CN116622737A - Pinus massoniana PmAP2/ERF gene and its expression protein and application - Google Patents

Abstract

The invention discloses the PmAP2/ERF gene of masson pine, its expressed protein and its application, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the PmAP2/ERF gene of Pine massoniana of the present invention is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. The invention uses pine seedlings as materials, obtains the PmAP2/ERF gene of pine pine by cloning, constructs its plant expression vector pBI121-PmAP2/ERF and transfers it into the leaves of Populus japonicus, and obtains transgenic plants. After 7 days of continuous drought, the relative water content of leaves of transgenic lines was higher than that of wild-type lines; under drought stress, the net photosynthetic rate of transgenic lines was higher than that of wild-type lines; The rate and stomatal conductance decrease slowly, and transgenic lines have higher transpiration rate than wild-type lines in later period.

Description

Pinus massoniana PmAP2/ERF gene and its expression protein and application

technical field

The invention belongs to the technical field of plant genetic engineering, and more specifically relates to the PmAP2/ERF gene of the masson pine and its expressed protein and application.

Background technique

The arid environment severely restricts the growth and reproduction of plants. Masson pine (Pinus massoniana) is a pioneer coniferous tree species for afforestation in harsh environments in my country. It has excellent characteristics such as drought resistance, cold resistance, and barren tolerance. Masson pine plays an irreplaceable role in industrial timber, forest product processing, forest resource development and ecological service functions. But the molecular mechanism of masson pine response to drought is still unclear. Therefore, excavating the drought-resistant genes of P. massoniana at the molecular level and revealing its drought-resistance regulation mechanism are of great significance for improving the tolerance of P. massoniana to drought stress and expanding the cultivation range of P. massoniana.

In plants, abscisic acid-mediated signaling pathways play a central role in plant stress responses. Drought triggers the production of abscisic acid (ABA) when plants lack water. Accumulated ABA binds to PYR/PYL/RCAR receptors to form dimers that bind protein phosphatase 2C (PP2C). PP2C releases sucrose nonhydrolytic protein kinase 2 (SNF1-related protein kinase 2), phosphorylates corresponding transcription factors, regulates the expression of ABA-responsive genes, causes stomatal closure, and improves plant drought resistance. In addition to the ABA signal transduction pathway, plant hormone signal transduction pathways such as gibberellin, jasmonic acid, and ethylene also play an important role in the response of plants to drought stress. Among these signal transduction pathways, transcription factors play a key role in activating or repressing the expression of defense genes. In addition, transcription factors can also regulate the interaction between different signaling pathways, so that plants can better adapt to the stress environment and improve the stress resistance of plants. Some members of the AP2/ERF transcription factor family are involved in these pathways. The AP2/ERF (APETALA2/ERF) transcription factor family is one of the largest plant transcription factor families and has attracted extensive attention from researchers. The ERF subfamily is an important transcription factor in the AP2/ERF family that is closely related to abiotic stress. Current studies have shown that the Arabidopsis ERF1 gene can participate in the JA, ET, and ABA signal transduction pathways and activate the expression of stress resistance genes. Arabidopsis overexpressing ERF1 significantly improved drought resistance, and transgenic plants could reduce leaf water loss by reducing stomatal aperture. DREB subfamily transcription factors are a class of transcription factors related to abiotic stress. The DREB subfamily can specifically bind to DRE/CRT cis-acting elements, activate the expression of downstream stress resistance genes, and improve plant resistance to drought stress independent of the ABA signal transcription pathway. Taken together, multiple metabolic pathways are involved in plant defense against abiotic stress. Regulating the expression of related genes can improve the adaptability of plants to adverse environments. In recent years, there have been many studies on the molecular mechanism of drought stress, but less research on the molecular mechanism of drought stress in coniferous trees.

Contents of the invention

For the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide P. massoniana PmAP2/ERF gene; Another technical problem to be solved by the present invention is to provide the expression protein of P. massoniana PmAP2/ERF gene; The present invention also will The technical problem to be solved is to provide the application of PmAP2/ERF gene of masson pine for breeding of masson pine.

