Soybean stem specific promoter Pgc17 and application thereof
By cloning the soybean stem-specific promoter Pgc17, the problem of lack of specific expression of soybean stem part is solved, and the specific gene expression of soybean stem part is achieved, improving the accuracy and efficiency of improving the quality of soybean stem.
Patent Information
- Application Number
- CN202510566188.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-07-18
AI Technical Summary
The lack of stem-specific promoters of soybeans in the prior art leads to uncontrolled gene expression of transgenic soybeans in undesired tissues, increasing plant metabolic burden and poor growth.
By screening the soy transcriptome database, specific primers were designed, stem-specific promoter Pgc17 was cloned from the genome of the soy variety Williams 82, and a recombinant vector was constructed to fuse it with the red fluorescent protein reporter DsRed, and introduced it into the soybean plant to achieve stem-tissue specific expression.
The specific expression of soybean stems was successfully achieved, gene accumulation in unexpected tissues was avoided, timing, localization and quantitative control of gene expression were improved, and the quality improvement of soybean stems was promoted.
Smart Images
Figure CN120330191A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant genetic engineering, and particularly relates to a stem-specific promoter Pgc17 of soybean and its application. Background Art
[0002] Soybean is an important food and oil crop in the world. The stem tissue of soybean is related to the transport of nutrients during the growth process of soybean. In addition, the soybean stem contains rich proteins and dietary fibers during the mature period, which can be used as animal feed, biomass dyes, and industrial raw materials, etc. The isolation and identification of stem-specific promoters are of great significance for improving crop stem development and stem quality using engineering techniques.
[0003] In-depth research on the structure, function, and expression pattern of promoters provides important support for elucidating the regulation mechanism of gene expression. Currently, most of the promoters used for crop genetic improvement belong to constitutive strong promoters, such as the CaMV 35S promoter. Since it is constitutively expressed and drives the high expression of foreign genes in all tissues and developmental stages of transgenic plants, it will increase the metabolic burden and high energy consumption of plants, have an adverse impact on the growth of crops, and lead to poor agronomic traits of transgenic crops. There are certain obstacles in the application of constitutive promoters in genetic engineering breeding. Tissue-specific promoters can drive the expression of downstream genes in specific types of tissues, avoid the problem of large accumulation of proteins in non-target tissues, and can precisely control the timing, location, and quantification of the expression of target genes, greatly accelerating the process of plant improvement. Summary of the Invention
[0004] The purpose of the present invention is to provide a stem-specific promoter Pgc17 of soybean.
[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:
[0006] The present invention provides a stem-specific promoter Pgc17 of soybean, and the nucleotide sequence of the stem-specific promoter Pgc17 is shown as SEQ ID NO.1.
[0007] The present invention provides a primer group for cloning the above-mentioned stem-specific promoter Pgc17. The primer group includes Pgc17-F1 and Pgc17-R1. The sequence of Pgc17-F1 is shown as SEQ ID NO.2, and the sequence of Pgc17-R1 is shown as SEQ ID NO.3.
[0008] The present invention provides a kit containing the above-mentioned primer group.
[0009] The present invention provides a method for constructing the stem-specific promoter Pgc17, comprising the following steps: Using the promoter sequence of the soybean Glyma.15G271000.1 gene as a template, performing PCR amplification with the above primer set to obtain a sequence fragment of the stem-specific promoter Pgc17.
[0010] The present invention provides a recombinant vector containing the above stem-specific promoter Pgc17.
[0011] The present invention provides a method for constructing the recombinant vector, comprising the following steps: Ligating the digested pCAMBIA3300 vector with the above stem-specific promoter Pgc17 to obtain the recombinant vector.
[0012] The present invention provides a recombinant bacterium containing the above recombinant vector.
[0013] The present invention provides the application of the stem-specific promoter Pgc17, or the kit, or the recombinant vector, or the recombinant bacterium in the tissue-specific expression of plant stems.
