Use of overexpression of auxin transporter ptopin1a gene in increasing biomass of populus tomentosa

Abstract

Provided is the use of overexpression of the auxin transporter PtoPIN1a gene in increasing the biomass of Populus tomentosa. The nucleotide sequence of the auxin transporter PtoPIN1a gene is as shown in SEQ ID No. 1. By means of using a genetic engineering method, wild-type Populus tomentosa is transformed by means of Agrobacterium tumefaciens infection to obtain a tissue-specific PtoPIN1a overexpressing transgenic plant, and subsequently, a plant with a relatively high expression level is selected, by means of positive identification, for phenotype observation. Results show that compared with the wild type in the same period, the PtoPIN1a transgenic plant has an increased plant height, a thicker stem and a significantly increased aboveground biomass. By means of the provided technical cultivation method, a high-biomass high-quality poplar variety is cultivated. The method is of great significance for future molecular breeding to cultivate excellent poplar varieties.

Description

Application of overexpression of auxin transporter PtoPIN1a gene in increasing biomass of Populus tomentosa Technical Field

[0001] The present invention relates to the field of plant biotechnology, and in particular to application of overexpressing an auxin transporter protein PtoPIN1a gene in increasing the biomass of Populus tomentosa. Background Art

[0002] As my country's environmental protection strategy progresses, its timber production has been declining. my country boasts large areas of poplar plantations. As an important economic forest, poplar wood has significant economic value in industries such as energy, construction, and papermaking. Simultaneously, plantations have become the world's primary source of timber processing and utilization. Vigorously developing plantations is a future trend in forestry and an inevitable consequence of changes in forest resource structure and timber supply. Increasing timber yield has become a primary goal of current forest genetic breeding.

[0003] Poplar (Populus L.) is an important economic and energy plant, and is also a model plant for studying wood development. The poplar has a very tall and straight appearance, with a trunk that stands upright and reaches the sky. It is one of the important plant choices for my country's afforestation and the Three North Shelterbelt Project. It not only effectively prevents and controls sandstorms, but also greatly increases vegetation coverage, allowing my country's environment to be further optimized and improved. In addition, poplar is also one of the very good choices for home building materials. Homes made of poplar are not only very strong and durable, but can also absorb toxic substances in the air. Therefore, further increasing the biomass of Populus tomentosa is of great significance to our production and life.

[0004] Overexpressing related genes through tissue-specific promoters can increase wood biomass. Wood thickening primarily results from secondary development of plant stems, with the vascular cambium differentiating outward to form the phloem and inward to form the xylem. Cells released from the cambium expand in size, thicken their cell walls, and ultimately develop into mature xylem cells. Xylem development is regulated by multiple factors, including plant hormones, carbohydrates, nitrogen, and meteorological factors. Auxin, a key plant hormone involved in wood formation, plays a crucial role in xylem development. Currently, no studies have reported whether the auxin transporter gene, PtoPIN1a, plays a role in xylem development.

[0005] Summary of the Invention

[0006] In view of this, one of the objects of the present invention is to provide an application of overexpressing the auxin transporter protein PtoPIN1a gene in increasing the biomass of Populus tomentosa; a second object of the present invention is to provide a method for overexpressing the auxin transporter protein PtoPIN1a gene to increase the biomass of Populus tomentosa.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. Application of overexpression of auxin transporter PtoPIN1a gene in increasing biomass of Populus tomentosa.

[0009] As a more preferred technical solution of the present invention, the amino acid sequence of the gene encoding the auxin transporter PtoPIN1a is shown as SEQ ID No.2.

[0010] As a more preferred technical solution of the present invention, the nucleotide sequence encoding the auxin transporter PtoPIN1a gene is shown as SEQ ID No.1.

[0011] 2. A method for increasing the biomass of Populus tomentosa by overexpressing the auxin transporter PtoPIN1a gene, comprising the following steps:

[0012] (1) Construction of plant overexpression vector: amplify the Populus tomentosa auxin transporter PtoPIN1a gene, connect it to a plant expression vector, and then transform Agrobacterium;

[0013] (2) Genetic transformation of Populus tomentosa: Wild-type Populus tomentosa leaves were transformed using the Agrobacterium-mediated leaf disc method;

[0014] (3) Molecular detection of transgenic plants: Using the genomic DNA of transgenic plants as a template, a gene segment between the backbone vector and the target gene fragment is amplified to screen for high biomass poplars.

