Application of overexpressed PtoPFD3.1 gene in improving biomass of populus tomentosa

Abstract

The invention discloses application of an overexpressed PtoPFD3.1 gene in improvement of biomass of populus tomentosa. The nucleotide sequence of the PtoPFD3.1 gene is shown as SEQ ID No.2. A genetic engineering method is adopted, wild populus tomentosa is transformed through an agrobacterium tumefaciens infection method to obtain a constitutive overexpressed PtoPFD3.1 transgenic plant, and a plant with high expression quantity is screened out through positive identification for phenotypic observation. The result shows that compared with a wild type in the same period, the average internode length of the transgenic plant over-expressing PtoPFD3.1 is increased, the fresh weight and dry weight of the overground part are increased, and the overground biomass is remarkably improved. And further section observation also finds that the xylem cell layer number and the xylem ratio of the transgenic plant are obviously improved, and the wood development is promoted. According to the cultivation technical method provided by the invention, the high-quality poplar variety with high biomass is cultivated. The method has great significance in molecular breeding and cultivation of excellent poplar in the future.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, and particularly to the application of overexpressing the PtoPFD3.1 gene in improving the biomass of Populus tomentosa. Background Art

[0002] Wood plays an important role in human production and life. Natural and renewable wood is one of the world's four major raw materials. Wood is often used in papermaking, house construction, furniture manufacturing, packaging, etc., and has important economic value. In addition, wood can not only be used to replace fossil fuels, but also be used as an ideal raw material to replace plastics and chemical products.

[0003] China has the largest plantation area in the world. Plantations are the main source of carbon sinks, significantly increasing the carbon storage in forest ecosystems. The area of poplar plantations in China is extensive, but the wood properties of poplar plantations are not good, mainly manifested as low wood strength, low wood density, poor wood stiffness performance, easy warping, deformation and cracking of boards, etc., which affect the application range of wood. Therefore, it is of great significance to directionally cultivate poplar plantation wood with fast growth, high productivity and excellent properties. At present, China's wood production is insufficient, mostly relying on imports, which is difficult to meet the industrial needs. It is necessary to identify key target genes that can improve wood production in forest trees, so as to achieve efficient breeding of poplars and enhance the innovation ability of the seed industry.

[0004] The production of wood is mainly related to the secondary development of plant stems. The vascular cambium differentiates outward to form phloem and inward to form xylem. The cells divided and differentiated from the cambium increase in volume and thicken their cell walls, and finally develop into mature xylem cells. During plant morphogenesis, the direction of the cell division plane and the direction of cell expansion are strictly controlled. Different from animal cells, plant cells are restricted within rigid cell walls and cannot quickly change their shapes or migrate. Microtubules and actin filaments are two key components of the plant cytoskeleton and play important roles in processes such as cell division, cell expansion, cell differentiation and cell communication. Prefoldins (PFDs), also known as "genes involved in the microtubule biogenesis complex", are involved in the folding of nascent actin and tubulin monomers in the cytoplasm. At present, whether PFDs genes are involved in regulating the process of xylem development during wood development is still unknown. Summary of the Invention

[0005] In view of this, one of the purposes of the present invention is to provide the application of overexpressing the PtoPFD3.1 gene in improving the biomass of Populus tomentosa; the second purpose of the present invention is to provide a method for improving the biomass of Populus tomentosa.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] 1. Application of overexpressing PtoPFD3.1 gene in improving biomass of Populus tomentosa, wherein the nucleic acid sequence of the PtoPFD3.1 gene is as shown in SEQ ID NO.2.

[0008] 2. Preferably, the improvement of the biomass of Populus tomentosa is to increase the average internode length, aboveground biomass, secondary xylem area or number of xylem cell layers.

[0009] 3. Method for improving biomass of Populus tomentosa by overexpressing PtoPFD3.1 gene, comprising the following steps: introducing the PtoPFD3.1 gene into Populus tomentosa plants by genetic engineering methods to obtain plants overexpressing the PtoPFD3.1 gene, which are Populus tomentosa with improved biomass.

