Application of knockout PtoABAPT2 gene in improvement of poplar biomass and method

Abstract

The invention discloses application of a knockout PtoABAPT2 gene in increasing poplar biomass, and belongs to the technical field of plant genetic engineering. It is found for the first time that PtoABAPT2 can regulate and control the plant height, stem diameter, xylem cell layer number and cell wall thickness of the poplar, the PtoABAPT2 is edited through the CRISPR / Cas9 technology, a transgenic plant with the plant height, the stem diameter, the xylem cell number and the cell wall thickness remarkably increased is obtained, the total biomass of the transgenic plant is remarkably increased, and the transgenic plant has the advantages of being high in yield, high in yield and good in application prospect. Therefore, the wood yield and quality are improved, the wood production age limit is shortened, the production cost is reduced, and the wood utilization rate is improved. The invention provides a new gene resource for forest tree gene engineering, lays a foundation for wood genetic improvement and directed molecular breeding of forest trees, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to application of knocking out a PtoABAPT2 gene in increasing poplar biomass, and also to a method for increasing poplar biomass. Background Art

[0002] Wood is the most important renewable resource on earth and the main raw material for industrial products such as pulp, paper, and furniture. Improving the productivity of artificial forests is one of the important ways to achieve the national strategic goals of "carbon peak and carbon neutrality". At present, my country's demand for wood is about 800 million cubic meters, and its external dependence exceeds 50%, making it the world's largest wood importer. With the global ban on logging of natural forests, my country's wood supply security faces severe challenges. Improving the productivity of artificial forests is of great strategic significance to ensuring my country's wood security. Improving wood production and quality through genetic improvement of existing major fast-growing tree species is an important way to solve the contradiction between market supply and demand. Poplar itself, as an economic fast-growing tree species widely distributed around the world, has the characteristics of fast maturity, strong adaptability, wide distribution, many varieties, easy hybridization, easy improvement of genetics, and easy reproduction, so it is widely used in the construction of artificial fast-growing forests. Compared with conventional hybrid breeding methods, molecular technology breeding methods provide a fast and effective way. With the development of genetics, genomics and molecular biology techniques in recent years, poplar has become a model plant for studying wood formation, seasonal changes of perennial plants, growth and development, flowering, sex determination and biological interactions. Therefore, using molecular biology and genetic engineering techniques to increase poplar wood yield has important theoretical significance and application value.

[0003] Wood biomass refers to the total amount of biomass contained in the woody part of a tree, and is an important indicator for assessing forest productivity and carbon storage capacity. The assessment of wood biomass mainly refers to the following important indicators: 1) Plant height: Plant height reflects the growth of trees in the vertical direction. Different tree species have different genetic characteristics, which determine their growth rate and maximum height potential. 2) Stem diameter: It reflects the growth of trees in the horizontal direction. The increase in stem diameter is closely related to the growth rate of trees. There are also differences in the growth rate of stem diameter among different tree species, which is related to their genetic characteristics. 3) Number of xylem layers: Xylem is the main component of the stem of a tree, and the number of xylem layers is closely related to the growth of the tree and the quality of the wood. 4) Cell wall thickness: As an important place for photosynthetic carbon storage in plants, the cell wall is the most important component of the forest carbon pool. The cell wall is the main component of wood and is the main influencing factor of the timber yield of fast-growing forests. The increase in cell wall thickness is usually accompanied by an increase in wood yield. At the same time, the cell wall is the main load-bearing part of wood. The increase in its thickness will improve the mechanical properties of wood, and also help to improve the durability and stability of wood, making it more resistant to erosion and damage from the external environment. Therefore, the thickness of the cell wall plays a decisive role in the structural properties and development and utilization of wood.

[0004] In summary, plant height, stem diameter, number of xylem layers and cell wall thickness are all important indicators for evaluating wood yield and quality. The increase of these indicators means the improvement of wood yield and quality. In practical applications, appropriate methods can be selected to improve the above indicators, thereby increasing the total biomass of forest trees, wood yield and quality.

[0005] S-acylation is an important protein post-translational modification process, which reversibly covalently links an acyl group to a cysteine ​​residue through a thioester bond. Through this post-translational modification process, the properties of the protein are changed. S-acylation plays a role in regulating the transport, stability and activity of target proteins. Through proteomics methods, hundreds of proteins have been identified with S-acylation modifications, indicating that S-acylation is involved in regulating plant development or stress response. The ABAPT family is involved in the regulation of protein de-S-acylation modification. Studies have shown that ABAPT8-mediated de-S-acylation of RIN4 leads to increased cell death in Arabidopsis and tobacco leaves, and ABAPT11 de-S-acylates most BSK family members, thereby integrating BR and SA signals to control plant development. However, to date, there has been no report on the improvement of wood yield by poplar ABAPT family genes. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide an application of knocking out the PtoABAPT2 gene in increasing poplar biomass; a second object of the present invention is to provide a method for increasing poplar wood yield.