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

The nucleotide sequence of PmAP2/ERF gene of Pinus massoniana is shown in SEQ ID NO.1.

The amino acid sequence of the protein expressed by PmAP2/ERF gene of Pine massoniana is shown in SEQ ID NO.2.

Containing the carrier of the masson pine PmAP2/ERF gene described in claim 1.

The carrier of the PmAP2/ERF gene of Pine massoniana is a plant expression carrier.

The plant expression vector is pBI121-PmAP2/ERF.

The application of the PmAP2/ERF gene of masson pine in improving the drought resistance of Populus pubescens comprises the following steps:

1) Construct the plant expression vector of PmAP2/ERF gene of Pine massoniana;

2) transforming the plant expression vector of the constructed Pinus massoniana PmAP2/ERF gene into Populus japonicus;

3) Cultivate and screen Populus japonicus plants with improved drought resistance.

The application of PmAP2/ERF gene in Pinus massoniana in improving the photosynthetic rate of Populus argentina.

The application of PmAP2/ERF gene of Pinus massoniana in improving the transpiration rate of Populus sinensis.

The application of PmAP2/ERF gene of Pine massoniana in improving the stomatal conductance of Populus argentina.

Compared with the prior art, the beneficial effects of the present invention are:

The invention uses the pine seedlings as materials, obtains the PmAP2/ERF gene of the pine masson by cloning, constructs its plant expression vector pBI121-PmAP2/ERF on this basis, and transfers it into the leaves of Populus montana to obtain transgenic plants. After 7 days of continuous drought, the relative water content of the leaves of the transgenic plants was higher than that of the wild-type plants; with the prolongation of the drought treatment, the photosynthetic rate of the wild-type plants decreased significantly, and there was almost no photosynthetic activity after 7 days of drought, while the transgenic plants The net photosynthetic rate of the line was higher than that of the wild type, and remained at a certain level on the seventh day. The transpiration rate and stomatal conductance of the lines decreased slowly, and the transgenic lines had a higher transpiration rate than the wild-type lines in the late stage, and the transgenic lines and the stomatal conductance both dropped to the lowest point in the late drought treatment, while the transgenic lines The drought resistance ability of the strain is improved.

Description of drawings

Figure 1 is a graph showing the specific expression of PmAP2/ERF genes in different organs of Pinus massoniana (young leaf YL, old leaf OL, xylem x, young stem YS, old stem OS and root R);

Fig. 2 is a change diagram of PmAP2/ERF gene transcription level in Pinus massoniana under dehydration treatment;

Figure 3 is a graph showing the changes in PmAP2/ERF gene transcription levels in Pinus massoniana under ABA stress;

Fig. 4 is the subcellular localization map of PmAP2/ERF protein of masson pine in tobacco leaf cells;

Fig. 5 is the PCR analysis figure (1: positive control, 2: negative control, 3-7: transgenic line) of transgenic poplar DNA;

Fig. 6 is the relative expression level figure of PmAP2/ERF gene in transgenic poplar (2: OE-2, 5: OE-5, 6: OE-6, 8: OE-8, 9: OE-9, 10: OE -10, 11: OE-11, 15: OE-15);

Fig. 7 is the leaf shape figure of transgenic poplar line (OE) and wild-type poplar line (WT) under drought stress;

Fig. 8 is a comparative figure of leaf relative water content of transgenic poplar line (OE) and wild-type poplar line (WT) under normal conditions and drought conditions;

Fig. 9 is a comparison chart of net photosynthetic rate duration curves of transgenic poplar lines (OE) and wild-type poplar lines (WT) under drought stress;

Fig. 10 is a graph comparing transgenic poplar strains (OE) and wild-type poplar strains (WT) in transpiration duration curves under drought stress;

Figure 11 is a comparison of stomatal conductance duration curves of transgenic poplar lines (OE) and wild-type poplar lines (WT) under drought stress

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific examples. Unless otherwise specified in the following examples, the technical means used are conventional means well known to those skilled in the art.