[0015] As a more preferred technical solution of the present invention, in step (1), the method for amplifying the auxin transporter protein PtoPIN1a gene is: using poplar cDNA as a template and the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers to perform PCR amplification, electrophoresis detection, and recovery to obtain the auxin transporter protein PtoPIN1a gene.

[0016] As a more preferred technical solution of the present invention, in step (1), the plant overexpression vector is obtained by connecting the nucleotide sequence shown in SEQ ID NO.1 into the Asc1 restriction site of the pCAMBIA1300-LMX5 vector by homologous recombination; the pCAMBIA1300-LMX5 vector is constructed by using EocR I and Kpn I to cut off the CaMV35S constitutive promoter in the pCAMBIA1300 vector, and connecting the LMX5 promoter shown in SEQ ID NO.5 into the EocR I and Kpn I restriction sites of the pCAMBIA1300 vector to obtain a specific plant expression vector driven by the xylem-specific promoter LMX5.

[0017] As a more preferred technical solution of the present invention, in step (3), the amplification primer sequences are shown as SEQ ID NO.6 and SEQ ID NO.7.

[0018] The beneficial effects of the present invention are as follows: the present invention cloned the auxin transporter PtoPIN1a gene from poplar, used genetic engineering methods, and transformed the wild-type white poplar by Agrobacterium tumefaciens infection to obtain tissue-specific PtoPIN1a overexpressing transgenic plants. The plants with higher expression levels were screened by subsequent positive identification for phenotypic observation. The results showed that compared with the wild-type plants of the same period, the transgenic plants of PtoPIN1a had increased plant height, thicker stems, and significantly increased aboveground biomass. This indicates that the PtoPIN1a protein positively regulates the differentiation of poplar wood, promotes xylem development, and thereby increases the proportion of xylem, the number of xylem layers, and the amount of wood biomass, laying the foundation for the future molecular breeding of high-quality poplars. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0020] Figure 1 shows the PCR detection (DNA detection) of transgenic plants;

[0021] Using DNA from wild-type and transgenic proLmx5-ptoPIN1a as templates, screening was performed by amplifying a 1368 bp segment of the gene between the backbone and the fragment; M represents the marker DL5000; - represents a non-transgenic wild-type plant;

[0022] Figure 2 is the expression analysis of transgenic plants (RNA detection);

[0023] WT is the wild-type Populus tomentosa, proLmx5-ptoPIN1a-L1, proLmx5-ptoPIN1a-L5, proLmx5-ptoPIN1a-L10, and proLmx5-ptoPIN1a-L12 represent different lines of PtoPIN1a transgenic plants, among which lines L10 and L12 have the highest expression levels;

[0024] Figure 3 shows the biomass analysis of overexpression transgenic plants;

[0025] A is the overall aboveground phenotype of wild-type Populus tomentosa and overexpression plants; B is the dry weight and fresh weight of the aboveground parts of wild-type plants and overexpression plants; C is the plant height of wild-type Populus tomentosa and overexpression plants; D is the number of internodes of wild-type Populus tomentosa and overexpression plants; E is the stem thickness of wild-type Populus tomentosa and overexpression plants; F is the phenotypic analysis of the xylem proportion of wild-type Populus tomentosa and overexpression plants; G is the statistical analysis of the xylem proportion of wild-type Populus tomentosa and overexpression plants; H is the xylem phenotypic analysis of wild-type Populus tomentosa and overexpression plants; I is the statistical analysis of the number of xylem layers of wild-type Populus tomentosa and overexpression plants. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0028] Example 1. Design of PCR primers and PCR amplification to obtain target fragments

[0029] 1. PCR primer design

[0030] First, we logged into PHYTOZOME (http: / / www.phytozome.com) and retrieved the CDS sequence of the poplar PtoPIN1a gene (SEQ ID NO. 1, 1–37 bp representing the 5' UTR, 38–1867 bp representing the CDS, and 1868–1922 bp representing the 3' UTR). The amino acid sequence encoded by the PtoPIN1a gene is shown in SEQ ID NO. 2. We designed primers specific for the PtoPIN1a CDS sequence with homology arms. The upstream primer sequence for the PtoPIN1a fragment with homology arms is: 5′-gcatctttccccaccaaccggcgcgTCTGCCAAAGTTTTGGGTTTTC-3′ (SEQ ID No. 3), and the downstream primer sequence is: 5′-gatcggggaaattcgagctcGGCGCGAGATGGGGTCTGCATGGATTC-3′ (SEQ ID No. 4). These primers were synthesized by BGI (Beijing). All the reaction reagents used in PCR were products of TaKaRa. The target fragment was amplified by PCR using a high-fidelity enzyme. The amplification system is shown in Table 1 below:

[0031] Table 1. PCR amplification system

[0032] The reaction system can be appropriately expanded according to the amount of product required.