[0014] The present invention further provides a method for constructing a transgenic soybean plant, comprising the following steps:
[0015] (1) Infecting a target soybean plant with the above recombinant bacterium, and then inducing adventitious buds;
[0016] (2) Inoculating the adventitious buds onto a bud elongation medium and culturing until the bud length reaches 3 - 5 cm, and then inoculating onto a rooting medium and continuing the culture to obtain a transgenic soybean plant with tissue-specific expression in the stem.
[0017] Preferably, step (1) is carried out in an induction medium, which uses water as a solvent and consists of components with the following mass concentrations: B5 salts 3.1 - 3.4 g / L, sucrose 28 - 32 g / L, MES 0.4 - 0.65 g / L, BAP 1.5 - 1.8 mg / L, cefamycin 240 - 260 mg / L, Timentin 90 - 110 mg / L, glufosinate 5 - 6 mg / L, and agar powder 6 - 10 g / L.
[0018] Preferably, the shoot elongation medium uses water as a solvent and consists of components with the following mass concentrations: MS salts 4.2 - 4.6 g / L, sucrose 28 - 32 g / L, MES 0.4 - 0.65 g / L, aspartic acid 48 - 52 mg / L, L-glutamic acid 48 - 52 mg / L, IAA 0.08 - 0.12 mg / L, GA3 0.4 - 0.6 mg / L, zeatin riboside 0.9 - 1.1 mg / L, cefamycin 240 - 260 mg / L, Timentin 90 - 110 mg / L, glufosinate 5 - 6 mg / L, and agar powder 6 - 10 g / L.
[0019] Preferably, the rooting medium uses water as a solvent and consists of components with the following mass concentrations: MS salts 4.2 - 4.6 g / L, sucrose 18 - 22 g / L, MES 0.4 - 0.65 g / L, aspartic acid 48 - 52 mg / L, L-glutamic acid 48 - 52 mg / L, IBA 0.8 - 1.2 mg / L, and phytagel 2 - 4 / L.
[0020] By adopting the above technical solutions, the present invention has the following beneficial effects:
[0021] In the present invention, the stem-specific expression gene Glyma.15G271000.1 was screened based on the soybean transcriptome database. Specific primers were designed through genomic sequence analysis, and the stem-specific promoter Pgc17 fragment of this gene was successfully cloned from the genomic DNA of soybean variety Williams 82. The obtained Pgc17 promoter sequence was constructed into a fusion expression vector pCAMBIA3300 - Pgc17 - DsRed with the red fluorescent protein reporter gene DsRed, and transgenic soybean plants were obtained by genetic transformation technology and introduced into the target plant soybean.
[0022] Experiments showed that the Glyma.15G271000.1 gene had the highest expression abundance in soybean stems, and the DsRed fluorescence signal was specifically limited to the stems of transgenic soybean plants. Compared with the prior art, the identified Pgc17 promoter in the present invention has significant stem tissue specificity advantages, overcomes the current shortage of endogenous specific promoters in soybeans, and has important application value in analyzing gene functions and genetic improvement. Description of the Drawings
[0023] Figure 1 It is a diagram of the RT-qPCR detection results of the Glyma.15G271000.1 gene in different tissue parts;
[0024] Figure 2 It is a diagram of the PCR gel electrophoresis results for cloning the promoter Pgc17;
[0025] Figure 3 It is the prediction result diagram of the cis - acting elements of the stem - specific promoter Pgc17;
[0026] Figure 4 It is the composition of the recombinant vector pCAMBIA3300 - Pgc17 - DsRed;
[0027] Figure 5 It is the red fluorescence result diagram of different parts of the pCAMBIA3300 - Pgc17 - DsRed transgenic soybean plants. Specific implementation manners
[0028]
[0029] The present invention also provides a primer set for cloning the above-mentioned stem-specific promoter Pgc17. The primer set includes Pgc17-F1 and Pgc17-R1. The sequence of Pgc17-F1 is shown as SEQ ID NO.2, and the specific sequence is 5’-GCAACAGGAACAATGTGACC-3’; the sequence of Pgc17-R1 is shown as SEQ ID NO.3, and the specific sequence is 5’-TGCTGCTTTTGGTATCCAA-3’.
