Application of populus tomentosa PtoHsfB1 gene in enhancing plant height and biomass

Abstract

The invention provides application of a poplar PtoHsfB1 gene in plant growth, and belongs to the technical field of gene engineering. The function of promoting plant growth of the populus tomentosa gene PtoHsfB1 is found for the first time. Experimental results show that the expression quantity of the PtoHsfB1 gene in the transgenic populus tomentosa is greatly improved compared with that of non-transgenic populus tomentosa, the height, dry weight and fresh weight of a transgenic plant are higher than those of a non-transgenic plant, ABA synthesis and signal transduction can be reduced through overexpression of the gene PtoHsfB1, growth and development of the populus tomentosa are promoted, and the yield of the populus tomentosa is increased. Therefore, a new genetic resource is provided for improvement and molecular breeding of the poplar and other woody plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of the PtoHsfB1 gene in enhancing the plant height and biomass of poplar trees. Background Art

[0002] Populus tomentosa, belonging to the family Salicaceae Mirb. and the genus Populus, is an important native tree species widely distributed in most parts of China. In addition, Populus tomentosa is also the largest planted forest tree species in northern China. Its characteristics include rapid growth, thick and straight trunk, strong environmental adaptability, and long lifespan (the lifespan of Populus tomentosa can exceed 500 years). Based on these characteristics, Populus tomentosa has many uses, such as wood production, papermaking and plywood manufacturing, ecological restoration, soil erosion control, etc. As an important industrial raw material forest, the growth rate and biomass of forest trees are the key factors determining their economic benefits.

[0003] Abscisic acid (ABA) is a plant hormone with a sesquiterpene structure that inhibits the elongation of plant height and roots and promotes leaf abscission. Its synthesis pathway is mainly regulated by NCED, ZEP1, ABA2, and AAO, and the main process of the signal transduction pathway is ABA→PYR / PYL→PP2C→SnRK2→AREB / ABF. Inhibiting the production of ABA will be beneficial to promoting plant growth. Therefore, in genetic engineering, the understanding of promoting poplar growth can be improved by inhibiting the formation and signal transduction of abscisic acid, and further improve the biomass of poplar.

[0004] The Hsf transcription factor family is the heat shock transcription factor family, which is ubiquitous in eukaryotic cells such as fungi, plants, and animals. The Hsf transcription factor family includes three subfamilies, namely the A subfamily, the B subfamily, and the C subfamily. There have been many studies on the HSF transcription factor family under heat stress, and most of the studies focus on the HsfA subfamily. The research on the B and C subfamilies is relatively weak. The A subfamily often acts as a transcriptional activator to play a positive regulatory role under heat stress. Except for HsfB5 in the HsfB subfamily, all B-class Hsfs feature the core tetrapeptide "-LFGV-" including a repression domain in the C-terminal domain, and often act as repression motifs by interacting with hitherto unknown corepressors in the transcriptional machinery. There are few studies on the regulation of plant growth and development by the HsfB subfamily. Studies have shown that Arabidopsis heat shock transcription factors HsfB1 and HsfB2b act as inhibitors of heat-induced Hsfs expression. Transgenic seedlings overexpressing HsfB1 are much smaller than the seedlings of hsfb1 mutants and wild-type plants. Pro35S:HsfB1 plants have small rosettes, small and wrinkled leaves, and bent roots, but positively regulate acquired thermotolerance. Studies in grapes have shown that the thermotolerance of plants overexpressing HsfB1 is positively correlated with the HsfB1 expression level. In addition, overexpression of Hsf4 (PagHsfB1) in Populus alba × Populus glandulosa significantly inhibits plant growth. Overexpression of the heat shock transcription factor HsfB1 gene inhibits the growth of Arabidopsis and Populus alba × Populus glandulosa, but there is a lack of specific research on the functional consistency of HsfB1 in different plants. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of the PtoHsfB1 gene in increasing plant height and plant biomass. The PtoHsfB1 gene can inhibit ABA production and signal transduction, thereby promoting plant growth.