[0033] The reaction conditions were as follows: initial denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds; annealing at 58°C for 10 seconds; extension at 72°C for 20 seconds, for 36 cycles; and a final extension at 72°C for 10 minutes. PCR products were analyzed by electrophoresis on 1%-2% agarose gels based on their size.

[0034] After electrophoresis, the target bands are observed. Only when the bands are clear and bright can the target fragments be recovered from the gel. For this process, use the BioFlux Gel Recovery Kit, which consists of four components: ① Extraction Buffer (yellow pH <= 7.0), ② Wash Buffer, ③ Elution Buffer, and ④ Spin Columns. The specific steps are as follows.

[0035] 1) Use a clean, sharp blade to cut the agarose gel containing the target DNA fragment and place it into a 1.5 or 2.0 mL centrifuge tube; (Try to remove the gel as much as possible during the cutting process)

[0036] 2) Add an appropriate volume of ① in a ratio of 1:3 (mass: volume in microliters);

[0037] 3) Place the centrifuge tube containing the gel and the mixture in step 1 in a 50-60°C water bath until the gel melts (mix thoroughly every 2-3 minutes during the melting process);

[0038] 4) If the target gene fragment is smaller than 500 bp, add an equal volume of isopropanol at a 1:1 ratio and mix thoroughly. (If the fragment is larger than 500 bp and smaller than 4 kb, no isopropanol is required.)

[0039] 5) After the agarose gel melts, transfer the entire mixed liquid to step (4), centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid in the collection tube;

[0040] 6) Then, add 500 μL of ① to ④, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube;

[0041] 7) Add 750 μL of ② to ④, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube;

[0042] 8) Repeat step (7);

[0043] 9) Centrifuge again at 12,000 rpm for 5 minutes to remove as much of ② as possible to prevent residues from inhibiting downstream reactions.

[0044] 10) Add 30 μL-50 μL of ③, water, or TE solution to ④ and let it stand at room temperature for 1 min;

[0045] 11) Centrifuge at 12000 rpm for 1 minute and collect the solution in the centrifuge tube; (Repeat once ②)

[0046] 12) After gel recovery, take 1 μL of the product for electrophoresis and observe the band size and brightness. Store the recovered product at -20°C.

[0047] Example 2: Construction of proLmx5-ptoPIN1a vector and transformation of Agrobacterium GV3101

[0048] 1. Construction of proLmx5-ptoPIN1a vector

[0049] The invention utilizes EocR I and Kpn I to cut out the CaMV35S constitutive promoter in the pCAMBIA1300 vector, and connects the LMX5 promoter shown in SEQ ID NO.5 into the EocR I and Kpn I restriction sites of the pCAMBIA1300 vector to obtain a specific plant expression vector driven by the xylem-specific promoter LMX5. The resulting vector is named pCAMBIA1300-proLMX5.

[0050] Inoculate 10 μL of the pCAMBIA1300-LMX5 vector into LB liquid medium containing the appropriate antibiotic and incubate at 37°C in a shaker at 200 rpm for amplification. Perform plasmid extraction from the overnight culture, digest it with Asc I, dephosphorylate it, and purify the product to obtain the vector backbone. Use a homologous recombination kit to ligate the gel-recovered fragment obtained in Example 1 into the Asc I restriction site of the pCAMBIA1300-LMX5 vector. Finally, transform the ligated fragment into competent E. coli DH5α cells, amplify the target gene fragment by PCR, and verify by enzyme digestion to obtain transformants, which are then stored. This completes the plant overexpression vector driven by the tissue-specific Lmx5 promoter.

[0051] 1) First, pick a single colony that tests positive for the bacteria and inoculate it into LB liquid culture medium containing the corresponding antibiotics. Place it in a shaker at 37°C and 200 rpm for amplification.

[0052] 2) E. coli plasmid extraction mainly uses the plasmid extraction kit of BioFlux, which mainly consists of the following 7 components: ①Resuspension buffer, ②Lysis buffer, ③Neutralization Buffer, ④Wash Buffer, ⑤Elution Buffer, ⑥RNase solution, and ⑦Spin columns.

