Salt-induced expression promoter PagVQ1Pro and application thereof

Abstract

The invention relates to a salt induced expression promoter PagVQ1Pro and application thereof, and relates to the technical field of plant genetic engineering, and the nucleotide sequence of the promoter PagVQ1Pro is shown as SEQ ID NO.8. The invention also relates to a preparation method of the salt induced expression promoter PagVQ1Pro. A salt stress induction experiment is carried out on a poplar transgenic line PagVQ1Pro-OE4, 6 and 10 of the overexpression promoter PagVQ1Pro, it is known that under the salt induction condition, the GUS dyeing activity in leaves of the transgenic line of the overexpression poplar promoter PagVQ1Pro is remarkably enhanced, and the result shows that the promoter PagVQ1Pro is subjected to salt induction expression.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a salt-induced expression promoter PagVQ1Pro and its application. Background Art

[0002] Populus L., as an important fast-growing tree species for afforestation and greening and a bioenergy tree species, is widely planted globally. It grows rapidly, has strong adaptability, and has a wide range of wood uses. In particular, it plays an important role in soil and water conservation and maintaining the ecological environment. However, during the growth process of Populus, it is often restricted by various abiotic and biotic stresses such as salinity, drought, pests, and diseases. These stresses seriously affect the normal growth and yield of Populus, limiting its extensive planting and application in adversity areas.

[0003] The VQ gene family, as a protein family unique to plants, has been proven to be involved in the regulation of plant responses to abiotic stresses such as drought and salinity. Members of the VQ gene family play important signal transduction and regulatory roles in plants, can respond to stress and activate downstream defense mechanisms, thereby improving plant stress resistance. In species such as Arabidopsis thaliana, rice, and grape, VQ genes have been deeply studied and proven to be involved in their respective growth and development and stress regulation. However, research on VQ genes in Populus is relatively scarce, especially on the functional research of Populus VQ genes in stress resistance (salt tolerance). Therefore, in-depth research and exploration of the functions of Populus VQ genes and their related genes are of great significance for improving the stress resistance of Populus, expanding its planting range, and application fields. Summary of the Invention

[0004] The purpose of the present invention is to provide a salt-induced expression promoter PagVQ1Pro and its application to solve the above problems.

[0005] The present invention achieves the above purpose through the following technical solutions:

[0006] The present invention provides a salt-induced expression promoter PagVQ1Pro, and the nucleotide sequence of the promoter PagVQ1Pro is as shown in SEQ ID NO.8.

[0007] The present invention also provides an overexpression vector, which contains the promoter PagVQ1Pro as described above.

[0008] As a further optimized solution of the present invention, the overexpression vector is pMDC164.

[0009] The present invention also provides a genetically engineered bacterium, which contains the above overexpression vector.

[0010] As a further optimization scheme of the present invention, the genetically engineered bacterium is Agrobacterium.

[0011] The present invention also provides an application of the salt-inducible expression promoter PagVQ1Pro in the salt stress-induced expression of plants.

[0012] As a further optimization scheme of the present invention, the promoter PagVQ1Pro drives the expression of the target gene under salt stress in plants.

[0013] As a further optimization scheme of the present invention, the plant is Populus.

[0014] The present invention also provides a method for obtaining the salt-inducible expression promoter PagVQ1Pro as described above. Using the nucleotide sequence shown in SEQ ID NO.1 as a template, the nucleotide sequence of PagVQ1Pro is cloned by DNA molecular cloning technology. The primer sequences for DNA molecular cloning are as follows:

[0015] SEQ ID NO.2: GCTAATGAAGGCAGTGAG;

[0016] SEQ ID NO.3: CGAGGCACTGGTCTTCAC.