[0010] Preferably, in the present invention, the introduction of the PtoPFD3.1 gene into Populus tomentosa plants by genetic engineering methods is to ligate the nucleotide sequence shown in SEQ ID NO.2 into the PCXSN-Flag vector at the site after cutting off ccdB with XcmⅠ.

[0011] Preferably, in the present invention, SEQ ID NO.2 is obtained by PCR amplification using the sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers and Populus tomentosa cDNA as a template.

[0012] Preferably, the introduction method in the present invention is mediated by Agrobacterium tumefaciens.

[0013] The beneficial effects of the present invention are as follows: The present invention discloses the application of overexpressing PtoPFD3.1 gene in improving biomass of Populus tomentosa. By using gene editing methods, transgenic plants are obtained after transforming wild-type Populus tomentosa by Agrobacterium tumefaciens infection method, and transgenic lines overexpressing PtoPFD3.1 are screened through subsequent positive identification and fluorescence quantitative PCR. The results show that compared with the wild-type at the same time, the transgenic plants overexpressing PtoPFD3.1 have significantly increased average internode length, significantly increased fresh weight and dry weight of stems and leaves aboveground, and significantly improved aboveground biomass without affecting plant height; and compared with the wild-type, the number of secondary xylem layers in the transgenic plants overexpressing PtoPFD3.1 increases and the proportion of xylem increases. It shows that PtoPFD3.1 can significantly promote the development of secondary xylem of Populus tomentosa and improve wood yield. The present invention provides good materials for cultivating high-quality poplar varieties with high biomass. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0015] Figure 1For vector maps (A: PCXSN-FLAG vector map; B: PCXSN-FLAG-PtoPFD3.1 map);

[0016] Figure 2 For qRT-PCR detection of transgenic plants (WT is wild-type Populus tomentosa, 35S pro :PFD3.1 are transgenic plants, and L1, L2, L5, and L6 represent different transgenic lines);

[0017] Figure 3 For 35S pro :PFD3.1 transgenic plant biomass analysis (A shows the overall above-ground phenotypes of wild-type Populus tomentosa and 35S pro :PFD3.1 transgenic plants; B shows the plant heights of wild-type Populus tomentosa and 35S pro :PFD3.1 transgenic plants; C shows the average internode lengths of wild-type Populus tomentosa and 35S pro :PFD3.1 transgenic plants; D shows the determination of fresh and dry weights of the above-ground parts of wild-type Populus tomentosa and 35S pro :PFD3.1 transgenic plants).

[0018] Figure 4 For 35S pro :PFD3.1 transgenic plant secondary xylem phenotype analysis (A shows the secondary xylem phenotypes of wild-type Populus tomentosa and 35S pro :PFD3.1 transgenic plants; B shows the secondary xylem phenotypes of wild-type Populus tomentosa and 35S pro :PFD3.1 transgenic plants; C shows the statistical analysis of the number of secondary xylem layers in wild-type plants and 35S pro :PFD3.1 transgenic plants; D shows the phenotype analysis of the proportion of secondary xylem in wild-type Populus tomentosa and 35S pro :PFD3.1 transgenic plants). Specific implementation manners

[0019] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments given are not intended to limit the present invention.

[0020] Example 1, Method for preparing culture media and reagents

[0021] (1) Culture media

[0022] The method for preparing the culture media is as follows:

[0023] ① Bacterial culture media:

[0024] LB medium (formula for 1 L): 5 g of yeast extract, 10 g of peptone, 10 g of sodium chloride. For solid medium, add 12 - 15 g of agar and sterilize at 121 °C under high pressure for 20 min.

[0025] YEP medium (formula for 1 L): 10 g of yeast extract, 10 g of peptone, 5 g of sodium chloride. For solid medium, add 12 - 15 g of agar and sterilize at 121 °C under high pressure for 20 min.

[0026] ② Plant medium (formula for 1 L):

[0027] WPM resuspension: 1.991 g of WPM powder, 1 mL of calcium salt, 30 g of sucrose. Adjust the pH to 5.80 - 5.85 with KOH, sterilize at 121 °C under high pressure for 20 min, and add AS to a final concentration of 100 μM when cooled to 50 °C.