Example 1

Masson pine seedlings were placed in mild drought (65 ± 5%), moderate drought (50 ± 5%) and severe drought (35 ± 5%) for 60 days, and normal water supply (80 ± 5%) was set. for the control group. The RNA of 4 treatments of Pinus massoniana seedlings was extracted, each treatment had 3 biological replicates, and sent to the company for high-throughput sequencing. According to the results of transcriptome sequencing, a differentially expressed gene PmAP2/ERF was screened out.

1. Extraction of total RNA

Extract the total RNA of Pinus massoniana seedlings according to the instructions of the polysaccharide and polyphenol plant total RNA extraction kit (Novizan), and the specific operation steps are as follows:

Add an appropriate amount of masson pine needle powder ground with liquid nitrogen, add 500 μL Buffer PRL preheated at 65°C (add 5% β-mercaptoethanol before use), and immediately vortex vigorously for 30-60sec to fully lyse, then place in a 65°C water bath 5min, invert 1-2 times during the period to help lysis; centrifuge at 12000rpm for 10min after lysis, transfer the supernatant to a new 1.5mL RNase-free centrifuge tube, add 0.5 times the volume of the supernatant in absolute ethanol, and immediately mix by pipetting Evenly; transfer the above mixed solution to II column (lower receiving collection tube), centrifuge at 12000rpm for 2min, discard the filtrate; put the column into a new collection tube, add 500μL Buffer PRL Plus, centrifuge at 12000rpm for 30sec, collect the filtrate; add 0.5 times the filtrate volume of absolute ethanol, blow and mix immediately; transfer the above mixed solution to the IV column (lower receiving tube), centrifuge at 12000rpm for 2min, discard the filtrate; add 700μL Buffer PRW1 to the IV column, and place it at room temperature for 1min. Centrifuge at 12000rpm for 30sec, discard the filtrate; add 500μL Buffer PRW2 to the IV column (add 48mL of absolute ethanol before use), centrifuge at 12000rpm for 30sec, discard the filtrate, repeat the operation once; put the empty column back into the collection tube, centrifuge at 12000rpm for 2min, ventilate Open the cover and place in the cabinet for 2-5min to completely volatilize the residual ethanol; transfer the adsorption column to a new 1.5mL centrifuge tube, add 30-100μL of ddH ₂ O dropwise to the center of the adsorption column membrane, place at room temperature for 2min, 12000rpm Centrifuge for 1 min to elute RNA.

2. cDNA synthesis

Using the extracted total RNA as a template, the cDNA chain is obtained by reverse transcription. The following operations need to be performed on ice. The specific operation steps are as follows:

The sample reaction system is as follows: total RNA 1 μg, Oligo(dT)18 (0.5 μg/μL) 1 μL, 2×TS ReactionMix 10 μL, RT/RI Enzyme Mix 1 μL, gDNA Remover 1 μL, RNase H ₂ O supplemented to 20 μL, using a pipette Gently blow and mix with the gun, centrifuge briefly to the bottom of the PCR tube; react at 42°C for 30 minutes, and react at 85°C for 5sec to inactivate the enzyme and gDNA Remover in the reaction system, obtain cDNA, and store it in the refrigerator at -20°C for later use.

3. Cloning of the target gene

According to the PmAP2/ERF sequence screened from the transcriptome data, specific primers were designed, and the PmAP2/ERF gene fragment was cloned using the synthesized P. massoniana cDNA as a template. PmAP2/ERF open reading frame cloning primer sequences are as follows:

F: 5'-ATGTATATGCATGTGCACGTCTTATC-3',

R: 5'-TTATTTTAATGCCAATGCGGTTCG-3'.

The PCR reaction system (20μL) is: 10μL 2×Hieff Gold PCR Master Mix, 1 μL Forward primer, 1 μL Reverse primer, 1 μL cDNA, 7 μL ddH ₂ O.

The PCR reaction program was: denaturation at 98°C for 10s, annealing at 57°C for 5s, extension at 72°C for 10s, 35 cycles.

