 ,

[0120] Example 2: P PfD14 Plant expression vector pBI-P PfD14 Genetic transformation and transgenic plant screening in Arabidopsis thaliana

[0121] Preparation of plant expression vector pBWA(V)HG-P PfD14 Suspend the EHA105 bacterial suspension of Agrobacterium tumefaciens in an equal volume of 5% sucrose solution. Pour the turbid sucrose solution into a culture dish with a diameter of 15 cm. Add Silwet L-77 with a final concentration of 0.02% (volume ratio) before transformation and mix well. Gently immerse the entire inflorescence of Arabidopsis to be transformed in sucrose, and take out the inflorescence of the plant after 15 seconds. Wrap the transformed plants in black plastic bags and culture them in a plant growth box.

[0122] Among them, Silwet L-77 was purchased from Beijing Wuzhou Yuanye Science and Technology Trade Center.

[0123] The next day, open the plastic bag and perform transformation again after one week. Harvest the seeds after about one month of cultivation and dry them in an incubator or under sunlight for 4 days. Disinfect the T0 generation seeds harvested from the transformation with 70% (volume ratio) alcohol and 0.01% (mass ratio) mercuric chloride for 3 minutes and 10 minutes respectively, then wash 6 times with distilled water and blow evenly onto the surface of MS solid screening medium containing kanamycin (50 mg / L). After placing in the dark at 4°C for 4 days, transfer to a plant growth incubator for cultivation. Screen positive seedlings based on kanamycin resistance.

[0124] When the leaves grow large enough (3-4 leaves), take a few green seedling leaves, extract DNA, and perform PCR positive detection. The reaction system is 25 μL, containing: 1×PCR buffer, MgCl21.5mmol / L, dNTP 0.2mmol / L, primer concentration of 0.5mol / L, Pfu enzyme 1.5 units, and template about 100ng;

[0125] The primers used were:

[0126] P PfD14 The nucleotide sequence of S is shown in SEQ ID NO.5:

[0127] 5'-ACCGCCTGCAGGTCTATAGAaaaaaaaaaaaaggcatagtaactatttgaacatatgacc g-3'.

[0128] P PfD14 The nucleotide sequence of A is shown in SEQ ID NO.6:

[0129] 5'-GATCTACCATGGTCAAGTTGtatttccttccaaatttacaaaaattcaagaaaccttttga-3'.

[0130] The cycle parameters were set as follows: 98℃ denaturation for 10s, 68℃ annealing for 30s, 72℃ extension for 2min, 35 cycles; 72℃ extension for 10min. The results showed that transgenic positive seedlings were obtained.

[0131] The transgenic positive seedlings were cultured, and after the green seedlings grew two true leaves, they were transplanted into vermiculite. After the plants grew inflorescences, a true leaf was taken to extract genomic DNA using the SDS method and perform PCR identification. The primer sequence used for PCR identification was P PfD14 S and P PfD14 A;

[0132] The PCR reaction system was as follows: 1 μL (about 50 ng) of genomic DNA template, 25 μL of BiorunPfu PCR Mix, 2.5 μL of each 10 μmol / L primer, and sterile water to 50 μL.

[0133] The reaction procedure was: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 50℃ annealing for 45s, 72℃ extension for 2min, 30 cycles, and 72℃ extension for 5min. The PCR reaction products were detected by 1% agarose gel electrophoresis. The results showed that: PfD14 Plant expression vector pBWA(V)HG-P PfD14 The T-DNA segment has been successfully transferred into Arabidopsis thaliana to obtain positive seedlings.

[0134] Example 3: Paulownia odorifera P PfD14 Functional analysis of promoters

[0135] The present invention cloned the Paulownia odorifera P for the first time. PfD14 The promoter sequence was sequenced and its function was analyzed. The positive seedlings screened in the Arabidopsis transformation and PCR detection steps in Example 2 were used. Different tissues were taken for GUS staining.

[0136] The staining process is as follows: soak the sample in GUS stain solution, vacuum for 5 minutes, and keep at 37°C overnight. The next day, decolorize with alcohol-acetic acid solution (volume ratio of 1:1) until the leaves become colorless, then rinse with 50% alcohol 4 times, and take pictures with a stereo microscope (OLYMPUS SZX16). Different parts of the plant were taken for staining during the seedling stage. The parts of the plant that are stained blue are the parts where the GUS gene is expressed.

[0137] The staining results are as follows Figure 4 As shown: In Arabidopsis, only the root is stained blue, and no blue appears in other tissues. This shows that the GUS gene driven by this promoter is mainly expressed in the root, but not in other tissues.

[0138] The above experimental results show that Paulownia leucophylla P PfD14 The promoter has the following biological function: the GUS gene driven by the promoter is specifically expressed in the root of Arabidopsis thaliana, but not in other tissues. PfD14 The GUS gene under regulation has certain temporal and spatial expression characteristics, which indicates that the P. PfD14 The promoter has the function of driving the expression of the downstream reporter gene GUS in the recipient plant. Under the regulation of this promoter, the GUS gene can be mainly expressed in the roots of the transgenic plants, but not expressed at all in other tissues.

[0139] This promoter with tissue-specific expression has application value in plant genetic engineering. This precise tissue-specific expression pattern not only greatly increases the accumulation of target genes in the roots, but also effectively avoids unnecessary energy loss and significantly improves the efficiency of genetic manipulation.

[0140] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0141] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A promoter of the Paulownia davidiana PfD14 gene for driving gene-specific expression, characterized in that: The sequence interval of the promoter includes the 3' end located at the 1st bp upstream of the ATG coding sequence of the Paulownia tomentosa SEQ ID NO.1 gene, and the 5' end located at the 1988 bp upstream of the ATG coding sequence of the Paulownia tomentosa SEQ ID NO.1 gene.

2. The promoter according to claim 1, characterized in that The promoter also includes a 5'UTR sequence.

3. The promoter according to claim 2, characterized in that .The length of the 5'UTR sequence is 296 bp.

4. The promoter according to claim 2, characterized in that The 5'UTR sequence is a sequence of 1 bp to 296 bp upstream of the ATG coding sequence of the gene of Paulownia candidum SEQ ID NO.

1.

5. The promoter according to any one of claims 1 to 4, characterized in that The nucleotide sequence of the promoter is shown in SEQ ID NO.

2.

6. The promoter according to claim 5, characterized in that The promoter can be induced by any one or more of light, low temperature, abscisic acid and gibberellin.

7. A recombinant vector or transgenic recombinant bacterium containing the promoter according to claim 1.

8. The recombinant vector or genetically modified recombinant bacterium according to claim 7, characterized in that: In the recombinant vector, the promoter drives the transcription of the Paulownia alba PfD14 gene.

9. The recombinant vector or genetically modified recombinant bacterium according to claim 8, characterized in that: The recombinant vector is pGreen-Pf14-pro-Luc.

10. Use of the promoter according to claim 1 in plant breeding, characterized in that: The promoter is used to initiate the expression of the target gene in the transgenic plant; The plants include tobacco and Arabidopsis; The target gene includes the GUS gene.

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