[0017] The beneficial effects of the present invention are as follows:

[0018] Through salt stress induction experiments on the transgenic Populus lines PagVQ1Pro-OE4, 6, and 10 overexpressing the promoter PagVQ1Pro, it is known that under salt induction conditions, the GUS staining activity in the leaves of the transgenic lines overexpressing the Populus promoter PagVQ1Pro is significantly enhanced. This result indicates that the promoter PagVQ1Pro is induced by salt for expression. Description of the Drawings

[0019] Figure 1 It is an agarose electrophoresis diagram (WT: control group; transgenic Populus promoter lines PagVQ1Pro-OE4, 6, 10);

[0020] Figure 2 It is a process diagram of the salt solution immersion experiment for the control group WT and the transgenic Populus promoter lines PagVQ1Pro-OE4, 6, 10 in the salt induction experiment of Populus seedlings;

[0021] Figure 3 It is a salt induction staining result diagram of the leaves of the control group WT and the transgenic Populus promoter lines PagVQ1Pro-OE4, 6, 10 in the salt induction experiment of Populus seedlings. Detailed Embodiments

[0022] The present application will be further described in detail below with reference to the accompanying drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0023] 1. Materials

[0024] Unless otherwise specified, the methods used in this embodiment are all conventional methods known to those skilled in the art. The reagents and other materials used are all commercially available products unless otherwise specified.

[0025] 2. Methods

[0026] 2.1 Cloning of the promoter sequence

[0027] The nucleotide sequence containing the PagVQ1 promoter as shown in SEQ ID NO.1 was cloned from the poplar genome using the PagVQ1Pro gene cloning primers in Table 1 (SEQ ID NO.1 has a total of 2188 bases and contains a CDS fragment of the PagVQ1 gene. The purpose is to accurately clone the nucleotide sequence of the PagVQ1 promoter. The nucleotide sequence of the PagVQ1 promoter is as shown in SEQ ID NO.8). The PCR product was constructed into a T vector to obtain the PagVQ1Pro cloning vector, which was then preserved and sequenced. The successfully sequenced PagVQ1Pro cloning vector was used as a template for the next experiment.

[0028] Table 1 PagVQ1Pro gene cloning primer sequences

[0029]

[0030] 2.2 Construction of the overexpression vector of the promoter PagVQ1Pro

[0031] The overexpression vector of PagVQ1Pro was constructed using the Gateway technology as follows:

[0032] 2.2.1 Primer design and PCR amplification

[0033] Gateway adapters were added to the F and R ends of the primers in step 2.1 above. The primer sequences with the added gateway adapters are shown in Table 2, and cloning was performed using the PagVQ1Pro cloning vector as a template.

[0034] Table 2 Primer sequences for PCR amplification

[0035]

[0036] The PCR reaction system is shown in Table 3:

[0037] Table 3 PCR reaction system

[0038]

[0039]

[0040] Each component was added to a sterilized PCR tube in sequence. After mixing and centrifuging, it was placed in a gradient PCR instrument for PCR amplification reaction. The procedure was as follows: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 30 sec; annealing at 55°C for 30 sec; extension at 72°C for 1 min; final extension at 72°C for 10 min. Among them, the three steps of denaturation - annealing - extension were set for 35 cycles. After the reaction ended, 2.5 μl of 10× DNA Loading Buffer was added to the PCR tube, and agarose gel electrophoresis was carried out. Subsequently, the band position was observed in the gel imaging system.

[0041] 2.2.2. Gel recovery of PCR products

[0042] The gel block with the correct and bright position was cut off in a gel cutting instrument and placed into a sterilized 1.5 ml dorf tube. Gel recovery was carried out using a gel recovery kit (Axygene). The steps were as follows:

[0043] a. Add 400 μL of buffer DE-A to the centrifuge tube and heat it in a 75°C metal bath until the gel block melts;

[0044] b. After adding 200 μL of buffer DE-B and mixing, transfer it to a 2.0 ml preparation tube and centrifuge at 12000 rpm for 1 min;

[0045] c. Discard the waste liquid, add 500 μL of buffer W1, and centrifuge at 12000 rpm for 30 sec;

[0046] d. Discard the waste liquid, add 700 μL of buffer W2, and centrifuge at 12000 rpm for 1 min, repeat once;

[0047] e. Place the empty tube in the centrifuge and centrifuge at 12000 rpm for 1 min to completely remove the liquid in the preparation column;

[0048] f. Place the preparation tube into a sterilized 1.5 ml centrifuge tube and let it stand at room temperature for 20 min until the absolute ethanol completely evaporates. Add 25 μL of Elution buffer preheated at 65°C for elution, and finally obtain a solution containing the target gene PagVQ1Pro, and store it at -20°C.