Mix 5 μL of PCR product (PmAP2/ERF gene fragment obtained by cloning) with 1 μL of 6×Loading Buffer, put it into the prepared 1.2% agarose gel well, and electrophoresis in 1×TAE buffer at 200V for 20 minutes, and the electrophoresis detection is correct Finally, use the agarose gel recovery kit to recover the target fragment with the remaining PCR product and 6×Loading Buffer; take 4 μL of the gel-cut recovery product in a 1.5 mL centrifuge tube, add 1 μL -Blunt, mix gently with a pipette gun, and place the centrifuge tube on ice after reacting at room temperature for 15 minutes. Add 50 μL LDH5α Escherichia coli competent cells to the ligation product, flick and mix well, then ice bath for 25 min, heat shock at 42 °C for 45 sec, immediately place on ice for 2 min, then add 900 μL LB medium, 200 rpm, 37 °C recovery for 1 h; 5000 rpm Centrifuge for 1 min, discard 800 μL of supernatant, blow and aspirate and mix evenly, spread on a plate containing Kan, invert the plate and incubate overnight in a 37°C incubator; pick a single colony on the LB plate in 10 μL ddH ₂ O water the next day , after blowing and mixing, take 2 μL for positive detection, add 1 mL of Kan-containing LB liquid medium to the remaining bacterial solution, and incubate at 200 rpm at 37 °C. After the bacterial inspection, select the bacterial solution with the correct electrophoresis band and send it to the company for sequencing.

The positive detection reaction system (20 μL) is: 10 μL 2×Rapid Taq Master Mix, 1 μL M13-F, 1 μL M13-R, 2 μL bacterial solution, 6 μL ddH ₂ O.

The positive detection reaction program is: 95°C for 3min; 95°C for 15sec; 55°C for 15sec; 72°C for 5sec, 35 cycles; 72°C for 5min; 4°C∞.

According to the sequencing results, the sequence of the target gene is determined as shown in SEQ ID NO.1. The total length of the open reading frame of Pinus massoniana AP2/ERF is 798bp, named PmAP2/ERF, and the expressed protein sequence is shown in SEQ ID NO.2 .

Example 2

1. Specific expression of PmAP2/ERF gene in different organs of Pinus massoniana

The RNA of young leaves, old leaves, xylem, young stems, old stems and root tissues of 15-year-old Pinus massoniana was extracted and reverse-transcribed into cDNA. The specific method is as shown in Example 1. The specific expression of PmAP2/ERF gene in different organs of Pinus massoniana was detected by real-time fluorescence quantitative PCR (RT-qPCR). The primer sequences are as follows:

PmAP2/ERF-q-F: 5'-GCGCCAACAAATGTAGCGAA-3',

PmAP2/ERF-q-R: 5'-AATGCCAATGCGGTTCGTTC-3'.

qRT-PCR reaction system (10 μL): 1 μL cDNA, 5 μL SYBR Green Master Mix, 0.4 μL PmAP2/ERF-qF, 0.4 μL PmAP2/ERF-qR, 3.2 μL ddH ₂ O.

The reaction procedure is as follows: 95°C for 2 min; 95°C for 10 sec; 60°C for 30 sec; 72°C for 30 sec; 40 cycles.

The results are shown in Figure 1, and the expression level in young leaves was set to 1. The results showed that PmAP2/ERF was expressed in all tissues and had tissue specificity. The expression level was the highest in old leaves, and expressed in xylem The level is weak.

2. Response of PmAP2/ERF gene to drought stress

Masson pine seedlings are dehydrated (natural drought without watering) and 400mM abscisic acid (ABA) are sprayed on leaves respectively, and samples are taken at 0h, 2h, 4h, 6h, 8h respectively, and after the total RNA is extracted respectively according to the method in Example 1 , using the extracted total RNA as a template, the cDNA strand was obtained by reverse transcription. Real-time fluorescent quantitative PCR (RT-qPCR) technology was used to detect the specific expression of PmAP2/ERF gene under dehydration and ABA conditions of Pinus massoniana. The primer sequences are as follows:

PmAP2/ERF-q-F: 5'-GCGCCAACAAATGTAGCGAA-3',

PmAP2/ERF-q-R: 5'-AATGCCAATGCGGTTCGTTC-3'.

qRT-PCR reaction system (10 μL): 1 μL cDNA, 5 μL SYBR Green Master Mix, 0.4 μL PmAP2/ERF-qF, 0.4 μL PmAP2/ERF-qR, 3.2 μL ddH ₂ O.

