Application of overexpression of PtoCC1 gene in improving biomass of poplar

Abstract

The application discloses application of overexpression of PtoCC1 gene in biomass increase of poplar and belongs to the technical field of plant genetic engineering. The PtoCC1 gene is introduced into poplar, and the transgenic poplar overexpressing PtoCC1 is obviously higher and thicker compared with the wild type, and the number of xylem cells and biomass are obviously increased, which indicates that the PtoCC1 gene is a key regulation gene for regulating formation of poplar wood and has important application value in the fields of forest genetic engineering and clone forestry.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of overexpression of the PtoCC1 gene in increasing poplar biomass. Background Technology

[0002] Timber is one of the most important renewable resources on Earth and a primary raw material for industries such as pulp and paper manufacturing and furniture making. Improving timber yield and quality through genetic modification of existing fast-growing tree species is a crucial way to address market supply and demand imbalances. Poplar, as a globally widespread economic fast-growing tree species, is characterized by rapid growth, strong adaptability, wide distribution, numerous varieties, ease of hybridization, ease of genetic modification, and ease of propagation, making it widely used in the establishment of artificial fast-growing forests. Compared to conventional hybridization breeding methods, molecular breeding techniques offer a rapid and effective approach. With the development of genetics, genomics, and molecular biology techniques in recent years, poplar has become a model plant for studying wood formation, seasonal variation patterns in perennial plants, growth and development, flowering, sex determination, and biotic interactions. Therefore, utilizing molecular biology and genetic engineering techniques to improve poplar timber yield has significant theoretical and practical value.

[0003] Secondary cell walls are a major component of wood, while cellulose, as a major component of plant cell walls, is the most abundant biopolymer on Earth. Cellulose is synthesized by cell membrane-localized cellulose synthase (CesA) complexes (CSCs), which typically arrange themselves into hexameric rosette structures. Studies have shown that cellulose synthase complexes, guided by cortical microtubules, synthesize cellulose on the plasma membrane using uridine diphosphate glucose (UDP-glucose) as a substrate, through β-1,4 glycosidic bonds linked by dextran chains. These dextran chains then form microfibrils through intramolecular and intermolecular hydrogen bonding. These microfibrils are crucial for cell wall tensile strength, plant development, directed cell growth, and plant height. Studies in the model plant Arabidopsis thaliana have shown that cellulose synthase chaperone 1 (CC1) can interact with CesA proteins and microtubules. Changes in cellulose synthase activity in response to extracellular and intracellular signals are crucial for growth modification under dynamic environmental conditions. Furthermore, in vitro and in vivo analyses have shown that the cytoplasmic portion of the CC protein interacts with microtubules, promoting microtubule formation and dynamics.

[0004] Importantly, cortical microtubules (CSCs) and cortical microtubules are interdependent, as changes in one can affect the activity of the other. Given the close relationship between cortical microtubules and CSCs, previous studies have largely focused on verifying that salt stress affects cell wall synthesis, while damage to cellulose-related components alters salt tolerance. This study, conducted in Arabidopsis thaliana, elucidated the mechanism by which CC1 maintains sustained cellulose synthesis under salt stress by constructing CC1 knockout lines. CCs have been shown to be indispensable for the recovery of cortical microtubule array and CSC activity under salt stress, and microtubule interactions with CC1 are key to this function.

[0005] However, this study has significant limitations. First, it was conducted in Arabidopsis thaliana, lacking research on secondary cell wall cellulose synthesis. Increasing biomass or secondary cell wall cellulose in woody plants, especially fast-growing species, is a pressing issue that needs to be addressed. Second, only knockout lines were constructed; no overexpression of the CC1 gene was observed.

[0006] Therefore, there is an urgent need for a method to overexpress the CC1 gene in woody plants to study the effects of the CC1 gene on secondary development and yield of wood. Summary of the Invention

[0007] Therefore, one of the objectives of this invention is to provide an application of overexpressing the PtoCC1 gene in increasing poplar biomass.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] Application of overexpression of the PtoCC1 gene in increasing poplar biomass, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0010] The present invention preferably describes the application of the overexpression of the PtoCC1 gene in improving the height of poplar trees.

[0011] The present invention preferably describes the application of the overexpression of the PtoCC1 gene in increasing the stem diameter of poplar trees.

[0012] The present invention preferably relates to the application of the overexpression of the PtoCC1 gene in increasing intersegmental length.

[0013] The present invention preferably relates to the application of the overexpression of the PtoCC1 gene in increasing the number of xylem cell layers.

[0014] The present invention preferably relates to the application of the overexpression of the PtoCC1 gene in increasing the thickness of the xylem and phloem cell walls.