[0030] The present invention also provides a kit containing the above-mentioned primer set.
[0031] The present invention also provides a construction method of the stem-specific promoter Pgc17, which includes the following steps: using the promoter sequence of the soybean Glyma.15G271000.1 gene as a template, and performing PCR amplification with the above-mentioned primer set to obtain a sequence fragment of the stem-specific promoter Pgc17.
[0032]
[0033] In the present invention, the system for PCR amplification includes 25 μL of high-fidelity enzyme mix, 2 μL of template, 1 μL of primer Pgc17-F, 1 μL of primer Pgc17-R, and ddH2O is added to make up to 50 μL.
[0034] In the present invention, the program for PCR amplification is 95 °C for 3 min; (95 °C for 30 sec; 60 °C for 30 sec; 72 °C for 2 min;) 30 cycles; 72 °C for 10 min.
[0035] The present invention provides a recombinant vector containing the above-mentioned stem-specific promoter Pgc17.
[0036] The present invention provides a method for constructing the recombinant vector, which includes the following steps: ligating the digested pCAMBIA3300 vector with the above-mentioned stem-specific promoter Pgc17, and then the recombinant vector is obtained.
[0037] The present invention provides a recombinant bacterium containing the above-mentioned recombinant vector.
[0038] The present invention provides the application of the above-mentioned stem-specific promoter Pgc17, or the kit, or the recombinant vector, or the recombinant bacterium in the specific expression of plant stem tissues.
[0039] The present invention also provides a method for constructing a transgenic soybean plant, which includes the following steps:
[0040] (1) Infecting the target soybean plant with the above-mentioned recombinant bacterium, and then inducing adventitious buds;
[0041] (2) Inoculating the adventitious buds onto a bud elongation medium and culturing until the buds grow to 3 - 5 cm, and then inoculating them onto a rooting medium and continuing the culture, thus obtaining a transgenic soybean plant with specific expression in stem tissues.
[0042] In the present invention, step (1) is carried out for induction in an induction medium. The induction medium uses water as a solvent and preferably consists of components with the following mass concentrations: 3.1 - 3.4 g / L of B5 salts, 28 - 32 g / L of sucrose, 0.4 - 0.65 g / L of MES, 1.5 - 1.8 mg / L of BAP, 240 - 260 mg / L of cefamycin, 90 - 110 mg / L of Timentin, 5 - 6 mg / L of glufosinate, and 6 - 10 g / L of agar powder; further preferably, it is 3.21 g / L of B5 salts, 30 g / L of sucrose, 0.59 g / L of MES, 1.67 mg / L of BAP, 250 mg / L of cefamycin, 100 mg / L of Timentin, 5 - 6 mg / L of glufosinate, and 8 g / L of agar powder.
[0043] In the present invention, the bud elongation medium uses water as a solvent and is preferably composed of components including the following mass concentrations: MS salts 4.2 - 4.6 g / L, sucrose 28 - 32 g / L, MES 0.4 - 0.65 g / L, aspartic acid 48 - 52 mg / L, L-glutamic acid 48 - 52 mg / L, IAA 0.08 - 0.12 mg / L, GA3 0.4 - 0.6 mg / L, zeatin riboside 0.9 - 1.1 mg / L, cefamycin 240 - 260 mg / L, Timentin 90 - 110 mg / L, glufosinate 5 - 6 mg / L, and agar powder 6 - 10 g / L; more preferably, it is MS salts 4.43 g / L, sucrose 30 g / L, MES 0.59 g / L, aspartic acid 50 mg / L, L-glutamic acid 50 mg / L, IAA 0.1 mg / L, GA3 0.5 mg / L, zeatin riboside 1.0 mg / L, cefamycin 250 mg / L, Timentin 100 mg / L, glufosinate 5 - 6 mg / L, and agar powder 8 g / L.