[0053] 3) Add 1 mL of overnight culture to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 minute. Discard the supernatant and collect the cells (this process can be repeated multiple times to collect more cells, depending on actual needs).

[0054] 4) Add 250 μL of ① and resuspend the bacterial pellet (resuspend until there are no bacterial clumps);

[0055] 5) Add 250 μL of ② and gently invert 4-6 times (do not shake violently to prevent genomic DNA breakage);

[0056] 6) Add 350 μL of solution ③ and immediately gently invert the tube 4-6 times (a flocculent precipitate should appear in the tube). Centrifuge at 12,000 rpm for 10 min at room temperature.

[0057] 7) Transfer the supernatant from step 4) to step 7, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0058] 8) Add 650 μL of Wash Buffer (④) to the sample in step ⑦, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0059] 9) Repeat 8) once;

[0060] 10) Centrifuge at 12000 rpm / min for 5 min to remove as much of ④ as possible to prevent residues from inhibiting downstream reactions;

[0061] 11) Transfer the above ⑦ to a new 1.5 mL centrifuge tube, add 30-50 μl of ⑤, water or TE solution, let it stand at room temperature for 1 minute, and centrifuge at 12,000 rpm for 1 minute;

[0062] 12) The solution contains plasmid DNA. Collect the solution in the centrifuge tube. Repeat step 7, centrifuging again at 12,000 rpm for 1 minute.

[0063] 13) Plasmid DNA can be directly used in various downstream molecular experiments and stored at -20°C;

[0064] 2. Transformation of Agrobacterium tumefaciens GV3101 with proLmx5-ptoPIN1a plasmid

[0065] Extract the pCAMBIA1300-proLmx5-ptoPIN1a plasmid from the positive transformant E. coli according to the Plasmid Mini Kit extraction procedure. The specific steps are as follows:

[0066] ① Take the bacterial solution cultured to the logarithmic phase and place it in an EP tube. Centrifuge at 13400 rpm for 1 min to collect the bacteria and discard the supernatant.

[0067] ②Add 250 μL of pre-cooled Solution I containing RNase A and vortex to resuspend the bacteria.

[0068] ③Add 250 μL Solution II and gently invert the tube 5-6 times to mix thoroughly, then let it stand for 2 minutes.

[0069] ④ Add 350 μL Solution III, invert 5-6 times until precipitation appears, and centrifuge at 13400 rpm for 10 min.

[0070] ⑤ Take the supernatant and add it to the adsorption column (about 700 μL supernatant), place the adsorption column in a 2 mL collection tube, centrifuge at 10,000 rpm at room temperature for 1 min, and discard the liquid.

[0071] ⑥Add 500 μL HB Buffer, centrifuge at 10,000 rpm at room temperature for 1 min, and discard the liquid.

[0072] ⑦Add 700μL DNA Wash Buffer, centrifuge at 10000rpm at room temperature for 1min, and discard the liquid.

[0073] ⑧Repeat step 7, add 700 μL DNA Wash Buffer, centrifuge at 10,000 rpm at room temperature for 1 min, and discard the solution.

[0074] ⑨ Place the adsorption column in the collection tube and centrifuge the empty tube at 13400 rpm for 2 minutes.

[0075] ⑩ Place the adsorption column in a new centrifuge tube, add 30-50 μL of Elution Buffer, let it stand for 2 minutes, and centrifuge at 13,400 rpm for 1 minute. Store at -20°C until needed.

[0076] The operation of transforming Agrobacterium is as follows:

[0077] ① Take 5 μL of proLmx5-ptoPIN1a plasmid and add it to 200 μL of Agrobacterium GV3101 competent cells and mix well.

[0078] ② Immediately place on ice for 30 minutes, quickly put into liquid nitrogen for 2 minutes, and place in a 37℃ water bath for 5 minutes.

[0079] ③ Add 800 μL of empty YEP liquid culture medium, mix well, and culture at 28°C and 200 rpm for 4-6 hours.

[0080] ④ Centrifuge at 5000 rpm for 8 min, discard 900 μL of supernatant, mix the remaining 100 μL of bacterial solution, and evenly spread 100 μL of bacterial solution on YEP + 40 mg / L Rif + 50 mg / L Kan solid culture medium using a sterilized and cooled spreading stick. Incubate inverted at 28°C for 2 days.

[0081] ⑤The bacterium was named GV3101-proLmx5-ptoPIN1a, added with 25% glycerol and stored at -80°C for subsequent genetic transformation experiments.