[0028] WPM co - culture medium: 1.991 g of WPM powder, 1 mL of calcium salt, 30 g of sucrose, final concentration of NAA 1.0 mg / L. Adjust the pH to 5.80 - 5.85 with KOH, add 7.6 - 7.8 g of agar powder, sterilize at 121 °C under high pressure for 20 min, and add AS to a final concentration of 100 μM and ZT to a final concentration of 2.0 mg / L when cooled to 50 °C.

[0029] WPM selection medium: 1.991 g of WPM powder, 1 mL of calcium salt, 30 g of sucrose, final concentration of NAA 1.0 mg / L. Adjust the pH to 5.80 - 5.85 with KOH, add 7.6 - 7.8 g of agar powder, sterilize at 121 °C under high pressure for 20 min, and add ZT to a final concentration of 2.0 mg / L and Cef to a final concentration of 400 mg / L when cooled to 50 °C.

[0030] WPM budding medium: 1.991 g of WPM powder, 1 mL of calcium salt, 30 g of sucrose, final concentration of NAA 0.1 mg / L. Adjust the pH to 5.80 - 5.85 with KOH, add 7.6 - 7.8 g of agar powder, sterilize at 121 °C under high pressure for 20 min, and add ZT to a final concentration of 2.0 mg / L and Cef to a final concentration of 400 mg / L when cooled to 50 °C.

[0031] WPM rooting medium: 2.41 g of WPM powder (containing calcium salt), 30 g of sucrose. Adjust the pH to 5.80 - 5.85 with KOH, add 7.6 - 7.8 g of agar powder, sterilize at 121 °C under high pressure for 20 min, and add Cef to a final concentration of 400 mg / L when cooled to 50 °C.

[0032] (2) Other reagents

[0033] ① 50×TAE Electrophoresis Buffer (1L): Tris 242 g, glacial acetic acid 57.1 mL, Na2EDTA·2H2O 37.2 g. Adjust the pH to 8.0 and dilute to 1× before use.

[0034] ② Toluidine Blue Staining Solution: Dissolve 1 g of toluidine blue powder in 100 mL of distilled water and dissolve thoroughly. Dilute it at a ratio of toluidine blue: water = 3:5 before use.

[0035] Example 2: Gene Amplification and Vector Construction

[0036] To design primers for amplifying PtoPFD3.1, first, on the plant genome website PHYTOZOME (http: / / www.phytozome.com), the amino acid sequence (SEQ ID No.1) of the Populus trichocarpa PtrPFD3.1 (Potri.010G147600) gene was retrieved. According to the Populus tomentosa genome database provided by Sichuan University, the CDs sequence (SEQ ID No.2) of the homologous gene PtoPFD3.1 (P.x_tomentosa22724) in Populus tomentosa was found, and the encoded amino acids are as shown in SEQ ID No.3. Based on this sequence, the upstream primer 5′-CATGGCATCGTCGTCATCAAC-3′ (SEQ ID No.4) and the downstream primer 5′-TCATGAGTCTTTCTCGGCAG-3′ (SEQ ID No.5) were designed, and these primers were synthesized by BGI (Beijing). The PCR amplification system is as follows:

[0037] Table 1: PCR Amplification System

[0038]

[0039] 2×PrimeSTAR is a product of TaKaRa Company.

[0040] The PCR amplification procedure is as follows:

[0041] Table 2: PCR Amplification Procedure

[0042]

[0043] After PCR amplification, agarose gel electrophoresis was performed for detection, and the band with the target gene fragment size (579 bp) was gel-extracted. The gel extraction kit is a product of BioFlux Company, and the specific operation steps can be carried out according to the instruction manual. The recovered target gene fragment needs to add a base A at its 3' end, and the reaction system is as follows:

[0044] Table 3: A-adding Reaction System

[0045]

[0046] The 2×Taq Mix is a product of Aikery Biotech Co., Ltd.

[0047] React at 72°C for 1 h.

[0048] At the same time, the Escherichia coli strain of the required vector PCXSN-Flag( Figure 1 , A) was expanded in culture, and a small amount of plasmid was extracted. The plasmid miniprep kit used in the experiment was a product of BioFlux Co., Ltd., and the specific operation steps can be carried out according to the instructions. After obtaining the plasmid, it was digested overnight with the restriction endonuclease XcmⅠ at 37°C, followed by agarose gel electrophoresis and gel extraction.