[0044] In the present invention, the rooting medium uses water as a solvent and is preferably composed of components including the following mass concentrations: MS salts 4.2 - 4.6 g / L, sucrose 18 - 22 g / L, MES 0.4 - 0.65 g / L, aspartic acid 48 - 52 mg / L, L-glutamic acid 48 - 52 mg / L, IBA 0.8 - 1.2 mg / L, and phytagel 2 - 4 g / L; more preferably, it is MS salts 4.43 g / L, sucrose 20 g / L, MES 0.59 g / L, aspartic acid 50 mg / L, L-glutamic acid 50 mg / L, IBA 1.0 mg / L, and phytagel 3 g / L.
[0045] The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.
[0046] The soybean variety Williams 82 described in the present invention is obtained from the National Crop Germplasm Resources Platform (website: http: / / www.cgris.net / home).
[0047] Example 1 Transcription levels of the Glyma.15G271000.1 gene in different tissues
[0048] Total RNA was separately extracted from tissue samples of roots, leaves, stems, pods, seeds, etc. of soybean plants at the mature stage, transcribed into cDNA, and specific primers Glyma.15G271000.1-F1 and Glyma.15G271000.1-R1 were designed. Then, real-time quantitative PCR was performed using the specific primers to verify the expression levels of Glyma.15G271000.1 in different tissues and organs of soybean. The detection instrument was ABI PRISM 7500Fast Real-Time PCR System (Applied Biosystems, CA, USA), and the internal reference gene was GmACT6 (GenBank No. NM_001289231). The relative expression levels of the genes were detected by the 2 –ΔΔCT (Livak) method.
[0049] The sequence of the soybean Glyma.15G271000.1 gene of the present invention is shown in SEQ ID NO.6. The sequence of Glyma.15G271000.1-F1 is shown in SEQ ID NO.4, and the specific sequence is 5’-ACATTTGGTGATCGGGTGAAGC-3’; the sequence of Glyma.15G271000.1-R1 is shown in SEQ ID NO.5, and the specific sequence is 5’-CAATGTAGGGCTCGGTGGAA-3’.
[0050] The RT-qPCR program was as follows: 94°C, 10 min; (94°C, 45 s; 60°C, 45 s; 72°C, 30 s) for 35 cycles; 72°C, 10 min. The RT-qPCR system was 10 μL of SYBR Green PCR Master Mix, 2 μL of cDNA, 0.2 μL of primer Glyma.15G271000.1-F1, 0.2 μL of primer Glyma.15G271000.1-R1, and ddH2O was added to make up to 20 μL.
[0051] The results showed that the expression level of the Glyma.15G271000.1 gene was the highest in the stem, and almost no expression was detected in other tissues such as leaves and stems (such as Figure 1 ), which was consistent with the transcriptome data, confirming the specific expression characteristics of the Glyma.15G271000.1 gene in the stem tissue.
[0052] Example 2 Cloning of the promoter Pgc17 of the Glyma.15G271000.1 gene
[0053] Specific primers were designed according to the promoter sequence of soybean Glyma.15G271000.1 genome, and then PCR amplification was carried out. The amplification products were electrophoretically separated on 1% agarose gel (the electrophoresis results are as Figure 2 shown). The agarose gel containing the target fragment was cut and collected into a centrifuge tube, recovered using the Omega Gel Extraction Kit kit, and cloned onto the pEASY-Blunt vector (purchased from Beijing TransGen Biotech Co., Ltd.), transformed into competent cells DH5α, positive clones were obtained by antibiotic screening, and after verification by colony PCR, it was sent for sequencing to confirm that the inserted DNA fragment was 1623 bp. After the sequence comparison was correct, the inventor named this sequence fragment as the stem-specific promoter Pgc17.