[0049] 2.2.3. Construction of the vector by BP reaction

[0050] Clone the target gene PagVQ1Pro obtained in Step 2.2.2 into the intermediate vector pDONR207 through BP reaction. The reaction system is shown in Table 4:

[0051] Table 4 Reaction system of BP reaction

[0052]

[0053] Under the condition of 25 °C, after 6 h of ligation, perform gel recovery of PCR products (the method is the same as in Step 2.2.2), and then carry out the transformation and sequencing of Escherichia coli. The specific experimental steps are as follows:

[0054] a. Add 5 μL of the reaction product to 50 μL of Escherichia coli competent cells, bury them on ice for 30 min, heat shock them in a 42 °C metal bath for 1 min, and then bury them on ice for another 2 min;

[0055] b. Add 200 μL of LB in a laminar flow hood, place it in a shaker at 37 °C and 180 r / min for 1 h of shaking culture. Pipette 100 μl of the bacterial solution in the laminar flow hood and evenly spread it on the LB solid medium containing Kan antibiotic with a spreader;

[0056] c. Place the culture dish with the spread bacterial solution face up in an incubator at 37 °C for 30 min, then invert it and culture it for about 12 h, and observe the growth of the bacterial colonies;

[0057] d. Use a pipette tip to pick a single, well-grown colony and transfer it into 1 ml of liquid medium containing Kan antibiotic, place it in a shaker at 37 °C and 220 r / min for 4 - 5 h of subculture;

[0058] e. Send the shaken bacterial solution to a biological company for sequencing. According to the returned results, preserve the bacterial solution with correct construction (bacterial solution: glycerol = 1:1). The remaining bacterial solution can be continuously subcultured in large quantities and then the plasmid can be extracted and preserved, or the plasmid returned by the company can be preserved.

[0059] 2.2.4. Construction of the vector by LR reaction

[0060] After the sequencing of the intermediate vector constructed in Step 2.2.3 is correct, use the LR reaction to construct the target gene PagVQ1Pro into the overexpression vector pCAMDC164. The reaction system is shown in Table 5, with the vector pCAMDC164 without the ligated target gene as a control:

[0061] Table 5 Reaction system of LR reaction

[0062]

[0063] At a temperature of 25 °C, connect for 6 h, then perform gel recovery of the PCR product, transformation, and sequencing (the method is the same as the steps in 2.2); then extract the plasmid, and finally introduce it into the Agrobacterium competent cell GV3101. The specific steps are as follows:

[0064] (1) Take out the Agrobacterium competent (GV3101) from the ultra-low temperature refrigerator and thaw it on ice to a freeze-thaw state;

[0065] (2) Pipette 2.5 μl of the recombinant plasmid obtained in step 2.2.3 into 50 μl of Agrobacterium competent cells, bury it on ice for 5 min, quickly freeze it in liquid nitrogen for 5 min, heat shock it in a 37 °C metal bath for 5 min, and continue to bury it on ice for 5 min;

[0066] (3) Add 200 μl of LB liquid medium in the laminar flow hood, place it in a shaker at 28 °C and 200 r / min for shaking culture for 4 - 5 h;

[0067] (4) Take 100 μl of the bacterial solution and evenly spread it on the LB solid medium containing two antibiotics, Kan and Rif. First, place it upright in a 28 °C incubator for culture, and after 30 min, place it upside down and culture it in the dark for 2 - 3 days;

[0068] (5) Pick a single colony in the laminar flow hood into 1 ml of LB liquid medium containing Kan and Rif, and then place the shaking tube on a shaker at 28 °C and 220 r / min for shaking culture for 24 - 48 h;

[0069] (6) After the bacterial solution is shaken evenly, perform bacterial solution PCR (the PCR program is the same as step 2.2.1). The reaction system is shown in Table 6. At the same time, set positive and negative controls with ddH2O and plasmid to ensure the successful transformation of the recombinant plasmid. The successfully transformed bacterial solution is preserved and stored at -80 °C at low temperature.