[0049] The linearized vector was ligated to the target gene using SolutionⅠ. The specific reaction system is as follows:

[0050] Table 4. Ligation system

[0051]

[0052] React at 16°C for about 12 h. SolutionⅠ is a product of Takara Co., Ltd.

[0053] For example, the PCXSN-FLAG vector map was digested at the XcmⅠ restriction site, and the PtoPFD3.1 gene fragment was ligated into the vector PCXSN-FLAG to obtain the PCXSN-FLAG-PtoPFD3.1 map.

[0054] The ligation product was transformed into Escherichia coli competent cells (DH5α) by the following method:

[0055] 1) Take out the Escherichia coli DH5α competent cells from the -80°C refrigerator and thaw them on ice;

[0056] 2) In the laminar flow hood, add the ligation product to 100 μL of competent cells, gently mix, and incubate on ice for 30 min;

[0057] 3) Incubate in a 42°C water bath for 90 s;

[0058] 4) Quickly transfer to an ice bath for 1 - 2 min;

[0059] 5) In the laminar flow hood, add 800 μL of LB medium, place it in a shaker at 37°C and 200 rpm, and shake for 60 min;

[0060] 6) Centrifuge at 5000 - 6000 rpm in a high-speed centrifuge for 4 min;

[0061] 7) In a laminar flow hood, resuspend the cells with 100 μL of the supernatant and transfer them onto an LB solid medium containing kanamycin, then spread evenly.

[0062] 8) Incubate the plate upside down in an incubator at 37 °C for 12 - 16 h.

[0063] After single colonies grow on the LB medium, perform colony PCR detection to identify positive clones. The reaction system is as follows:

[0064] Table 5. PCR amplification system

[0065]

[0066] Pick a single colony with a sterile pipette tip and transfer it into the mixture system, then perform the following PCR program:

[0067] Table 6. PCR amplification program

[0068]

[0069] After the PCR reaction, perform agarose gel electrophoresis. Inoculate the positive clones into an LB liquid medium (containing kanamycin) for expansion culture, and then extract the plasmids.

[0070] Example 3. Genetic transformation of poplar

[0071] (1) Transformation of Agrobacterium competent cells (GV3101)

[0072] 1) Take out the Agrobacterium GV3101 competent cells from the -80 °C refrigerator and place them on ice to thaw. Add 5 μl of the plasmid, gently mix well, and incubate on ice for 5 min.

[0073] 2) Freeze in liquid nitrogen for 1 min, and then transfer to a 37 °C water bath for 5 min after freezing.

[0074] 3) After the water bath, incubate on ice for 5 min.

[0075] 4) Add 800 μl of YEP liquid medium (containing Rif: 20 mg / ml).

[0076] 5) Wrap it with plastic wrap and place it in a shaker at 28 °C and 200 rpm for 4 h.

[0077] 6) Centrifuge at 5000 rpm for 5 min using a high-speed centrifuge, discard the excess supernatant, resuspend the cells, and spread them evenly onto a YEP solid medium containing kanamycin.

[0078] 7) Incubate the plate upside down in an Agrobacterium incubator at 28 °C for 2 days.

[0079] After transferring the constructed PCXSN-Flag-PtoPFD3.1 vector into Agrobacterium tumefaciens GV3101, Agrobacterium was used to infect Populus tomentosa leaf discs, and then callus and multiple buds were induced, and finally regenerated plants were obtained.

[0080] (4) Transformation of Populus tomentosa by Agrobacterium-mediated leaf disc method

[0081] 1) Pick a single colony of Agrobacterium and inoculate it into 20 ml of YEP liquid medium. Incubate overnight at 28 °C with shaking at 200 rpm until the OD 600 is 0.4 - 0.6.

[0082] 2) Inoculate the Agrobacterium activated overnight into a conical flask at a ratio of 1:50 and culture until the OD 600 is 0.2 - 0.4.

[0083] 3) Add the Agrobacterium solution from the previous step into a 50 ml centrifuge tube (sterilized), centrifuge at 4000 rpm for 10 minutes, discard the supernatant, add 10 mL of WPM resuspension and mix well, then add 20 mL - 30 mL of WPM resuspension, transfer it into a round-mouth flask, and incubate at 28 °C with shaking at 200 rpm for 40 - 60 min.