[0006] The purpose of the present invention is also to provide a method for cultivating transgenic poplars that promote growth, so as to enhance the plant height and biomass of poplars, and provide new genetic resources for the molecular breeding of fast-growing timber forest varieties.

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

[0008] The present invention also provides the application of the PtoHsfB1 gene in poplar growth. The CDS coding sequence of the PtoHsfB1 gene is shown in SEQ ID NO.4.

[0009] The present invention also provides the application of the PtoHsfB1 gene in poplar growth. The amino acid sequence of the PtoHsfB1 protein is shown in SEQ ID NO.5.

[0010] Preferably, under normal growth conditions, overexpression of the PtoHsfB1 gene increases the plant height.

[0011] Preferably, the PtoHsfB1 gene can increase the fresh weight of plants under normal growth conditions.

[0012] Preferably, the PtoHsfB1 gene can increase the dry weight of plants under normal growth conditions.

[0013] Preferably, under normal growth conditions, the PtoHsfB1 gene can down-regulate the ABA synthesis-related genes ZEP1, NCED, ABA2, and AAO.

[0014] Preferably, under normal growth conditions, the PtoHsfB1 gene can down-regulate the ABA signal transduction-related genes PYR / PYL and AREB / ABF.

[0015] The present invention also provides a method for cultivating transgenic poplar trees that promote the growth of Populus tomentosa, including: transforming a biological material containing the PtoHsfB1 gene into a recipient Populus tomentosa material by the leaf disc method, and cultivating and screening poplar trees with overexpression of PtoHsfB1 for growth promotion. The CDS coding sequence of the PtoHsfB1 gene is shown in SEQ ID NO.4;

[0016] The biological material is any one of the following: A1) a recombinant expression vector containing the PtoHsfB1 gene; A2) a recombinant bacterium containing the PtoHsfB1 gene; A3) a recombinant bacterium containing the recombinant expression vector described in A1); A4) transgenic plant cells and transgenic plant tissues containing the gene; A5) transgenic plant cells or transgenic plant tissues containing the recombinant vector described in A1).

[0017] Compared with the prior art, the present invention discloses the following beneficial effects:

[0018] The present invention first discovers that the Populus tomentosa gene PtoHsfB1 has the function of promoting growth. Previous studies have shown that the HsfB1 gene in Arabidopsis thaliana and Populus alba × Populus glandulosa inhibits plant growth, and there is no relevant report on the consistency of the HsfB1 gene in Populus tomentosa with the previous research results.

[0019] The PtoHsfB1 (Potom07G0038500) gene was cloned from Populus tomentosa in this invention, enriching the research on Hsf transcription factor family genes in poplars. By constructing the overexpression vector pBI121-PtoHsfB1-GFP and transforming poplars, PtoHsfB1 overexpression lines were obtained, and research was carried out from the aspect of obtaining the function of PtoHsfB1, verifying the role of this gene in regulating plant growth, providing raw materials for the functional research of PtoHsfB1 in regulating plant growth rate, and having good application value. By conducting multiple subcultures on PtoHsfB1 overexpression lines and observing the phenotypes of poplar seedlings at one-month and three-month seedling ages, it is of great significance for clarifying the biological function of the PtoHsfB1 gene.

[0020] The discovery of the special gene PtoHsfB1 that controls the fast growth of poplars provides important theoretical guiding significance for exploring and identifying plant fast growth and deeply discussing the molecular mechanism of genes increasing plant biomass; it provides new genetic resources for forest tree molecular breeding, genetic improvement, and timber forest breeding. Brief Description of the Drawings

[0021] Figure 1 It is the amino acid sequence alignment result of the PtoHsfB1 (Potom07G0038500) gene and the known PtrHsfB1 gene of Populus trichocarpa;