The reaction procedure is as follows: 95°C for 2 min; 95°C for 10 sec; 60°C for 30 sec; 72°C for 30 sec; 40 cycles.

The results are shown in Figure 2, within 6 hours of dehydration, the expression of PmAP2/ERF showed an upward trend; at 6 hours of dehydration, the expression increased about six times, reached a peak, and then began to decrease.

The results are shown in Figure 3. After ABA treatment, the expression of PmAP2/ERF was significantly up-regulated first. After ABA treatment for 3 hours, the expression of PmAP2/ERF increased by about 2.5 times. After ABA treatment for 6 hours, the expression of PmAP2/ERF decreased sharply.

3. Subcellular localization

Take 1 mL of the E. coli liquid containing the pJIT166 carrier stored at -80°C in our laboratory for activation, take 1-4 mL of the overnight cultured liquid, and use the plasmid mini-extraction kit to extract the plasmid. According to the ORF sequence of the pJIT166 vector and PmAP2/ERF (remove the stop codon), the primers carrying restriction sites (Hind III and Xba I) were designed by CE-Design software. The primer sequences are as follows:

pJIT166-PmAP2/ERF-F:

5'-TGGAGAGGACAGCCCAAGCTTATGTATATGCATGTGCACGTCTTATC-3',

pJIT166-PmAP2/ERF-R:

5'-GCTCACCATGGATCCTCTAGATTTTAATGCCAATGCGGTTCG-3'.

The 35S::PmAP2/ERF-GFP fusion vector was constructed by homologous recombination.

Hind III and Xba I were used for double digestion, and the enzyme digestion system (20 μL) was as follows: 1 μg of plasmid, 1 μL of HindIII, 1 μL of Xba I, and ddH ₂ O supplemented to 20 μL.

The ligation system was as follows: 0.02 × base pairs (ng) of the cloning vector linearized vector, 0.04 × base pairs (ng) of the insert fragment, 2 μL 5 × CEIIBuffer, 1 μL Exnase II, ddH ₂ O supplemented to 10 μL. Place it in a PCR instrument at 37°C for 25 minutes and place it on ice immediately.

Add 50 μL LDH5α Escherichia coli competent cells to the ligation product, flick and mix well, then ice bath for 25 min, heat shock at 42 °C for 45 sec, immediately place on ice for 2 min, then add 900 μL LB medium, 200 rpm, 37 °C recovery for 1 h; 5000 rpm Centrifuge for 1 min, discard 800 μL of supernatant, blow and aspirate and mix evenly, spread on a plate containing Kan, invert the plate and incubate overnight in a 37°C incubator; pick a single colony on the LB plate in 10 μL ddH ₂ O water the next day , after blowing and mixing, take 2 μL for positive detection, add 1 mL of Kan-containing LB liquid medium to the remaining bacterial solution, and incubate at 200 rpm at 37 °C. After the bacterial inspection, the bacterial solution with the correct electrophoresis band was selected and sent to the company for sequencing, and the positive clones with correct sequencing results were screened out for expansion and culture, and the recombinant plasmid was extracted.