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

[0008] 1. Application of knocking out the PtoABAPT2 gene in increasing poplar biomass, the nucleotide sequence of the PtoABAPT2 gene is shown in SEQ ID NO.1.

[0009] Preferably, the knockout of PtoABAPT2 gene is used to improve the growth rate of poplar trees.

[0010] Preferably, the knockout PtoABAPT2 gene is used to increase the stem thickness of poplar trees.

[0011] Preferably, the knockout PtoABAPT2 gene is used to increase the number of xylem layers.

[0012] Preferably, the knockout PtoABAPT2 gene of the present invention is used to increase the thickness of xylem cell walls.

[0013] Preferably, the method for knocking out the PtoABAPT2 gene is to use CRISPR / Cas9 technology.

[0014] Preferably, the knockout of the PtoABAPT2 gene is to transform the CRISPR / Cas9 gene editing vector of the PtoABAPT2 gene into poplar, to obtain transgenic plants with edited mutations of the PtoABAPT2 gene, and the wood yield of the obtained transgenic plants is increased.

[0015] 2. A method for increasing the wood yield of poplar trees, comprising: transforming poplar trees with the CRISPR / Cas9 gene editing vector of the PtoABAPT2 gene to obtain transgenic plants with an edited mutation of the PtoABAPT2 gene.

[0016] Preferably, the method for transforming poplar is mediated by Agrobacterium.

[0017] Preferably, the Agrobacterium of the present invention is Agrobacterium GV3101.

[0018] The beneficial effect of the present invention is that: the present invention uses Populus tomentosa as a material and uses pYLCRISPR / Cas9-DH as a vector skeleton to construct a four-target CRISPR-Cas9 gene knockout vector. After knocking out the PtoABAPT2 gene, a new type of poplar variety with significantly increased plant height, stem diameter, xylem cell number, cell wall thickness and total biomass was successfully created, indicating that the PtoABAPT2 gene is a key regulatory gene for regulating the formation of poplar wood, and has important application value in the fields of forest genetic engineering and asexual forestry. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 1 Schematic diagram for constructing the plant knockout vector for the PtoABAPT2 gene;

[0021] Figure 2 Schematic diagram of DNAMAN alignment for identification of positive lines of PtoABAPT2 gene knockout plants;

[0022] Figure 3 Schematic diagram of the comparison of macroscopic phenotypes and stem thickness between PtoABAPT2 gene knockout plants and WT;

[0023] Figure 4 Microscopic observation and statistics of stem tissue cross-sections of PtoABAPT2 knockout plants. Toluidine blue staining and imaging of the tissue cross-sections of the 10th internode of the stem of WT and PtoABAPT2 knockout plants (objective lens 10×, scale bar 200μm) and statistics of the number of xylem cell layers (n>100, unit: layer);

[0024] Figure 5 Scanning electron microscopy images of xylem cells from the 10th internode of the stem of WT and PtoABAPT2 gene knockout plants after gold spraying (5000×, scale bar 10 μm) and statistics of cell wall thickness (n>100, unit μm). DETAILED DESCRIPTION

[0025] The present invention is 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 implement it, but the embodiments are not intended to limit the present invention.

[0026] Example 1: Construction method of PtoABAPT2 gene knockout plant vector

[0027] The CRISPR-Cas9 knockout vector of PtoABAPT2 was constructed. The coding sequence of PtoABAPT2 is shown in SEQ ID NO.1. Four suitable knockout target sites were determined within the coding region of PtoABAPT2 using the target design website. Then, the T1-T4 target sites were sequentially connected to the Cas9 vector backbone of pYLCRISPR / Cas9-DH to construct a four-target Cas9 knockout vector. The vector map is shown in the attached figure. Figure 1 The specific experimental method is as follows:

[0028] (1) Use the targetDesign website to select appropriate target sites;

[0029] ABAPT5-T1:5'-TGGAAAGAAAGCGAACTTCG-3'(SEQ ID NO.2);

[0030] ABAPT5-T2:5'-TCGCCTTCGCATTAATCTTA-3'(SEQ ID NO.3);