[0054] The primer group designed in the present invention includes Pgc17-F1 and Pgc17-R1. The sequence of Pgc17-F1 is as shown in SEQ ID NO.2, and the specific sequence is 5’-GCAACAGGAACAATGTGACC-3’; the sequence of Pgc17-R1 is as shown in SEQ ID NO.3, and the specific sequence is 5’-TGCTGCTTTTGGTATCCAA-3’.
[0055] The PCR amplification system of the present invention is: 25 μL of high-fidelity enzyme mix, 2 μL of template, 1 μL of primer Pgc17-F1, 1 μL of primer Pgc17-R1, and ddH2O was added to make up to 50 μL. The PCR amplification program of the present invention is as follows: 95 °C, 3 min; (95 °C, 30 sec; 60 °C, 30 sec; 72 °C, 2 min;) 30 cycles; 72 °C, 10 min.
[0056] The system of the colony PCR amplification of the present invention includes 10 μL of 2X PCR MasterMix, 1 μL of template, 1 μL of forward primer, 1 μL of reverse primer, and ddH2O was added to make up to 20 μL. The program of the colony PCR amplification is 95 °C, 3 min; (95 °C, 30 sec; 60 °C, 30 sec; 72 °C, 2 min;) 30 cycles; 72 °C, 10 min. The forward primer used is the same as the sequence of Pgc17-F1, and the reverse primer is the same as the sequence of Pgc17-R1.
[0057] Example 3 Analysis of cis-acting elements of promoter Pgc17
[0058] The PlantPAN 4.0 online software was used to analyze the cis-acting elements of the promoter Pgc17 sequence obtained in Example 2.
[0059] The results showed that the Pgc17 promoter sequence contains cis - acting elements such as circadian, CAT - box, G - Box, and Box4 (as Figure 3 shown).
[0060] The sequence of the circadian is shown in SEQ ID NO.7, and the specific sequence is AAAATATCT; the sequence of the CAT - box is shown in SEQ ID NO.8, and the specific sequence is GCCACT; the sequence of the G - Box is shown in SEQ ID NO.9, and the specific sequence is CACGTG; the sequence of the Box 4 is shown in SEQ ID NO.10, and the specific sequence is ATTAAT.
[0061] Example 4 Obtaining of Transgenic Soybeans Containing the pCAMBIA3300 - Pgc17 - DsRed Recombinant Vector
[0062] After double - digesting the pCAMBIA3300 vector (purchased from Tsingke Biotechnology Co., Ltd., Beijing) with EcoRⅠ and HindⅢ, it was ligated with the red fluorescent protein reporter gene DsRed, and pCAMBIA3300 - DsRed was obtained through optimization.
[0063] The Pgc17 fragment of the Glyma.15G271000.1 promoter cloned on the pEASY - Blunt vector was seamlessly inserted into the pCAMBIA3300 - DsRed expression vector to obtain the pCAMBIA3300 - Pgc17 - DsRed recombinant vector (as Figure 4 ).
[0064] Agrobacterium - mediated transformation was used. The pCAMBIA3300 - Pgc17 - DsRed recombinant vector was introduced into Agrobacterium tumefaciens EHA105 (purchased from Tsingke Biotechnology Co., Ltd., Beijing) to obtain recombinant Agrobacterium. The specific transformation process is as follows:
[0065] (1) Pick a single colony of Agrobacterium tumefaciens EHA105 and inoculate it into 5 mL of YEP liquid medium (50 - 100 mg / L spectinomycin, 25 mg / L rifampicin), and culture it overnight at 28°C. The next day, expand the culture in 50 mL of YEP medium until the OD 600nm reaches 0.6 - 0.8. After centrifuging the bacteria at 3000 rpm for 10 min, resuspend them in the liquid co - culture medium (the components are: 0.321 g / L of B5 salts, 30 g / L of sucrose, 3.9 g / L of MES, 1.67 mg / L of BAP, 0.25 mg / L of GA3, 400 mg / L of cysteine, 154.2 mg / L of DTT, and 200 μmol / L of AS, pH 5.4), and adjust the OD 600nm to 0.5 for use.