[0022] Figure 2 It is a schematic diagram of the construction process of the pBI121-HsfB1-GFP vector; among them, A is the structural schematic diagram of the pBI121-GFP vector, and B is the fusion cloning method; 35S: cauliflower mosaic virus 35S promoter; GFP is the green fluorescent tag; NOS: terminator;

[0023] Figure 3 It is the flow chart of Agrobacterium-mediated genetic transformation of Populus tomentosa leaf discs;

[0024] Figure 4 It is the identification and expression level analysis results of PtoHsfB1 overexpression transgenic poplar positive plants; among them, A is the identification result of PtoHsfB1 overexpression transgenic poplar positive plants, and the target band size is 855bp; B is the detection result of the PtoHsfB1 expression level in PtoHsfB1 overexpression transgenic poplar plants;

[0025] Figure 5Plant height results of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants; A is the phenotype of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants one month after transplantation into the soil, and the scale bar is 5 cm; B is the phenotype of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants three months after transplantation into the soil

[0026] Figure 6 Plant height of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants. A is the plant height of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants one month after transplantation into the soil; B is the plant height of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants three months after transplantation into the soil.

[0027] Figure 7 Fresh weight of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants. A is the fresh weight of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants one month after transplantation into the soil; B is the fresh weight of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants three months after transplantation into the soil.

[0028] Figure 8 Dry weight of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants. A is the dry weight of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants one month after transplantation into the soil; B is the dry weight of wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants three months after transplantation into the soil.

[0029] Figure 9 Expression levels of genes related to the ABA synthesis pathway in wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants.

[0030] Figure 10 Expression levels of genes related to the ABA signal transduction pathway in wild-type Populus tomentosa (WT) and PtoHsfB1 (OE) overexpressing plants. Detailed implementation manners

[0031] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0036] Example 1 Extraction, cloning and sequencing of the PtoHsfB1 (Potom07G0038500) gene

[0037] (1) Populus tomentosa 'Yixian Female Clone' (College of Biological Sciences and Biotechnology, Beijing Forestry University) was used as the experimental material. Total RNA of Populus tomentosa leaves was extracted using the Total RNA Isolation Kit (purchased from Vazyme, China). The determination of nucleic acid concentration was carried out on an IMPLEN Nano Photometer-N50 series ultra-micro ultraviolet spectrophotometer (Germany). RNA with an RNA260 / 280 ratio between 1.9 and 2.1, a 260 / 230 ratio greater than 2.0, and a concentration greater than 500 ng / mL was used for the next analysis. cDNA synthesis was performed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (purchased from Vazyme, China). Using 1 μg of total RNA as a template, it was mixed with 4 μL of 4×gDNA wiper Mix and DEPC-water to a total volume of 16 μL, incubated at 42°C for 2 min, and then quickly cooled on ice for 2 - 3 min; then 4 μL of 5×Hiscript II qRT Super Mix II was added and mixed to a total volume of 20 μL; then incubated at 50°C for 15 min and 85°C for 5 sec. After diluting each cDNA to 200 μL, it was stored at -20°C for later use.

[0038] The cloning primers PtoHsfB1-F and PtoHsfB1-R were used to amplify the target band. Amplification was carried out using the KOD OneTM PCR Master Mix (TOYOBO). The PCR reaction conditions were: denaturation at 98°C for 10 s; annealing at 60°C for 5 s, extension at 68°C for 10 s, for 35 cycles, and stored at 4°C. Then the PCR product was cloned into the pBI121 vector. The recovery of the target fragment was carried out according to the Axygen DNA Gel Extraction Kit (Corning Life Sciences Co., Ltd.). The ligation system of the target fragment and the linearized pBI121 vector was: 1 μL of the target fragment, 4 μL of the linearized pBI121 vector, 5 μL of 2×CE Mix (purchased from Vazyme, China), and ligated at 50°C for 30 min. The ligation product was transformed into DH5α competent cells by heat shock. The bacterial solution was spread on an LB solid plate containing 50 mg / L Kan antibiotic. After growing for about 10 - 12 h, single colonies were selected for colony PCR, and the primers were 35S and PtoHsfB1-R. The positive colonies were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0039] The primer information is as follows:

[0040] PtoHsfB1-F: 5'-GAGAACACGGGGGACTCTAGAATGGCGCAGAGGTCAGCTC-3'

[0041] (SEQ ID NO.1);

[0042] PtoHsfB1-R: 5'-GGACTGACCACCCGGGGATCCATTACAGATCTTGATTTCCTTTGCAC-3‘

[0043] (SEQ ID NO.2);

[0044] 35S: 5’-GACGCACAATCCCACTATCC-3’ (SEQ ID NO.3).

[0045] (2) PtoHsfB1 gene sequence analysis

[0046] After sequencing, it was found that the full length of the nucleotide sequence amplified by this gene was 855 bp (SEQ ID NO.4). Using the DNAMAN software for amino acid sequence alignment, it was found that this target band was highly homologous to Populus trichocarpa PtrHsfB1 at the amino acid level (similarity 98.60%), which was consistent with the results of the alignment on NCBI ( Figure 1 ). The amino acid sequence encoded by the PtoHsfB1 gene is shown in SEQ ID NO.5.

[0047] SEQ ID NO.4:

[0048] ATGGCGCAGAGGTCAGCTCCGGCGCCGTTTTTGACGAAGACCTACCAGCTGGTGGATGATCCAAGCACTGATGATGTGATATCATGGAATGAAACCGGCACAACCTTTGTTGTCTGGAAAACTGCTGATTTTGCCAAGGATTTGCTTCCTAATTATTTCAAACACAACAACTTCTCTAGCTTTGTTCGCCAGCTCAATACCTATGGCTTTAGAAAAATCGTGCCGGACAAATGGGAATTCGCCAACGAGAATTTCCGGCGAGGACAGAAAGAGCTCCTCGCTGAGATACGCCGCCGAAAGACAGCGGCTCCGTCTCCGACAACCCAAACCTCGCCAGCCGGAAAATCCGGAGGCGCATCCTCTTCGTCCAACTCCGGAGAGGACCTGGGGTCCACGTCAACGTCATCACCGGATTCGAAGAACCCGGGGTCTGTCGAAACTGCGGCGACACAAGTCGCCGACTTATCGATCGAGAACGAGCAGTTGAAGAAAGACAACGACGTCCTGAGCAGTGAACTCGAGCAAGCTAAGAAACAATGTGGGGAGTTGATTAATTTTTTGACGGAGTATGTTAAGGTGAGCCCTGATCAAATCAATCGCATCATTGGATGTGGTGGGTCCACCTGTAATGGTGAGGCCGATGCTGGCGATAATCAAAGTGAAGATGATGATGATGAAAATACGAGTGATGACACTGATCATGATGATACCGATCATGATGGTGACGGTGGGGAAGGGGGGGGCTTGAAATTGTTTGGGGTTTTGTTGAAGTGTCAAAACAAGAAAAAGAGGGGCCGTGATGAGAAAATGGGGCTCGGGGGGCCCCGTGCAAAGGAAATCAAGATCTGTAATTGA

[0049] SEQ ID NO.5:

[0050] MAQRSAPAPFLTKTYQLVDDPSTDDVISWNETGTTFVVWKTADFAKDLLPNYFKHNNFSSFVRQLNTYGFRKIVPDKWEFANENFRRGQKELLAEIRRRKTAAPSPTTQTSPAGKSGGASSSSNSGEDLGSTSTSSPDSKNPGSVETAATQVADLSIENEQLKKDNDVLSSELEQAKKQCGELINFLTEYVKVSPDQINRIIGCGGSTCNGEADAGDNQSEDDDDENTSDDTDHDDTDHDGDGGEGGGLKLFGVLLKCQNKKKRGRDEKMGLGGPRAKEIKICN

[0051] Example 2 Obtaining of Poplar Transgenic Plants Overexpressing Pto HsfB1 (Potom07g0038500)