[0082] Example 3: Genetic transformation of Populus tomentosa

[0083] The transformation material is wild-type Populus tomentosa leaves, and the genetic transformation method mediated by Agrobacterium tumefaciens is adopted.

[0084] 1) Activation of Agrobacterium

[0085] Agrobacterium GV3101 containing the recombinant plasmid was inoculated onto YEP solid medium (containing 50 mg / L Kan + 40 mg / L Rif) and cultured at 28°C for 1-2 days; a single colony was picked and inoculated into YEP liquid medium containing 50 mg / L Kan and 40 mg / L Rif, and cultured at 28°C with shaking until the OD600 reached 0.8-1.0; 200 μL of the viable bacterial liquid was transferred to fresh YEP medium and cultured with shaking at 28°C for 6-8 hours. When the OD600 reached 0.6-0.8, the cells were collected by centrifugation, resuspended in 30 ml of WPM liquid medium supplemented with AS, and cultured in a shaker at 28°C for 1-2 hours before use for transformation.

[0086] 2) Transformation of Agrobacterium

[0087] Select sterile wild-type poplar material in a tissue culture flask as the background for transformation. First, cut an appropriate amount of tender green leaves and, in an appropriate amount of sterile water, cut them into small pieces using a sterile scalpel blade (ensuring smooth cuts to facilitate transformation growth). Transfer an appropriate amount of cut leaves to the resuspended bacterial solution and infect for 10 minutes, shaking every 5 minutes to ensure that the cut wounds are fully exposed to the resuspended bacterial solution.

[0088] 3) Co-cultivation of Populus tomentosa

[0089] The infected leaf material was carefully removed with tweezers and the bacterial liquid was absorbed. The leaf was spread flat on WPM co-culture medium (2.0 g / L WPM powder + 30 g / L sucrose + 7.8 g agar + 100 μmol AS + 1.0 mg / L NAA + 2.0 mg / L ZT) with the back of the leaf facing down. The culture was placed at room temperature (25°C) and cultured in the dark for 2 days.

[0090] 4) Selective cultivation of Populus tomentosa

[0091] After 2 days, the transformed leaves were transferred to WPM selection medium (WPM powder 2.0 g / L + sucrose 30 g / L + agar 7.8 g + kan 50 mg / L + NAA 1.0 mg / L + ZT 2.0 mg / L + Cef 400 mg / L) and cultured at room temperature 25°C in the dark for 3 to 4 weeks, during which the medium was replaced every 5 days (during the selection culture period, it is necessary to observe the degree of changes in the leaves more frequently).

[0092] Note: 1L WPM selection medium is added with 2ml cef and 500ul kanamycin antibiotics, 2ml ZT

[0093] 5) Bud culture of Populus tomentosa

[0094] When white dot-like loose callus tissue appears around the leaves, it is moved to WPM budding medium (WPM powder 2.0g / L + sucrose 30g / L + agar 7.8g + kan 50mg / L + NAA 0.1mg / L + ZT 2.0mg / L + Cef 400mg / L) and induced to sprout on a light culture rack under the conditions of 2000-10000Lux and room temperature of 25℃. The time is about 4-5 weeks. During the budding culture period, it is still necessary to observe the changes in callus growth. If the callus is found to be not growing or browning, the culture medium should be replaced in time.

[0095] 6) Rooting culture of Populus tomentosa

[0096] When the callus tissue in the budding medium produces adventitious buds with a length of about 3 to 4 cm, they are cut off and transferred to WPM rooting medium (WPM 2.0 g / L + sucrose 30 g / L + agar 7.8 g + kan 50 mg / L + NAA 0.1 mg / L + Cef 400 mg / L). Under normal circumstances, the rooting time of the wild type is about 7 days, while the rooting time of the resistant ones is slower.

[0097] 7) Transplantation of transgenic Populus tomentosa

[0098] When the rooted seedlings grow to about 10 cm and the root system is relatively developed, take out the seedlings, wash the agar on the roots with water, mark them, and transplant them into the greenhouse for cultivation (note at this time that the seedlings removed from the tissue culture bottles need a period of adaptation to the external environment, so they need to be sealed with plastic wrap, and the film should be slowly removed after they have adapted).

[0099] Example 4. DNA extraction and PCR molecular detection of transgenic plants

[0100] 1. Poplar DNA extraction

[0101] The CTAB method is mainly used to extract poplar genomic DNA. The specific experimental method is as follows:

[0102] 1) Before DNA extraction, prepare enough 1.5 mL EP tubes and add 300 μL of preheated CTAB lysis buffer (containing 15 μL of β-mercaptoethanol) to each tube. Also add 2-3 magnetic beads (the beads ensure that the tissue is fully disrupted).