Transfer the recombinant plasmid into the Agrobacterium competent cell GV3101, as follows: Take 10 μL of the recombinant plasmid and add it to 100 μL of Agrobacterium competent in a mixed state of ice and water, gently tap the bottom of the tube to mix, and then place it on ice, liquid nitrogen, etc. , 37°C water bath, and ice bath for 5 minutes each; add 700 μL LB liquid medium and place it in a constant temperature shaker at 28°C for 2.5 hours; absorb 100 μL supernatant and spread it on a medium containing 50 mg·L- ¹ Kan and 25 mg·L- ¹ Rif Put it upside down on an LB plate and culture it in an incubator at 28°C for 2 days; pick a single colony with good growth status for PCR detection, expand the positive monoclonal colony, and inoculate 500 μL of overnight activated bacteria in 50 mL of LB liquid medium solution (50mg/LKan, 25mg/LRif), cultured at 28°C with shaking at 220rpm/min until the OD ₆₀₀ value was 0.7-0.8, 5000rpm/min, discarded the _supernatant , and collected the cells; pH = 5.6) and 200 μM acetosyringone (AS) in sterile water to resuspend the bacterial pellet, mix the same amount of the two target bacterial liquids with p19 Agrobacterium at a ratio of 1:1, and adjust the OD600 value to about 1.0 , room temperature, and dark light for 3-4h.

Using the pressure injection method, use a 10mL syringe (without a needle), inject the suspended Agrobacterium liquid into the young leaves of 3-week-old wild Nicotiana benthamiana, hold the front of the leaves with your hand, and inject the Agrobacterium slowly through osmotic pressure Tissue gaps on the back of the leaves, avoiding the veins, and finally mark the injected tobacco leaves; pour an appropriate amount of water into the bottom of the pot to keep it slightly moist. After 1 day of culture in the dark, it was transferred to light culture for 1 day, followed by microscopic observation under a laser confocal microscope. Injection of 35S::GFP was used as negative control.

The results are shown in Figure 4. It was observed under the confocal laser scanning microscope that the fluorescent signal of the 35S::PmAP2/ERF-GFP fusion protein was localized in the nucleus, while the fluorescent signal of the 35S::GFP protein was distributed in the whole cell. Specific localization, the results showed that PmAP2/ERF was specifically localized in the nucleus.

Example 3

1. Obtaining of transgenic poplar plants

Primers were designed according to the XbaI and BamHI restriction sites on the pBI121 vector and the open reading frame of PmAP2/ERF. The primer sequences are as follows:

pBI121-PmAP2/ERF-F;

5'-GAGAACACGGGGGACTCTAGAATGTATATGCATGTGCACGTCTTATC-3',

pBI121-PmAP2/ERF-R:

5'-GGACTGACCACCCGGGGATCCTTATTTTAATGCCAATGCGGTTC-3'.

According to the aforementioned method, XbaI and BamHI were used to double-enzyme-digest the vector, and the ORF was connected to the vector after digestion to obtain the plant expression vector pBI121-PmAP2/ERF. After sequencing, the recombinant plasmid was extracted from the successfully constructed plant expression vector, and used The method described in example 2 transfers the recombinant plasmid into the Agrobacterium competent cell GV3101, and adopts the method mediated by Agrobacterium tumefaciens to transform the leaves of the 30d wild-type Populus japonicus tissue culture seedlings. The transformation method is as follows:

1) Prepare the bacterial liquid: inoculate a single colony normally grown on the solid medium into LB liquid medium containing 1 μL Kan (50mg/L), culture on a temperature-controlled shaker at 28°C and 200rpm for 12h, and take 0.8mL of the bacterial colony The solution was inoculated in 50 mL liquid medium containing Kan (50 mg/L) and Rif (40 mg/L) until the OD ₆₀₀ value was 0.5, centrifuged at 3000 rpm at room temperature for 10 min, discarded the supernatant, and used an equal volume of sugar-free MS liquid added with AS Culture medium resuspended bacteria.

2) Infestation of leaves: take 30-40d grown and flat and stretched leaves of Populus japonicus tissue culture seedlings, remove the midrib with scissors, cut out wounds along the edge of the leaves, trim them into square leaves of about 1 cm, and put the cut leaf discs into The Agrobacterium bacteria liquid prepared above was sealed, and cultured for 15 minutes with gentle shaking on a shaker at 100 rpm at 25°C.

3) Co-cultivation: Take out the leaf disk with sterile tweezers, place it on a sterile filter paper, suck out part of the residual bacterial solution, and spread the positive piece of the leaf on the differentiation medium plate containing AS neatly and evenly, and then place it at 25°C for cultivation The dark culture was carried out in the box for 2 days, and then light culture was carried out for 2-3 days under light regulation.