[0052] (1) Construction of Plant Overexpression Vector

[0053] Design primers for PtoHsfB1, adding homologous arms with BamHl and XbaI restriction sites at the front and back of the primers respectively. Double digest the pBI121-GFP vector with BamHI and XbaI. The digestion system is as follows: pBI121 (100 ng / μL) 10 μL, 10X FastDigest 2 μL, BamHl 1 μL, XbaI 1 μL, supplemented with sterile water to a final volume of 20 μL, and digest at 37 °C for 50 min. After 1% agarose gel electrophoresis, recover the single linearized vector fragment. Use 2×CE Mix enzyme (Vazyme) to ligate the PCR product obtained above into the pBI121-GFP linearized vector, transform it into Escherichia coli competent DH5α, screen positive clones and identify them by sequencing, so as to obtain the Populus tomentosa PtoHsfB1 transgenic overexpression vector, named pBI121-PtoHsfB1-GFP. The construction process is as Figure 2 shown. This vector contains the gene sequence resistant to kanamycin, and the promoter for overexpression of PtoHsfB1 is the CAMV 35S promoter. Extract the plasmid from the correctly sequenced Escherichia coli, transform it into Agrobacterium tumefaciens GV3101, and then conduct positive identification. The identified positive Agrobacterium strains are streaked on plates for preservation. The method for preparing Agrobacterium plates is: 10 g / L tryptone + 5 g / L yeast extract + 5 g / L NaCl + 50 mg / L kanamycin + 50 mg / L rifampicin + 15 g / L agar.

[0054] (2) Genetic Transformation of Populus tomentosa

[0055] The pBI121-PtoHsfB1-GFP fusion vector was transformed into Populus tomentosa by Agrobacterium-mediated leaf disc infection method, and adventitious buds were screened. The specific steps are as follows (the transformation process is shown in Figure 3 ):

[0056] a. Culture of Populus tomentosa tissue culture seedlings and pre-culture of Populus tomentosa leaves. The culture medium for Populus tomentosa tissue culture seedlings is 1 / 2MS medium + 0.4 mg / L IBA + 20 g / L sucrose + 7 g / L agar (pH 5.8 - 6.2). After the shoot tips of Populus tomentosa are cultured in the rooting medium for one month, select healthy Populus tomentosa tissue culture seedlings, and cut the fully expanded leaves at the 3-5 leaf positions as the materials for pre-culture of Populus tomentosa leaves. The culture medium for pre-culture of Populus tomentosa leaves is MS + 0.2 mg / L 6-BA + 0.1 mg / L NAA + 0.01 mg / L TDZ + 20 g / L sucrose + 7 g / L agar (pH 5.8 - 6.2). Cut off the leaf base and leaf tip of the cut Populus tomentosa leaves with a sterile blade, and make 3-5 cuts perpendicular to the main vein. Place the cut leaves on the culture medium for pre-culture of Populus tomentosa leaves and culture them under light for 2 - 3 days. The culture temperature is 25°C, the light intensity is 12000 lux, the relative humidity is 70%, and the light cycle is 16 h light / 8 h dark.

[0057] b. Activation of Agrobacterium tumefaciens. Re-streak and activate the Agrobacterium tumefaciens stored on the plate. Pick a single colony and inoculate it into LB medium (50 mg / L kanamycin and 50 mg / L rifampicin), and culture it at 28°C and 200 rpm until the OD600 is about 0.5 - 0.7, then add 80 mM / L AS.

[0058] c. Infection. Put the pre-cultured leaves into a conical flask containing Agrobacterium tumefaciens solution and infect for 15 min, shake it every 2 min, take out the leaves after 15 min, and blot the surface bacterial solution with sterile filter paper.

[0059] d. Co-culture. Place the Populus tomentosa leaves blotted with bacterial solution on the co-culture medium MS + 0.2 mg / L 6-BA + 0.1 mg / L NAA + 0.01 mg / L TDZ + 20 g / L sucrose + 7 g / L agar (pH 5.8 - 6.2), and culture them in the dark at 25°C for 2 - 3 days.