[0103] 2) Disrupt the tissue using a tissue disruptor for 10 minutes. After observing the disruption, add 200 μL of preheated CTAB lysis buffer (containing β-mercaptoethanol) and incubate at 65°C in a water bath for 45 minutes, mixing by inversion every 10 minutes.

[0104] 3) After the water bath, centrifuge at 12,000 rpm for 10 minutes at room temperature;

[0105] The supernatant was transferred to a new 1.5 mL EP tube, and an equal volume of CTAB extraction solution (chloroform: isoamyl alcohol, 24:1, V / V) was added. The tube was shaken vigorously and then placed flat to emulsify for 10 min.

[0106] 4) Centrifuge at 12000 rpm for 10 min at room temperature;

[0107] 5) Transfer the supernatant to a new 1.5 mL EP tube and repeat step 4 to remove as much protein and other impurities as possible.

[0108] 6) Transfer the supernatant to a new 1.5 mL EP tube. Add an equal volume of pre-chilled isopropanol and mix thoroughly by inverting the tube. A white flocculent precipitate should form.

[0109] 7) Carefully remove the supernatant from the EP tube using a pipette, then rinse once with 1 mL of 75% ethanol. Centrifuge at 12,000 rpm for 5 minutes, and then pour off the supernatant (be careful not to pour out the white precipitate).

[0110] 8) Repeat step 7;

[0111] 9) Dry in a 37°C oven;

[0112] 10) When the white flocculent precipitate becomes transparent, add 50 μL of ddH2O (containing DNase) and digest at 37°C for 1 hour. Store at -20°C.

[0113] 11) The extracted DNA was detected by 1% agarose gel electrophoresis.

[0114] 2. PCR molecular detection

[0115] The template DNA was transgenic poplar genomic DNA. Transgenic positive plants were screened by amplifying a gene segment (1368 bp) between the backbone and the fragment. Specific primers were designed, and the sequences are as follows:

[0116] Plmx5-F: 5′-AACCCTCGAACTATTATCAT-3′ (SEQ ID No. 6);

[0117] PIN1a-R: 5'-ATCATTGAGTAGAAATCCGTG-3' (SEQ ID No. 7).

[0118] The PCR reaction system was the same as in the table below, and the amplified products were detected by 1% agarose gel electrophoresis.

[0119] The PCR reaction system is shown in Table 2 below:

[0120] Table 2

[0121] The reaction procedure is as follows: 94°C, 5 min (pre-denaturation); 94°C, 30 s (denaturation); annealing temperature according to the designed primer (generally 58°C), 30 s; 72°C, extension time (determined according to the fragment length, about 1 kb / min), for 36 cycles; 72°C, extension for 10 min; 2 μL of the amplified PCR product was taken for detection by agarose gel electrophoresis.

[0122] Example 5: Extraction of total RNA from poplar and reverse transcription into cDNA

[0123] 1. Total RNA extraction from poplar

[0124] First, prepare for RNA extraction. Wash the spoon, mortar, and pestle with sterilized DEPC water and dry them in an oven. The next day, remove the prepared mortar and pestle, add an appropriate amount of quartz sand, and burn them with 10 mL of anhydrous ethanol. Once cooled, extract the RNA.

[0125] During the experiment, all operations were performed using RNase-free pipette tips and 1.5 mL centrifuge tubes provided by AXYGEN. For the specific steps of RNA extraction, refer to the instructions of the Plant Total RNA Extraction Kit:

[0126] 1) First, add 500 μL of lysate to a 1.5 mL RNase-free centrifuge tube. Add approximately 15 μL of β-mercaptoethanol and mix well.

[0127] 2) Grind approximately 0.5g-2g of the prepared plant material into a powder under liquid nitrogen freezing conditions, then divide the powder into the tubes containing the lysis solution and shake vigorously to mix thoroughly.

[0128] 3) Let stand at room temperature for 5 minutes, then centrifuge at 12,000 rpm for 10 minutes. Precool the centrifuge to 4°C.

[0129] 4) After centrifugation, gently remove approximately 400 μL-500 μL of the supernatant (try to remove as much as possible) and add it to a new 1.5 mL centrifuge tube. Add 1 / 2 volume of anhydrous ethanol and quickly invert to mix. Slightly flocculent particles may be visible at this point.