[0015] The beneficial effects of this invention are as follows: Using Populus tomentosa as material, the PtoCC1 gene was cloned; simultaneously, an overexpression vector Pcxsn-35S::PtoCC1 was constructed. This gene is located after the promoter P35S. Driven by the promoter P35S, PtoCC1 can be efficiently expressed in poplar trees, thereby regulating the formation of poplar wood. Overexpression of the PtoCC1 gene successfully created a new poplar variety with high cellulose, high biomass, significantly increased number of xylem cell layers and fiber cell wall thickness, indicating that the PtoCC1 gene is a key regulatory gene for regulating the formation of poplar wood and has important application value in the fields of forest tree genetic engineering and clonal forestry. Attached Figure Description

[0016] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0017] Figure 1 A schematic diagram illustrating the construction of a plant overexpression vector for the PtoCC1 gene;

[0018] Figure 2 Macroscopic phenotype and statistical analysis of PtoCC1 overexpression (A: Overall macroscopic phenotype photography of the two positive overexpression lines of WT and PtoCC1; B: Measurement of plant height, relative growth and relative biomass based on marked internodes in A; C: Comparison of internode lengths of 13-16 in A; Comparison of complete cross-sections taken from the lower ends of internodes 8, 12, 16, 20, and 24; D: Stem diameter measured using vernier calipers in the middle sections of internodes 9, 13, 17, 21, and 24).

[0019] Figure 3 Microscopic observation and statistical analysis of cross-sectional sections of stem tissue from PtoCC1-overexpressing plants (A: Cross-sectional sections of tissue from different stem nodes of WT and PtoCC1-overexpressing plants under an optical microscope, stained with toluidine blue (10X objective, 200μm scale bar); B: Statistical analysis of the number of xylem cell layers in different internodes of each line (n>100, unit: layers); C: Xylem fiber cell imaging of cross-sectional sections of tissue from different stem nodes of WT and PtoCC1-overexpressing plants under a scanning electron microscope after gold sputtering (5000X, 10μm scale bar); D: Statistical analysis of cell wall thickness of xylem fiber cells in different internodes of each line (n>100, unit: μm); E: Phloem fiber cell imaging under a scanning electron microscope). Detailed Implementation

[0020] 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 and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0021] Example 1: Construction of a plant overexpression vector for the PtoCC1 gene

[0022] The method for constructing a plant overexpression vector for the PtoCC1 gene is as follows:

[0023] Based on the 960 bp CDS sequence of PtoCC1, as shown in SEQ ID NO.5, the nucleotide sequence was ligated to the Pcxsn-35S vector backbone via homologous recombination, with the BamHI restriction site, resulting in the vector Pcxsn-35S::PtoCC1. The E. coli strain used was DH5α, and the primers are shown below. A schematic diagram of the vector construction is attached. Figure 1 As shown.

[0024] Forward primer: 5'-ACGAACGATACTCGAGGGATGCACGCCAAGACAGACT C-3' (SEQ ID No. 1).

[0025] Reverse primer: 5'-CGGGGAAATTCGCTAGTATCATATGTGCAAGCCTCCTTGAG-3' (SEQ ID No. 2).

[0026] Example 2: Genetic transformation of Populus tomentosa

[0027] In this study, wild-type Populus tomentosa was used for genetic transformation. The genetic transformation of Populus tomentosa was carried out using Agrobacterium-mediated leaf disc infection. Hygromycin was used as the screening marker for transgenic Populus tomentosa.

[0028] 1) Culture of Agrobacterium

[0029] The constructed plant expression vector Pcxsn-35S::PtoCC1 was transformed into Agrobacterium GV3101. Then, Agrobacterium GV3101 containing the recombinant plasmid was inoculated onto YEP solid medium (containing the corresponding antibiotic) and cultured upside down at 28°C. Single clones were picked and inoculated into YEP liquid medium containing the corresponding antibiotic and cultured at 28°C until the OD600 reached 0.6–0.8. One live bacterial culture was transferred to fresh YEP medium at a ratio of 1:100 and cultured at 28°C until the OD600 reached 0.6–0.8. The cells were collected by centrifugation at 4°C, resuspended in 30 mL of WPM liquid medium supplemented with acetylsyleugenol, and cultured in a shaker at 28°C for 1 h.

[0030] 2) Agrobacterium-mediated leaf disc infection

[0031] Take sterile leaves from tissue culture seedlings and cut them into 0.5 × 0.5 cm pieces on a clean bench. 2Place the leaf discs of the desired size into the pre-suspended Agrobacterium bacterial solution and immerse them for 10 minutes. Gently shake the bacterial solution every 2-3 minutes to ensure that the leaf discs are fully immersed.

[0032] 3) Co-cultivation of Populus tomentosa

[0033] The infected leaf discs were removed with sterilized tweezers, placed on sterilized filter paper, and the bacterial solution was blotted dry. The leaf discs were then laid flat on WPM co-culture medium and incubated in the dark at 25°C for 2 days.