[0060] e. Differentiation and screening: After 2 - 3 days of dark culture of the infected leaves, blot the bacterial solution on the leaf surface. Transfer them to the differentiation medium (MS + 0.5 mg / L 6-BA + 0.002 mg / L TDZ + 0.1 mg / L NAA) containing 50 mg / L kanamycin and 300 mg / L ticarcillin, and culture them at 25°C under the light cycle of 16 h / 8 h. Replace the fresh differentiation medium every 10 days.

[0061] f. Rooting screening: When the resistant buds of the leaves in the differentiation screening medium grow to about 2 cm, cut the resistant buds with a sterile blade and transfer them to the rooting medium containing 50 mg / L kanamycin and 200 mg / L cefotaxime (1 / 2 MS medium + 0.4 mg / L IBA + 20 g / L sucrose + 7 g / L agar (pH 5.8 - 6.2)) and wait for rooting.

[0062] (3) Identification of transgenic plants

[0063] Take the young leaves of the transformed plants, extract DNA by the conventional CTAB method, and identify positive seedlings by PCR amplification of the Kan gene on the vector. The primers used are:

[0064] 35S: 5’-GACGCACAATCCCACTATCC-3’ (SEQ ID NO.3)

[0065] PtoHsfB1-R: 5'-GGACTGACCACCCGGGGATCCATTACAGATCTTGATTTCCTTTGCAC (SEQ ID NO.2);

[0066] The negative control is the non-transgenic wild-type plant (WT), and the positive control is the pBI121-PtoHsfB1-GFP plasmid.

[0067] The specific steps of the conventional CTAB method are as follows: Take the leaves of young Populus tomentosa tissue culture seedlings with a length of 1 - 2 cm, place them in a pre-cooled mortar, add liquid nitrogen 2 - 3 times and grind thoroughly into fine powder, transfer to a 2 mL centrifuge tube, and add 1000 μL CTAB solution. Incubate in a metal bath at 65 °C for 30 min, gently shake once every 10 min, cool to room temperature for 2 min, then add 1000 μL chloroform and mix well by shaking. Centrifuge at 10000 rpm - 12000 rpm at room temperature for 10 min. Pipette about 600 μL of the supernatant into a new centrifuge tube, add 2 volumes of absolute ethanol, slowly shake up and down for 30 s, centrifuge at 10000 rpm - 12000 rpm for 10 min, wash the precipitate with 500 μL of 75% ethanol, centrifuge at 10000 rpm for 5 min, repeat the 75% ethanol wash once, dry at 45 °C, and dissolve with 30 - 50 μL ddH2O. The extracted Populus tomentosa genomic DNA is detected for concentration using a Nanodrop micro nucleic acid detector.

[0068] The detection results are as Figure 4 shown in A below; for both of the 2 transformed plants numbered 1 and 8, electrophoretic bands of the expected size (855 bp) can be amplified, while the wild-type control has no electrophoretic band, indicating that the Populus tomentosa genome already contains exogenous gene DNA fragments. Add OE in front of the numbers of the identified transgenic positive plants.

[0069] (4) qRT-PCR Identification of Transgenic Populus tomentosa Overexpressing PtoHsfB1

[0070] a. Extraction of Genomic RNA from Populus tomentosa Leaves

[0071] Use the TransZol up plus RNA kit (ER501-01, TransGen Biotech) to extract the total RNA from the leaves of WT and the positive PtoHsfB1 overexpression lines identified in step (3). Then use the IIQ RT SuperMix for qRNA (+gDNAwiper) kit (Vazyme Biotech Co., Ltd., China) to reverse transcribe the RNA into cDNA.