[0130] 5) Transfer the mixed liquid into a 1.5 mL collection tube with an adsorption column and centrifuge at 12,000 rpm for 1 min at 4°C.

[0131] 6) Discard the liquid in the collection tube, add 600 μL of PG buffer to the adsorption column, and centrifuge at 12,000 rpm for 1 min at 4°C.

[0132] 7) Pour off the liquid in the collection tube again and add 600 μL of Wash Buffer to the adsorption column to wash away impurities. Centrifuge at 12,000 rpm for 1 min at 4°C.

[0133] 8) Repeat step 7);

[0134] 9) Centrifuge at 12000 rpm for 5 min to remove as much wash buffer as possible to prevent residual wash buffer from inhibiting downstream reactions.

[0135] 10) Transfer the adsorption column to a new 1.5 mL centrifuge tube and add 30 μL to 50 μL of RElution Buffer or DEPC water (pH > 7.0) to the center of the adsorption column membrane. Let it stand at room temperature for 2 minutes, then centrifuge at 12,000 rpm at 4°C for 1 minute.

[0136] 11) Pipette the eluate back into the adsorption column and centrifuge at 12,000 rpm for 1 minute at 4°C. The product is the total RNA of the sample, which needs to be promptly stored in a -80°C refrigerator for later use.

[0137] 2. Reverse transcription of total RNA from poplar to synthesize cDNA

[0138] Experiments were performed using a TakaRa reverse transcription kit according to the manufacturer's instructions. The kit contains the following seven components: ① gDNAEraser, ② 5x gDNAEraser Buffer, ③ PrimeScript RT Enzyme Mix I, ④ 5x PrimeScript Buffer 2 (for Real Time), ⑤ RT Primer Mix, ⑥ RNase-Free dH2O, and ⑦ EASY Dilution.

[0139] The total RNA extracted above was reverse transcribed to obtain single-stranded cDNA. The reaction product was stored in a -20°C refrigerator for later use. The specific reaction system is shown in Table 3 below:

[0140] Table 3

[0141] Reaction procedure: 37°C for 15 minutes; 85°C for 5 seconds; store at -20°C. The single-stranded cDNA obtained after inversion is used for quantitative fluorescence analysis of gene expression and transcriptome sequencing analysis.

[0142] Example 6: Analysis of expression levels in transgenic plants

[0143] First, DNA level detection was performed on unidentified transgenic plants obtained through genetic transformation of Populus tomentosa using the same method as in Example 4.

[0144] PCR testing of the 14 transgenic plants revealed that four (L1, L5, L10, and L12) had successfully transformed with the exogenous DNA. The results of agarose gel electrophoresis are shown in Figure 1.

[0145] Although the genetic transformation of Populus tomentosa was successful, the expression levels of the same gene varied due to individual differences between plants, so it was necessary to test the expression levels of the transgenic plants. In the present invention, four transgenic plants, L1, L5, L10, and L12, were selected for testing.

[0146] RNA was extracted from wild-type Populus tomentosa and the obtained transgenic positive plants, and then reverse transcribed into cDNA using the same method as in Example 5. Primers for the internal reference gene UBQ were designed:

[0147] qPCR-ptoPIN1a-F: 5′-TCTAAGAGGCACGCTCTTACAC-3′ (SEQ ID No. 8)

[0148] qPCR-ptoPIN1a-R: 5′-AGTACACAAGAGTTATGGGCA-3′ (SEQ ID No. 9)

[0149] cDNA from the wild-type and transgenic lines was amplified by PCR using the same PCR reaction system as in Table 3 of Example 5. Fluorescence quantitative PCR was used to detect the target gene PtoPIN1a using the same concentrations of cDNA from the wild-type and overexpressing lines, and the expression levels of PtoPIN1a in the different transgenic plants were analyzed. (Amplified products can be detected by electrophoresis on a 1% agarose gel.)

[0150] The fluorescence quantitative reaction system is shown in Table 4 below:

[0151] Table 4

[0152] The reaction conditions were as follows: 95°C, 30 s; 95°C, 5 s; 60°C, 1 min; 40 cycles of 95°C, 15 s; 60°C, 30 s; 95°C, 15 s.

[0153] The quantitative test results are shown in Table 5 below:

[0154] Table 5

[0155] Analysis of the quantitative detection results above revealed that transgenic plants L10 and L12 had higher expression levels. A bar graph of the expression levels of the PtoPIN1a gene in different transgenic plants is shown in FIG2 .