[0034] 4) Selection and cultivation of Populus tomentosa

[0035] After co-culturing for 2 days, the transformed explants were transferred to a selective medium that can induce callus and cultured at 25°C in the dark for 3–5 weeks, with the selective medium being replaced approximately every 5 days.

[0036] 5) Populus tomentosa bud induction culture

[0037] When white, loose callus tissue appears around the leaf margin, transfer the callus tissue to WPM budding medium on a clean bench and culture it at 25°C under light for about 4 to 5 weeks, replacing the WPM budding medium every 10 days.

[0038] 6) Rooting culture of Populus tomentosa

[0039] When the adventitious buds grow to about 5 cm, they are transferred to WPM rooting medium containing the corresponding antibiotics to induce rooting.

[0040] 7) Transplanting of Populus tomentosa

[0041] When the seedlings have a well-developed root system, remove them, rinse off the agar from the roots, transplant them into a greenhouse for cultivation, and cover them with plastic wrap to keep them warm and moist.

[0042] WPM resuspension: (WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS);

[0043] WPM co-culture medium: (WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS + 1.0 mg NAA + 2.0 mg ZT);

[0044] WPM selective medium: (WPM powder + 2.14 g + 30 g sucrose + 9 mg Hyg + 1.0 mg NAA + 2.0 mg ZT + 400 mg Cef);

[0045] WPM germination medium: (WPM powder + 2.14 g + 30 g sucrose + 9 mg Hyg + 0.1 mg NAA + 2.0 mg ZT + 400 mg Cef);

[0046] WPM rooting medium: (WPM + 30 g sucrose + 9 mg Hyg + 0.1 mg NAA + 400 mg Cef);

[0047] YEP medium (L): 10 g yeast extract, 10 g peptone, 5 g NaCl, pH 7.0, autoclaved at 121 °C for 20 minutes. For solid media, add 10-12 g agar powder before sterilization.

[0048] LB medium (L): 5 g yeast extract, 10 g peptone, 10 g NaCl, pH = 7.0, autoclaved at 121 ℃ for 20 min. For solid media, add 10-12 g agar powder before sterilization.

[0049] Example 3: Identification and macroscopic phenotypic observation of transgenic positive seedlings

[0050] I. Extraction of total RNA from Populus tomentosa and reverse transcription to synthesize cDNA

[0051] Soak the spatula, mortar, and pestle used for RNA extraction in DEPC water overnight, then autoclave and dry them for later use. Follow the instructions for the Axygen kit for RNA extraction. Store the obtained RNA at -80°C for later use. The specific steps are as follows:

[0052] 1) Quickly wrap the removed fresh plant tissue in aluminum foil and immerse it in liquid nitrogen for flash freezing;

[0053] 2) Grind the sample into powder thoroughly with liquid nitrogen in a mortar and pestle for RNase removal;

[0054] 3) Transfer the powder to AG buffer, shake thoroughly to mix until a homogenate is formed, and let stand at room temperature for 5-10 minutes.

[0055] 4) Centrifuge at 4℃, 12000 rpm / min, for 10 min;

[0056] 5) Transfer the supernatant to a new 1.5 mL EP tube, accurately estimate the volume of the supernatant, add 0.5 times the volume of anhydrous ethanol, and mix well;

[0057] 6) Transfer the mixture to a Spin Clum extraction column and centrifuge at 12000 rpm / min for 1 min;

[0058] 7) Discard the waste liquid in the collection tube, add 500 μL of PG buffer to the column, and centrifuge at 12000 rpm / min for 1 min;

[0059] 8) Discard the waste liquid, add 600 μL of wash buffer to the column, and centrifuge at 12000 rpm for 30 s;

[0060] 9) Repeat step (8) once;

[0061] 10) Discard the waste liquid and centrifuge the empty tube at 12000 rpm / min for 1 min to remove all liquid from the filter membrane;

[0062] 11) Add 30-50 μL of RElution buffer to the center of the membrane, let stand at room temperature for 2 min, centrifuge at 12000 rpm / min for 1 min to obtain total RNA;

[0063] 12) Take 1 μL of RNA sample and run it on agarose gel electrophoresis. Observe the integrity of the RNA band at 28 s and 18 s to detect the extraction quality.

[0064] cDNA synthesis follows PrimeScript TM The procedure was performed according to the instructions of the RT reagent Kit with gDNA Eraser (Takara), and the steps are as follows:

[0065] Genomic DNA erasure:

[0066] (1) Add 1-7 μL of RNA sample (total amount not exceeding 1 μg) according to the RNA concentration.

[0067] (2) Add 2 μL of 5× DNA digestion enzyme buffer and 1 μL of DNA digestion enzyme;

[0068] (3) Add RNase ddH2O to bring the total volume to 10 μL;

[0069] (4) The reaction conditions are 42℃, 2 min 30 s.