[0084] 4) Take sterile Populus leaves and cut them into squares with a side length of 4 - 6 mm.

[0085] 5) Put the cut leaf discs into the WPM resuspension after shaking culture in the third step and infect for 10 minutes. During this period, gently shake every 3 - 5 minutes.

[0086] 6) Take out the infected leaves, blot the attached bacterial liquid with sterile filter paper, and inoculate them on the WPM co-culture medium. Two days later, transfer the co-cultured explants to the selection medium and incubate them in the dark at 25 °C. Replace the selection medium every 7 - 10 days. When the transformed explants grow callus, transfer them to the shoot induction medium and incubate them under light at 25 °C. Replace the medium every 15 - 30 days. After one to two months, cut the multiple buds that have grown with sterile scissors and insert them into the rooting medium, and incubate them under light at 25 °C. After one month, complete plants will grow.

[0087] Example 4. Positive identification of transgenic plants

[0088] (1) RNA extraction

[0089] Extract the RNA of wild-type and transgenic plants using a polysaccharide and polyphenol plant total RNA extraction kit (the polysaccharide and polyphenol plant total RNA extraction kit is a product of BioFlux). The specific steps refer to the kit instructions (note: the lysis buffer used for extracting Populus RNA is Lysis AG).

[0090] (2) Reverse transcription of RNA into cDNA

[0091] After extracting the total RNA from wild-type and transgenic plants, the mRNA in it was reverse-transcribed into cDNA using a reverse transcription kit. The reverse transcription kit is the PrimeScript RT reagent Kit with gDNA Eraser, a product of TaKaRa Company for quantitative PCR. The specific reaction system and reaction program are shown in Table 7 and Table 8. TM RTreagent Kit with gDNA Eraser. The specific reaction system and reaction program are shown in Table 7 and Table 8.

[0092] Table 7. Reverse transcription system 1 (removing genomic DNA)

[0093]

[0094]

[0095] 42 °C, 2 min; 4 °C

[0096] Table 8. Reverse transcription system 2 (reverse transcription)

[0097]

[0098] 37 °C, 15 min; 85 °C, 5 s; 4 °C

[0099] The cDNA obtained after reverse transcription was stored at -20 °C, or directly diluted 1 - 2 times and used for fluorescence quantitative PCR.

[0100] (3) Fluorescence quantitative PCR

[0101] According to the CDs sequence of PtoPFD3.1 (SEQ ID No. 2), fluorescence quantitative PCR primers were designed. The upstream primer sequence is: 5'-ATGCTGGCTCAGTCTGTTTA-3' (SEQ ID No. 6), and the downstream primer sequence is: 5'-TAGATCCGCAACTAGAACTT-3' (SEQ ID No. 7). The primers were synthesized by BGI (Beijing), and at the same time, the quantitative primers for the housekeeping gene UBQ were synthesized. The upstream primer sequence is: 5'-GTTGATTTTTGCTGGGAAGC-3' (SEQ ID No. 8), and the downstream primer sequence is: 5'-GATCTTGGCCTTCACGTTGT-3' (SEQ ID No. 9).

[0102] The obtained cDNA was diluted 1 - 2 times.

[0103] The detection results are as Figure 2 shown. The results show that the expression level of the PtoPFD3.1 gene in the transgenic lines increased.

[0104] Example 5, Phenotypic Analysis and Histochemical Staining of Transgenic Plants

[0105] Created and successfully obtained 35S pro :PtoPFD3.1 transgenic materials. After 2 months of cultivation, the phenotypes of the above-ground parts (stems + leaves) of wild-type Populus tomentosa and transgenic plants L1 and L6 were observed and compared respectively. Preliminary observations found that the growth trends of transgenic plants and wild-type plants were relatively consistent, as shown in Figure 3 , A. Further statistics on the plant phenotypes found that transgenic plants overexpressing PtoPFD3.1 had no significant difference from the wild-type or were slightly shorter than the wild-type ( Figure 3 , B). The average internode length was longer ( Figure 3 , C), among which the internode length of transgenic plant L1 increased by 25.7% compared with the wild-type, and the internode length of transgenic plant L6 increased by 22.6%.