[0031] ABAPT5-T3:5'-AACCAGTAAGTCCTTTGAC-3'(SEQ ID NO.4);

[0032] ABAPT5-T4:5'-GTCAAAAGGACTTACTGGTT-3'(SEQ ID NO.5);

[0033] (2) The position:

[0034] B1':5'-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3'(SEQ ID NO.6);

[0035] B2:5'-AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3'(SEQ ID NO.7);

[0036] B2':5'-TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG-3'(SEQ ID NO.8);

[0037] B3:5'-AGCGTGggtctcGtcttGGTCCATCCACTCCAAGCTC-3'(SEQ ID NO.9);

[0038] B3':5'-TTCAGAggtctcTaagaCACTGGAATCGGCAGCAAAGG-3'(SEQ ID NO.10);

[0039] B4:5'-AGCGTGggtctcGagtcGGTCCATCCACTCCAAGCTC-3'(SEQ ID NO.11);

[0040] B4':5'-TTCAGAggtctcTgactCACTGGAATCGGCAGCAAAGG-3'(SEQ ID NO.12);

[0041] BL: 5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3' (SEQ ID NO. 13);

[0042] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID NO. 14);

[0043] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID NO. 15);

[0044] Anneal the adapter primer to complete double-stranded binding. The reaction conditions are: 95°C for 1 min, 94°C for 30 s, then cycle to 4°C for 30 s, with a decrease of 1°C in each cycle.

[0045] (3) The gRNA expression cassette was constructed by the cutting and ligation method. The cutting and ligation reaction system and reaction conditions are as shown in Tables 1-2:

[0046] Table 1. Cutting and ligating reaction system

[0047]

[0048] Table 2. Cutting and ligating reaction conditions

[0049]

[0050] (4) The first round of nested PCR amplification was performed to improve the efficiency of target junctions. The reaction system and reaction conditions were as shown in Tables 3-4:

[0051] Table 3. First round nested PCR reaction system

[0052]

[0053]

[0054] Table 4. First round nested PCR reaction conditions

[0055]

[0056] (5) In the second round of nested PCR amplification, restriction sites were added to the beginning and end of each gRNA expression cassette. The reaction system and reaction conditions are as shown in Tables 5-6:

[0057] Table 5. Second round nested PCR reaction system

[0058]

[0059] Table 6. Second round nested PCR reaction conditions

[0060]

[0061] (6) Connect each gRNA expression cassette to the pYLCRISPR / Cas9-DH expression vector while cutting and connecting. The reaction system and reaction conditions are as shown in Tables 7 to 8:

[0062] Table 7. Ligation reaction system

[0063]

[0064] Table 8. Ligation reaction temperature

[0065]

[0066] The ligation product after cutting and ligation is the Cas9 double knockout vector with four targets ( Figure 1 ), used to transform Escherichia coli, extract the plasmid of Escherichia coli, and then transform Agrobacterium for subsequent genetic transformation experiments.

[0067] Example 2: Genetic transformation of Populus tomentosa

[0068] The material used for genetic transformation of poplar in this study was wild-type Populus tomentosa. The genetic transformation of Populus tomentosa was carried out by Agrobacterium-mediated leaf disc infection, and the screening marker for transgenic poplar was hygromycin.

[0069] 1) Cultivation of Agrobacterium

[0070] The constructed plant knockout vector PtoABAPT2 was transformed into Agrobacterium GV3101, and then the Agrobacterium GV3101 containing the recombinant plasmid was inoculated into YEP solid culture medium (containing corresponding antibiotics) and inverted for culture at 28°C; a single clone of bacteria was picked and inoculated into YEP liquid culture medium containing corresponding antibiotics, and cultured at 28°C until OD600 was 0.6-0.8; a live bacterial liquid was transferred to fresh YEP culture medium at a ratio of 1:100, cultured at 28°C until OD600 was 0.6-0.8, centrifuged at 4°C, the bacteria were collected, resuspended with 30 mL of WPM liquid culture medium supplemented with acetosyringone, and placed in a shaker at 28°C for shaking culture for 1 h.

[0071] 2) Agrobacterium-mediated leaf disc infection

[0072] Take the sterile leaves of the tissue culture seedlings and cut them into 0.5×0.5cm pieces on the clean bench. 2 Place leaf discs of different sizes into the resuspended Agrobacterium solution and infect for 10 minutes. Gently shake the solution every 2 to 3 minutes to ensure that the leaf discs are fully infected.