[0066] (2) Agrobacterium infection
[0067] Use a scalpel to cut open the Jiyu 86 soybean seeds along the hilum part of the soybean seeds, remove the skin, make a slight scratch at the cotyledon node position, and then place the prepared explants in the resuspended Agrobacterium for 30 min. Transfer the infected explants to the co-culture medium (the components are 0.321 g / L of B5 salt, 30 g / L of sucrose, 3.9 g / L of MES, 1.67 mg / L of BAP, 0.25 mg / L of GA3, 400 mg / L of cysteine, 154.2 mg / L of DTT, 200 μmol / L of AS, and 5 g / L of agar powder, pH 5.4), and culture them in the dark at 23°C for 4 d.
[0068] (3) Adventitious bud induction
[0069] After 4 d of co-culture of the explants, transfer the explants to the induction medium (the components are 3.21 g / L of B5 salt, 30 g / L of sucrose, 0.59 g / L of MES, 1.67 mg / L of BAP, 250 mg / L of cefotaxime, 100 mg / L of Timentin, 5 - 6 mg / L of glufosinate, and 8 g / L of agar powder, pH 5.7). The adaxial surface of the cotyledon node and hypocotyl parts of the explants should be inserted into the medium facing upwards and at an angle of 45° to the horizontal plane, and culture them under the conditions of 25°C and a 16 / 8 h light / dark cycle for about 2 weeks. Take out the explants, cut off the excess hypocotyl part, leaving only 5 mm, and then transfer the explants to fresh induction medium and continue to culture for 2 weeks under the same culture conditions.
[0070] (4) Bud elongation
[0071] Transfer the induced cluster buds (removing the cotyledon tissue) to the bud elongation medium (the components are 4.43 g / L of MS salt, 30 g / L of sucrose, 0.59 g / L of MES, 50 mg / L of aspartic acid, 50 mg / L of L-glutamic acid, 0.1 mg / L of IAA, 0.5 mg / L of GA3, 1.0 mg / L of zeatin riboside, 250 mg / L of cefotaxime, 100 mg / L of Timentin, 5 - 6 mg / L of glufosinate, and 8 g / L of agar powder, pH 5.7) for culture, and the culture conditions are 25°C and a 16 / 8 h light / dark cycle. Subculture once every 2 weeks.
[0072] (5) Rooting
[0073] When the resistant buds grow to 3 - 5 cm in length, cut them off, soak them in IBA (1 mg / L) for 30 s, and then transfer them to a rooting medium (the components are MS salts 4.43 g / L, sucrose 20 g / L, MES 0.59 g / L, aspartic acid 50 mg / L, L - glutamic acid 50 mg / L, IBA 1.0 mg / L, and plant gel 3 g / L, pH 5.6) for further cultivation. Transplant them to the greenhouse to grow and set seeds when strong roots have grown.
[0074] Using the specific primers of promoter Pgc17, perform PCR detection on the T1 generation transgenic soybeans. After combining herbicide screening and obtaining transgenic soybeans containing pCAMBIA3300 - Pgc17 - DsRed, observe the red fluorescence.
[0075] Example 5
[0076] Using the LUYOR - 3415RG dual - wavelength fluorescence imaging system, perform tissue - specific expression analysis on the T1 generation transgenic soybeans of pCAMBIA3300 - Pgc17 - DsRed, and observe the red fluorescence in different parts of the transgenic soybeans of pCAMBIA3300 - Pgc17 - DsRed.
[0077] The results show that only the stems of the transgenic soybean plants have red fluorescence (as Figure 4 shown), indicating that the promoter Pgc17 is specifically expressed in the stem tissue.