[0072] b. Real-Time Fluorescent Quantitative PCR

[0073] To determine whether PtoHsfB1 is overexpressed in Populus tomentosa, use the above-extracted cDNA as a template and analyze the transgenic plants identified in step (3) by real-time fluorescent quantitative PCR (qRT-PCR). Use the SYBRGreen I dye method (AG11701, Accurate Biotechnology (Hunan) Co., Ltd.), and use the Populus tomentosa housekeeping gene PtoActin as an internal standard gene. Its primers are synthesized by Shanghai Sangon Biotech Co., Ltd. The qRT-PCR primers for PtoActin (LOC106418315) are: PtoActin-F: 5’-CTCCATCATGAAATGCGATG-3’ (SEQ ID NO.6);

[0074] PtoActin-R: 7-R1: 5’-AGCCGTCTCCAGCTCTTGC-3’ (SEQ ID NO.7).

[0075] The qRT-PCR primers for the PtoHsfB1 gene are:

[0076] PtoHsfB1-F: 5’-GCATCCTCTTCGTCCAACT-3’ (SEQ ID NO.8);

[0077] PtoHsfB1-R: 5’-TCACCTTGACATACTCCGTC-3’ (SEQ ID NO.9).

[0078] (This pair of primers is a qRT-PCR specific primer designed based on the nucleotide sequence obtained by sequencing in Example 1)

[0079] PCR program: Pre-denaturation at 95°C for 30 sec in the isothermal segment, then add the cycling segment. The number of cycles is 40 (95°C for 5 sec, 60°C for 34 sec), and then add the melting segment.

[0080] As Figure 4 shown in Figure B of [reference], the expression levels of PtoHsfB1 in OE-1 and OE-8 lines were both significantly higher than those of the wild-type (WT) control. The expression level of the OE-1 line reached more than 150 times that of the wild-type line, and the expression level of the OE-8 line was about 50 times that of the wild-type. This indicates that these two lines are independent transgenic lines overexpressing PtoHsfB1.

[0081] Example 3 Increase in plant height and biomass of transgenic Populus tomentosa overexpressing PtoHsfB1 (Potom07G0038500)

[0082] (1) Measurement of plant height of PtoHsfB1 overexpressing poplar plants

[0083] The plant heights of WT and PtoHsfB1 overexpressing plants in the greenhouse were measured. The measurement method was as follows: when the plants grew for one month, WT and the overexpressing lines OE-1 and OE-8 of PtoHsfB1 were selected, and a tape measure was used to measure the plant height, starting from the base of the plant stem to the highest point of the plant stem tip. The results are as Figure 5 and Figure 6 shown. The average heights of the two overexpressing transgenic lines were extremely significantly higher than those of WT. When the plants grew for three months, WT and the overexpressing line PtoHsfB1 were selected, and the measurement method was the same as that for the one-month-old greenhouse seedlings. The results are as Figure 5 and Figure 6 shown, and the difference in plant height between the overexpressing transgenic lines and WT was more significant.

[0084] (2) Biomass measurement of PtoHsfB1 overexpressing lines

[0085] The biomass of WT and PtoHsfB1 overexpressing plants in the greenhouse was measured. The method for measuring fresh weight was as follows: for the one-month-old and three-month-old greenhouse seedlings of WT and the OE-1 and OE-8 lines of HsfB1, the fresh weight of the above-ground part and the root fresh weight of the plants were weighed with an electronic balance, and the two weight results were added to calculate the total fresh weight. The results are as Figure 7 shown. The average fresh weights of the one-month-old and three-month-old greenhouse seedlings of the two overexpressing transgenic lines were significantly heavier than those of WT. The fresh plant materials with the weighed fresh weight were placed in an oven to dry, and the dry weight of the above-ground part and the root dry weight of the plants were weighed with an electronic balance, and the two weight results were added to calculate the total dry weight. The results are as Figure 8 shown. The average dry weights of the one-month-old and three-month-old greenhouse seedlings of the two overexpressing transgenic lines were significantly heavier than those of WT.