[0106] To explore the effect of overexpressing PtoPFD3.1 on plant biomass, the above-ground parts (stems + leaves) of wild-type Populus tomentosa and overexpressing transgenic plants were taken starting from the parts exposed above the ground under the same cultivation time and the same cultivation conditions. After sampling, the surface moisture was dried, the fresh weight (wet weight) was weighed, and counted. Different materials were put into kraft paper envelopes, marked, sealed, and then placed in an oven (60 °C) for dehydration and drying. Drying for more than two days has a better effect. At this time, the materials are easy to break. Take them out carefully, weigh the dry weight, and count. The results showed that the above-ground biomass of transgenic plants was significantly increased compared with the wild-type, as shown in Figure 3 , D, E. The fresh weights of transgenic plants L1 and L6 increased by 49.2% and 49.5% respectively compared with the wild-type, and the dry weights increased by 26.7% and 28.9%.

[0107] To further analyze the effect of overexpressing PtoPFD3.1 on plant wood development and yield, stem segments of transgenic plants and wild-type Populus tomentosa cultivated in soil for two and a half months were taken, and the stem segments were transversely sectioned (80 μm) using a vibratome, and the xylem development was observed by toluidine blue staining of the sections (toluidine blue staining for 15 - 30 s, washed twice with ddH2O, and observed under an optical microscope). The observation found that compared with wild-type Populus tomentosa, the secondary xylem area in transgenic plants overexpressing PtoPFD3.1 was larger than that of the wild-type ( Figure 4 , A, B), and the quantitative results showed that the proportion of secondary xylem in transgenic plants was significantly increased compared with the wild-type ( Figure 4, D), in which the transgenic plant L1 increased by 91.6% compared to the wild type, and L6 increased by 60.2%. Statistical analysis of the number of xylem cell layers found that the number of xylem cell layers in the wild type was 14 - 16 layers, the number of xylem cell layers in the PtoPFD3.1 overexpression line was 33 - 35 layers. The number of xylem cell layers in transgenic plant L1 was twice that of the wild type, and the number of xylem cell layers in transgenic plant L6 also increased by 88.2% compared to the wild type. Thus, it can be seen that the number of xylem cell layers in transgenic plants overexpressing PtoPFD3.1 increased significantly( Figure 4 , C). These results indicate that overexpression of PtoPFD3.1 can promote the secondary xylem development of poplar, thereby increasing the wood specific gravity of poplar. Compared with the wild type, the overexpression lines have more xylem cell layers and a higher proportion of xylem.

[0108] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Application of overexpression of PtoPFD3.1 gene in increasing biomass of Populus tomentosa, characterized by: The nucleic acid sequence of the PtoPFD3.1 gene is shown in SEQ ID NO.

2.

2. The use of the overexpressed PtoPFD3.1 gene in increasing the biomass of Populus tomentosa according to claim 1, characterized in that: The method of increasing the biomass of Populus tomentosa is to increase the average internode length, the aboveground biomass, the secondary xylem area or the number of xylem cell layers.

3. A method for increasing the biomass of Populus tomentosa, characterized in that: The method comprises the following steps: introducing the PtoPFD3.1 gene into Populus tomentosa plants by genetic engineering method to obtain plants over-expressing the PtoPFD3.1 gene, namely, Populus tomentosa with increased biomass.

4. The method for increasing the biomass of Populus tomentosa according to claim 3, characterized in that: The PtoPFD3.1 gene is introduced into Populus tomentosa plants by genetic engineering method, which is to connect the nucleotide sequence shown in SEQ ID NO.2 into the PCXSN-Flag vector and cut out the site after ccdB using XcmⅠ.

5. The method for increasing the biomass of Populus tomentosa according to claim 3, characterized in that: The SEQ ID NO.2 is obtained by PCR amplification using the sequences shown in SEQ ID NO.4 and SEQ ID NO.5 as primers and Populus tomentosa cDNA as a template.

6. The method for increasing the biomass of Populus tomentosa according to claim 3, characterized in that: The method of introduction is mediated by Agrobacterium.

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