4) Selective culture: After light culture, the transformed material was alternately washed 3 times with sterile water, sterile water plus cephalosporin water, and finally the cleaned material was transferred to a screening medium containing corresponding antibiotics and continued to grow until The callus was grown, and the medium was replaced every 7 days during this period.

5) Sprout cultivation: when small green buds grow on the screening medium, we transfer them to the medium for bud elongation to induce sprouting, and the induction time is about 20-25 days. , cleaned in time, and replaced with new medium.

6) Rooting culture: After 20 days of budding culture, clip the adventitious buds with Kan resistance and transfer them to the rooting medium for rooting culture of resistant plants, and rooting can take place in about 10 days.

7) Transplanting of transgenic plants: select the seedlings of wild type and overexpression strains that were rooted and cultured for 30 days, 3 plants per strain, open the tissue culture bottle after domestication and culture for 3 days, take out the plants, wash the agar at the root with clean water, and transplant Put it into the potting soil, place it in the greenhouse for cultivation, and water it every 2 days during this period.

2. Screening of transgenic positive plants

The DNA of the transgenic Populus montana plants obtained through preliminary screening was extracted using a plant genome DNA extraction kit (TIANGEN Company). For specific steps, refer to the instruction manual. Then the common 35s-F and pBI121-PmAP2/ERF-R were tested by PCR, and the results are shown in FIG. 5 .

35sF primers are as follows:

35s-F: 5'-TGAAGATAGTGGAAAAGGAAGGTG-3',

PCR reaction system: DNA template 100ng, forward and reverse primers 1 μL each, 2×Phanta Max Master Mix (Vazyme) 10 μL, ddH ₂ O to make up to 20 μL.

PCR reaction program: pre-denaturation at 95°C for 3 min; 35 amplification cycles including denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 45 s, extension at 72°C for 5 min, and storage at 4°C. Use 1.2% agarose gel and ultraviolet gel imaging system to observe and compare whether the size of the PCR product band is consistent with the size of the target fragment inserted in the recombinant plasmid.

According to the instructions of the polysaccharide polyphenol plant total RNA extraction kit, extract RNA from the candidate plants (1-month-old potted seedlings) with positive DNA levels, and use agarose gel electrophoresis and ultra-micro spectrophotometer to detect the integrity, concentration and purity of RNA . Synthesis of cDNA: 500ng of RNA template, 4 μL of 4×gDNA wiper Mix, 1 μL of ligo(dT) ₂₃ VN (10uM), RNase-freeddH ₂ O supplemented to 16 μL, pipette gun to mix gently, 42°C for 2min, and then Add 4 μL of 5×HiScriptIISelect qRT SuperMix II and mix well, 50°C for 15min, 85°C for 5s. Taking Populus montana EF1-α as an internal reference gene, EF1-αx-F (5'-GGCAAGGAGAAGGTACACAT-3') and EF1-α-R (5'-RCAATCACACGCTTGTCAATA-3') primers were used for PCR detection. qRT-PCR 10μL reaction system is: 5μL 2×ChamQ SYBR Color qPCR Master Mix (HighROX Premixed), 0.4μL (10uM) forward primer, 0.4μL (10uM) reverse primer, 40ng cDNA, RNase-free ddH ₂ O supplemented to 10 μL. The qRT-PCR reaction program was: 95°C for 30s, 40 cycles of 95°C for 5s, 60°C for 30s, 3 samples for each strain and 3 technical replicates. use Perform statistics.

The results are shown in Figure 6. There are differences in the expression levels of different transgenic lines. In the eight lines (OE-2, OE-5, OE-6, OE-8, OE-9, OE- 10. Among OE-11 and OE-15), the expression level of OE-2 was the highest, about 25 times that of the wild type (WT), followed by OE-8, which was about 15 times that of the wild type. Except for OE-15, The expression levels of other strains were all 1-10 times. Two transgenic lines, OE-2 and OE-8, with the highest expression levels were selected for subsequent experiments.