[0086] Expression levels of ABA-related genes in transgenic poplar plants overexpressing PtoHsfB1 (Potom07g0038500)

[0087] (1) Expression levels of genes related to the ABA synthesis pathway

[0088] Transcriptome sequencing was performed on the roots of one-month-old transgenic poplar plants overexpressing PtoHsfB1 and wild-type plants transplanted into the soil. The sequencing results showed that the expression levels of the key genes ZEP1, NCED, ABA2, and AAO for abscisic acid synthesis were significantly inhibited in the transgenic plants. The results are as Figure 9 shown.

[0089] (2) Expression levels of genes related to the ABA transduction pathway

[0090] Transcriptome sequencing was performed on the roots of one-month-old transgenic poplar plants overexpressing PtoHsfB1 and wild-type plants transplanted into the soil. The sequencing results showed that the expression levels of the key genes PYR / PYL and AREB / ABF for abscisic acid signal transduction were significantly inhibited in the transgenic plants. The results are as Figure 10 shown.

[0091] In summary, overexpression of the PtoHsfB1 gene may significantly increase plant height and biomass by inhibiting the synthesis and signal transduction of ABA.

[0092] The above-described embodiments are only used to describe the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of HsfB1 gene in enhancing plant height and biomass, characterized in that, The HsfB1 gene is the Populus tomentosa PtoHsfB1 gene; the CDS coding sequence of the PtoHsfB1 gene is shown in SEQ ID NO.

4. Under normal growth conditions, overexpression of the PtoHsfB1 gene can enhance the plant height and biomass of Populus tomentosa.

2. The application according to claim 1, characterized in that Under normal growth conditions, overexpression of the HsfB1 gene significantly increased the plant height during plant growth.

3. The application according to claim 1, wherein Under normal growth conditions, overexpression of the HsfB1 gene significantly increased the fresh weight and dry weight of the above-ground part of the plant during plant growth.

4. The application according to claim 1, characterized in that Under normal growth conditions, overexpression of the HsfB1 gene significantly increased the fresh weight and dry weight of the roots during plant growth.

5. The application according to claim 1, characterized in that Under normal growth conditions, overexpression of the HsfB1 gene decreased the transcriptional levels of the ABA synthesis genes ZEP1, ABA2 and AAO, reduced the synthesis of ABA, and thus promoted the growth and development of poplar.

6. Application of PtoHsfB1 in regulating the growth and development of poplar, characterized in that, Overexpression of the HsfB1 gene decreased the transcriptional level of the ABA synthesis rate-limiting enzyme gene NCED, reduced the synthesis of ABA, and thus promoted the growth and development of poplar.

7. The application according to claim 1, characterized in that Under normal growth conditions, overexpression of the HsfB1 gene decreased the transcriptional levels of the ABA signal transduction genes PYR / PYL and AREB / ABF, reduced the ABA signal transduction, and thus promoted the growth and development of poplar.

8. Application of the PtoHsfB1 transcription factor in regulating the growth and development of poplar, characterized in that, The PtoHSFB1 protein; the amino acid sequence of the PtoHsfB1 protein is shown in SEQ ID NO.

5. Overexpression of the PtoHsfB1 protein under normal growth conditions can enhance the plant height and biomass of Populus tomentosa.

9. A method for cultivating transgenic poplars that promote the growth of Populus tomentosa, characterized in that, Including: The biological material containing the PtoHsfB1 gene was transformed into the recipient Populus tomentosa material by the leaf disc method, and poplar with PtoHsfB1 overexpression promoting growth was cultivated and screened. The CDS coding sequence of the PtoHsfB1 gene is shown in SEQ ID NO.

4.

10. The biological material is any one of the following: A1) a recombinant expression vector containing the PtoHsfB1 gene; A2) a recombinant bacterium containing the PtoHsfB1 gene; A3) a recombinant bacterium containing the recombinant expression vector described in A1); A4) transgenic plant cells and transgenic plant tissues containing the gene; A5) transgenic plant cells or transgenic plant tissues containing the recombinant vector described in A1).

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