[0070] First-strand cDNA synthesis:

[0071] Add 4 μL of reverse transcription buffer, 1 μL of Oligo dT random primer, 1 μL of reverse transcriptase, and 4 μL of RNase-free water to the first step reaction system, making the final reaction volume 20 μL.

[0072] The reaction conditions were: 37℃, 15 min reverse transcription, -85℃, 5 s reaction termination, -4℃, 5 min cooling and storage.

[0073] II. Design of target gene-specific primers and PCR amplification to obtain the target fragment

[0074] To screen for overexpressing transgenic lines, quantitative primers were designed using quantitative real-time PCR (qPCR) technology, and specific cDNA sequences in the test samples were quantitatively analyzed using internal controls.

[0075] Forward primer: 5'-GGGTGCTAGTAAGCCACAAA-3' (SEQ ID No. 3);

[0076] Reverse primer: 5'-TCATATCAGTTGCCACTCCC-3' (SEQ ID No. 4).

[0077] After identification, the two positive overexpression lines of WT and PtoCC1 were propagated and cultured. Healthy tissue culture seedlings were then selected and hardened off together with wild-type WT seedlings of the same age for about two weeks. Internodes were then marked, and the seedlings were cultured in a greenhouse at 25℃ under long-day conditions (16 hours light / 8 hours darkness, light intensity 10000 lux) for 2-3 months. Macroscopic phenotypic observation and sampling were performed, and the results are as follows: Figure 2 As shown in Figure A. Then, the height of each plant, relative growth based on marked internodes, and relative biomass were measured. The results are shown in Figure A. Figure 2 As shown in Figure B, the lengths of internodes 13-16 of each plant were compared; complete cross-sections of the lower ends of internodes 8, 12, 16, 20, and 24 were also compared, and the results are shown below. Figure 2 As shown in C. The stem diameter was measured using vernier calipers at the midpoint of internodes 9 / 13 / 17 / 21 / 24, as shown below. Figure 2 As shown in Figure D. The above results indicate that the overexpression line is taller than the wild type, and the 13-16 internodes of the overexpression plants are longer, with larger cross sections and stem diameters than the wild type, suggesting that PtoCC1 can promote the growth of Populus tomentosa.

[0078] Example 4: Microscopic observation and statistical analysis of cross-sectional sections of stem tissue from PtoCC1-overexpressing plants.

[0079] The results of imaging of cross-sectional tissue sections from different stem nodes of WT and PtoCC1 overexpressing plants under an optical microscope after toluidine blue staining are as follows: Figure 3 As shown in Figure A. The statistical analysis of the number of xylem cell layers in different internodes of each strain is shown below. Figure 3 As shown in Figure B. Cross-sectional tissue sections of different stem nodes from WT and PtoCC1-overexpressing plants, imaged with xylem fiber cells after gold sputtering under a scanning electron microscope. Figure 3 As shown in Figure C. The statistical results of cell wall thickness of xylem fiber cells in different internodes of various lines are shown in Figure C. Figure 3 As shown in Figure D. Scanning electron microscopy imaging of phloem fibroblasts is shown below. Figure 3 As shown in E.

[0080] The results showed that transgenic poplar trees overexpressing PtoCC1 were significantly taller and thicker than wild-type trees, with a significant increase in the number of xylem cell layers and cell wall thickness. This indicates that the PtoCC1 gene is a key regulatory gene that controls the formation of poplar wood and has important application value in the fields of forest tree genetic engineering and clonal forestry.

[0081] The above-described embodiments are merely preferred embodiments provided to fully illustrate 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 all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The use of overexpression of PtoCC1 gene in increasing the biomass of poplar, characterized in that: The nucleotide sequence of the PtoCC1 gene is shown as SEQ ID NO.

5.

2. The use of overexpression of PtoCC1 gene to improve the biomass of poplar according to claim 1, characterized in that: Application of the overexpressed PtoCC1 gene in increasing the height of poplar.

3. The use of overexpression of PtoCC1 gene to improve the biomass of poplar according to claim 1, characterized in that: Application of the overexpressed PtoCC1 gene in increasing the stem diameter of poplar.

4. The use of overexpression of PtoCC1 gene to improve the biomass of poplar according to claim 1, characterized in that: Application of the overexpressed PtoCC1 gene in increasing the internode length.

5. The use of overexpression of PtoCC1 gene to improve the biomass of poplar according to claim 1, characterized in that: Application of the overexpressed PtoCC1 gene in increasing the number of xylem layers.

6. The use of overexpression of PtoCC1 gene to improve the biomass of poplar according to claim 1, characterized in that: Application of the overexpressed PtoCC1 gene in increasing the thickness of xylem and phloem cell walls.

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