Example 4

1. Leaf traits under drought

The wild-type line (WT) of 2-month-old Populus montana and the screened transgenic lines (OE-2 and OE-8) of 2-month-old Populus montana were treated with short-term drought (without watering for 7 consecutive days).

The results are shown in Figure 7. After 7 days of continuous drought, the dead leaves of the wild-type lines (WT) were more severe, while the leaves of the transgenic lines (OE-2 and OE-8) were still plump, and the leaves of the transgenic lines (OE-8) were still plump. -2 and OE-8) had higher leaf relative water content (RWC) than the wild-type line (WT) (Fig. 8).

2. Photosynthetic index

Choose sunny weather, adopt CIRAS-3 photosynthetic instrument to randomly select three strains of wild-type poplar and transgenic poplar OE-2, OE-8 each three strains at about 10:00 in the morning, carry out photosynthetic determination, the main index of determination includes net photosynthetic rate ( Pn), transpiration rate (Tr), and stomatal conductance (Gs), each measurement was repeated three times, and a buffer bottle was used during the measurement to keep the CO ₂ concentration in a relatively stable state.

The results are shown in Figure 9. With the prolongation of drought treatment, the photosynthetic rate of wild-type lines (WT) was significantly reduced, and there was almost no photosynthetic activity after seven days of drought, and the photosynthetic rate of transgenic lines (OE-2 and OE-8) The net photosynthetic rate also showed a decreasing trend, but it was higher than that of the wild type, and remained at a certain level on the seventh day, indicating that the transgenic poplar could also produce energy substances required for physiological activities through photosynthesis under drought conditions.

The results are shown in Figure 10. Under drought stress, the transpiration rates of the wild-type lines (WT) and the transgenic lines (OE-2 and OE-8) showed a significant downward trend, but it can be seen that the transgenic lines (OE-2 and OE-8) decreased transpiration rate more slowly, and later transgenic lines (OE-2 and OE-8) had higher transpiration rates than wild-type lines (WT).

The results are shown in Figure 11. Under drought stress, the stomatal conductance of wild-type lines (WT) and transgenic lines (OE-2 and OE-8) showed a significant downward trend, and the transgenic lines (OE-2 and OE-8) were higher than those of the wild-type strain (WT), indicating that under drought conditions, the water loss of the wild-type strain (WT) was faster, and the transpiration rate of the wild-type strain (WT) Both stomatal conductance and stomatal conductance were reduced to the lowest point, and it was difficult to maintain normal physiological activities. However, the drought resistance of transgenic lines (OE-2 and OE-8) was improved, and normal physiological activities could still be maintained in the later stage of drought treatment.

Claims (10)

1. The nucleotide sequence of the Pinus massoniana PmAP2/ERF gene is shown as SEQ ID NO. 1.

2. The Pinus massoniana PmAP2/ERF gene of claim 1, wherein the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2.

3. A vector comprising the pinus massoniana PmAP2/ERF gene of claim 1.

4. A vector of the PmAP2/ERF gene of pinus massoniana according to claim 3, wherein the vector is a plant expression vector.

5. The vector of the Pinus massoniana PmAP2/ERF gene according to claim 4, wherein the plant expression vector is pBI121-PmAP2/ERF.

6. The use of the pinus massoniana PmAP2/ERF gene according to claim 1 in improving drought resistance of mountain new poplar.

7. The use of the pinus massoniana PmAP2/ERF gene according to claim 6 for improving drought resistance of aspen, comprising the steps of:

1) Constructing a plant expression vector of a Pinus massoniana PmAP2/ERF gene;

2) Transforming the constructed plant expression vector into mountain new poplar leaves;

3) Cultivating and screening to obtain mountain new Yang Zhizhu with improved drought resistance.

8. The use of the pinus massoniana PmAP2/ERF gene of claim 1 in increasing rate of shanxin Yang Guangge.

9. The use of the pinus massoniana PmAP2/ERF gene of claim 1 in increasing rate of shanxin Yang Zhengteng.

10. The use of the pinus massoniana PmAP2/ERF gene of claim 1 in improving the conductivity of shanxin Yang Qikong.