[0156] After two months of cultivation, the aboveground phenotypes (stems and leaves) of wild-type Populus tomentosa and transgenic plants L10 and L12 were observed and compared. The results showed that the transgenic plants grew better and had more lush aboveground parts than the wild-type plants, as shown in Figure 3, A.

[0157] To highlight the importance of the ptoPIN1a gene, we selected the L1 line with the highest expression level for subsequent phenotypic statistical analysis. This included transgenic biomass, plant height, stem diameter, number of internodes, internode length, xylem percentage, and number of xylem layers. The results showed that, compared with wild-type Populus tomentosa, overexpressing ptoPIN1a resulted in increased plant height, minimal change in internode number, and increased stem thickness in transgenic plants, as shown in Figures C, D, and E of Figure 3.

[0158] The increase in culm thickness, combined with the previously observed more vigorous growth of the transgenic plants, suggests that the aboveground biomass of the transgenic plants was increased. To investigate the effect of overexpressing ptoPIN1a on plant biomass, the aboveground parts (stems and leaves) of wild-type and transgenic plants expressing ptoPIN1a were harvested starting from the aboveground portion under the same culture conditions for the same time period.

[0159] After sampling, dry the surface moisture, weigh the fresh weight (wet weight), and count.

[0160] Place the different materials into kraft paper envelopes, label them, seal them, and place them in an oven (60°C) for dehydration and drying. Drying for two or more days is optimal, as the materials will break easily at this stage. Carefully remove them, weigh them, and count them, as shown in Table 6.

[0161] Table 6

[0162] According to the above data, after overexpression of ptoPIN1a, the aboveground biomass of transgenic plants increased significantly, and the statistical results are shown in Figure 3, B.

[0163] As mentioned above, the transgenic plants showed significantly increased plant height and stem diameter compared to wild-type Populus tomentosa, suggesting an increase in the proportion of internal xylem and the number of xylem layers. Using an oscillating microtome, transgenic material (internode 10) was cross-sectioned. Preliminary observations indicate that the proportion of xylem in the transgenic plants was higher than that in the wild-type, and the number of xylem layers was greater. However, more detailed statistical analysis is still needed. As shown in Figure 3 (F, G, H, I), the phenotypic statistics are shown in Table 7.

[0164] Table 7

[0165] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Application of overexpressing auxin transporter PtoPIN1a gene in improving biomass of Populus tomentosa.

2. The application according to claim 1, characterized in that, The amino acid sequence encoding the auxin transporter PtoPIN1a gene is shown as SEQ ID No.

2.

3. The application according to claim 2, characterized in that, The nucleotide sequence encoding the auxin transporter PtoPIN1a gene is shown as SEQ ID No.

1.

4. A method for improving the biomass of Populus tomentosa by overexpressing the auxin transporter gene PtoPIN1a, characterized in that, It includes the following steps: (1) Construct a plant overexpression vector: Amplify the auxin transporter PtoPIN1a gene of Populus tomentosa, connect it with a plant expression vector, and then transform Agrobacterium. (2) Genetic transformation of Populus tomentosa: Transform the leaves of wild-type Populus tomentosa by the Agrobacterium-mediated leaf disc method. (3) Molecular detection of transgenic plants: Using the genomic DNA of transgenic plants as a template, screen by amplifying a segment of gene between the backbone vector and the target gene fragment to screen out Populus with high biomass.

5. The method according to claim 4, characterized in that, In step (1), the method for amplifying the auxin transporter PtoPIN1a gene is: Using Populus cDNA as a template, performing PCR amplification with the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers, detecting by electrophoresis, and recovering to obtain the auxin transporter PtoPIN1a gene.

6. The method according to claim 4, characterized in that In step (1), the plant overexpression vector is obtained by ligating the nucleotide sequence shown in SEQ ID NO.1 into the Asc1 restriction site of the artificially modified pCAMBIA1300-LMX5 vector through homologous recombination. The construction method of the pCAMBIA1300-LMX5 vector is to cut off the CaMV35S constitutive promoter in the pCAMBIA1300 vector with EocR I and Kpn I, and ligate the LMX5 promoter shown in SEQ ID NO.5 into the EocR I and Kpn I restriction sites of the pCAMBIA1300 vector to obtain a specific plant expression vector driven by the xylem-specific promoter LMX5.

7. According to the method described in claim 4, in step (3), the amplification primer sequences are shown as SEQ ID NO.6 and SEQ ID NO.7.

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