[0078] In summary, it can be seen that the technical solution of the present invention successfully cloned the stem - specific promoter Pgc17 fragment from the genomic DNA of the soybean variety Williams 82, and further obtained transgenic soybean plants containing the expression vector pCAMBIA3300 - Pgc17 - DsRed. In the present invention, it was verified that the Glyma.15G271000.1 gene has the highest expression abundance in soybean stems, and the DsRed fluorescence signal is specifically limited to the stems of the transgenic soybean plants, that is, the Pgc17 promoter has significant stem - tissue - specific advantages.
[0079] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A stem-specific promoter Pgc17 of soybean, characterized in that, The nucleotide sequence of the stem-specific promoter Pgc17 is shown in SEQ ID NO.
1.
2. A primer set for cloning the stem-specific promoter Pgc17 described in claim 1, characterized in that The primer set includes Pgc17-F1 and Pgc17-R1. The sequence of Pgc17-F1 is shown in SEQ ID NO.2, and the sequence of Pgc17-R1 is shown in SEQ ID NO.
3.
3. A kit containing the primer set according to claim 2.
4. A method for constructing the stem-specific promoter Pgc17 according to claim 1, characterized in that, It includes the following steps: Using the promoter sequence of the soybean Glyma.15G271000.1 gene as a template, performing PCR amplification with the primer set according to claim 2 to obtain a sequence fragment of the stem-specific promoter Pgc17.
5. A recombinant vector containing the stem-specific promoter Pgc17 according to claim 1.
6. The method for constructing the recombinant vector according to claim 5, characterized in that It includes the following steps: Connecting the digested pCAMBIA3300 vector with the stem-specific promoter Pgc17 according to claim 1 to obtain it.
7. A recombinant bacterium containing the recombinant vector according to claim 5.
8. The application of the stem-specific promoter Pgc17 according to claim 1, or the kit according to claim 3, or the recombinant vector according to claim 5, or the recombinant bacterium according to claim 7 in the tissue-specific expression of plant stems.
9. A method for constructing a transgenic soybean plant, characterized in that, It includes the following steps: (1) Infecting the target soybean plants with the recombinant bacterium according to claim 7, and then inducing adventitious buds. (2) Inoculating the adventitious buds into a bud elongation medium and culturing until the bud length reaches 3 - 5 cm, and then inoculating them into a rooting medium and continuing the culture to obtain transgenic soybean plants with tissue-specific expression in stems.
10. The construction method according to claim 9, characterized in that, Step (1) is carried out in an induction medium. The induction medium uses water as a solvent and consists of components with the following mass concentrations: B5 salts 3.1 - 3.4 g / L, sucrose 28 - 32 g / L, MES 0.4 - 0.65 g / L, BAP 1.5 - 1.8 mg / L, cefotaxime 240 - 260 mg / L, Timentin 90 - 110 mg / L, glufosinate 5 - 6 mg / L, and agar powder 6 - 10 g / L. The bud elongation medium uses water as a solvent and consists of components with the following mass concentrations: MS salts 4.2 - 4.6 g / L, sucrose 28 - 32 g / L, MES 0.4 - 0.65 g / L, aspartic acid 48 - 52 mg / L, L-glutamic acid 48 - 52 mg / L, IAA 0.08 - 0.12 mg / L, GA3 0.4 - 0.6 mg / L, zeatin riboside 0.9 - 1.1 mg / L, cefotaxime 240 - 260 mg / L, Timentin 90 - 110 mg / L, glufosinate 5 - 6 mg / L, and agar powder 6 - 10 g / L. The rooting medium uses water as a solvent and consists of components with the following mass concentrations: MS salts 4.2 - 4.6 g / L, sucrose 18 - 22 g / L, MES 0.4 - 0.65 g / L, aspartic acid 48 - 52 mg / L, L-glutamic acid 48 - 52 mg / L, IBA 0.8 - 1.2 mg / L, and phytagel 2 - 4 g / L.