[0070] Table 6 Reaction system of bacterial solution PCR

[0071]

[0072] 3 Poplar genetic transformation

[0073] 3.1 Genetic transformation steps

[0074] a. Add the Agrobacterium obtained in step 2.2.4 above to the LB medium containing the corresponding Kan and Rif and shake it vigorously to make its OD600 value reach about 0.6;

[0075] b. In the laminar flow hood, place the cut leaves of 84K poplar with wounds into the bacterial solution in the previous step and shake it on a low-speed horizontal shaker for about 20 min;

[0076] c. Drain the infected leaves of excess bacterial solution and place them in differentiation medium, incubate in the dark at 24℃ for 2 days;

[0077] d. After 2 days of dark culture, transfer the leaves to the screening medium containing hygromycin and timentin for screening. Generally, resistant buds will appear in about 20 days.

[0078] e. Cut the resistant seedlings and transfer them to the rooting medium containing the same resistance. After rooting, expand the culture medium and detect the expression level.

[0079] 3.2. Identification of transgenic poplars by PCR

[0080] Using the GUS gene primer pair GUS-F / GUS-R (as shown in Table 7), PCR was used to identify whether the pCAMDC164 vector fragment had been successfully transferred into the resistant plants. The successfully transferred resistant plants PagVQ1Pro-OE4, 6, and 10 were selected, which were the poplar promoter transgenic lines with overexpression of PagVQ1Pro. At the same time, non-transgenic poplar (WT) was used as a negative control. The PCR identification results are shown in Figure 7. Figure 1 shown.

[0081] Table 7 GUS transgenic identification related primers

[0082]

[0083] 3.3 Salt induction experiment

[0084] 3.3.1. Preparation of culture medium

[0085] The tissue culture seedlings of the poplar promoter transgenic lines overexpressing PagVQ1Pro (PagVQ1Pro-OE4, 6, 10) and the control group (WT) were propagated, and the culture medium formula was: 1 liter system formula (MS: 2.215 g; sucrose 10 g; agar: 8 g).

[0086] 3.3.2 Salt induction experimental process

[0087] 1) Pull out the 4-week-old poplar seedlings with their roots and place them in a 200 mM NaCl solution (see Figure 2 , the roots of poplar seedlings were immersed in salt solution) and soaked at 0, 3, 6, 12, and 24 hours respectively, and leaves were taken from each seedling in each period;

[0088] 2), Place the leaves taken at each time period in a 2 ml centrifuge tube, add GUS solution, soak the whole leaf in the GUS solution, and after 24 hours of dark culture treatment, put the leaf into a centrifuge tube containing 70% ethanol, and then put it into a 95 °C water bath for 10 - 15 minutes to remove the chlorophyll in the leaf, and observe the leaf color; Principle of GUS staining: The GUS gene encodes β-glucuronidase (GUS), which is a hydrolase that can catalyze the hydrolysis of many β-glucoside ester substances. It can decompose 5-bromo-4-chloro-3-indolyl-glucronide (abbreviated as X-Gluc) into a blue substance.

[0089] The decolorization results are as Figure 3 shown: Under salt induction conditions, the GUS staining activity was significantly enhanced in the leaves of the poplar promoter transgenic lines overexpressing PagVQ1Pro, indicating that the promoter PagVQ1Pro is induced by salt for expression.

[0090] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A salt-induced expression promoter PagVQ1Pro, characterized in that, The nucleotide sequence of the promoter PagVQ1Pro is shown in SEQ ID NO.

8.

2. An overexpression vector, characterized in that, The overexpression vector contains the promoter PagVQ1Pro as described in claim 1.

3. An overexpression vector according to claim 2, characterized in that, The overexpression vector is pMDC 164.

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacterium contains the overexpression vector as described in any one of claims 2-3.

5. A genetically engineered bacterium according to claim 4, characterized in that, The genetically engineered bacterium is Agrobacterium.

6. Use of a salt-induced expression promoter PagVQ1Pro as described in claim 1 in plant salt stress-induced expression.

7. The application according to claim 6, wherein The promoter PagVQ1Pro drives the expression of a target gene under plant salt stress induction.

8. The application according to claim 7, characterized in that, The plant is Populus.

9. A method for obtaining the salt-induced expression promoter PagVQ1Pro as described in claim 1, characterized in that, Using the nucleotide sequence shown in SEQ ID NO.1 as a template, the nucleotide sequence of PagVQ1Pro is cloned by DNA molecular cloning technology, wherein the primer sequences for DNA molecular cloning are as follows: SEQ ID NO.2: GCTAATGAAGGCAGTGAG; SEQ ID NO.3: CGAGGCACTGGTCTTCAC.

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