[0073] 3) Co-cultivation of Populus tomentosa

[0074] Pick out the infected leaf disc with sterilized tweezers, place it on sterilized filter paper, absorb the bacterial solution, spread the leaf disc flat on WPM co-cultivation medium, and culture it in the dark at 25℃ for 2 days.

[0075] 4) Selective cultivation of Populus tomentosa

[0076] After 2 days of co-cultivation, the transformed explants were transferred to a selective medium capable of inducing callus tissue and cultured at 25°C in the dark for 3 to 5 weeks, during which time the selective medium was replaced every 5 days.

[0077] 5) Populus tomentosa bud induction culture

[0078] When white loose callus tissue appears around the leaf margin, move the callus tissue to WPM budding medium on the clean bench and culture it at 25℃ under light for about 4 to 5 weeks, replacing new WPM budding medium every 10 days.

[0079] 6) Rooting culture of Populus tomentosa

[0080] When the adventitious buds grow to about 5 cm, they are transferred to WPM rooting medium containing corresponding antibiotics to induce rooting.

[0081] 7) Transplantation of Populus tomentosa

[0082] When the root system of the seedlings is relatively developed, take out the seedlings, rinse off the agar on the roots, transplant them into the greenhouse for cultivation, and cover them with plastic wrap to keep them warm and moist.

[0083] WPM resuspension: (WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS);

[0084] WPM co-culture medium: (WPM powder + 2.14 g + 30 g sucrose + 100 μmol AS + 1.0 mg NAA + 2.0 mg ZT);

[0085] WPM selection medium: (WPM powder + 2.14g + 30g sucrose + 9mg Hyg + 1.0mg NAA + 2.0mg ZT + 400mg Cef);

[0086] WPM germination medium: (WPM powder + 2.14g + 30g sucrose + 9mg Hyg + 0.1mg NAA + 2.0mg ZT + 400mg Cef);

[0087] WPM rooting medium: (WPM+30g sucrose+9mg Hyg+0.1mg NAA+400mg Cef);

[0088] YEP medium: 10g yeast extract, 10g peptone, 5g NaCl, pH 7.0, sterilized at 121℃ for 20 minutes. For solid medium, add 10-12g agar powder before sterilization.

[0089] LB medium: 5 g yeast extract, 10 g peptone, 10 g NaCl, pH = 7.0, sterilize at 121°C for 20 min. For solid medium, add 10-12 g agar powder before sterilization.

[0090] Example 3: Identification of transgenic positive seedlings

[0091] DNA was extracted from 30 transgenic seedlings using the CTAB method. The specific steps are as follows:

[0092] 1) Take a 10 mL centrifuge tube, add 3 mL CTAB solution (100 M pH 8.0 Tris-HCl, 20 mM EDTA, 1.4 M sodium chloride, 2% PVP (polyvinyl pyrrolidone), sterilized at 121°C for 20 minutes and stored at room temperature) and 90 μL β-mercaptoethanol (final concentration 3%), and preheat at 65°C;

[0093] 2) Grind about 2.6 g of fresh poplar leaves into powder in liquid nitrogen, transfer to the CTAB extraction solution in step 1), and mix well;

[0094] 3) Incubate in a 65°C water bath for 45 minutes, shaking vigorously every 15 minutes to mix;

[0095] 4) After standing at room temperature for 5 minutes, add an equal volume of chloroform:isoamyl alcohol (24:1) solution, mix vigorously by inversion, and then place flat to emulsify for 10 minutes;

[0096] 5) Centrifuge at 1000 rpm for 10 minutes, pipette the supernatant into a new centrifuge tube, and repeat step 4) once;

[0097] 6) Centrifuge at 1000 rpm for 10 minutes, aspirate the supernatant into a new centrifuge tube, add an equal volume of -20°C pre-cooled isopropanol, gently invert to mix, and centrifuge at 12000 rpm for 10 minutes;

[0098] 7) Pour off the supernatant and wash the precipitate twice with 500 μL 75% ethanol solution;

[0099] 8) Pour off the supernatant as much as possible and dry the precipitate in a 37°C constant temperature oven until it becomes transparent;

[0100] 9) Add 50 μL of sterile water containing RNase to dissolve the precipitate and digest the RNA;

[0101] Then, primers were designed to amplify the DNA fragment containing the PtoABAPT2 knockout target, and the target DNA was recovered using a gel recovery kit. The specific steps are as follows:

[0102] 1) Cut the agarose gel containing the target band and put it into a 2 mL centrifuge tube;

[0103] 2) Add 3 times the volume of gel extraction buffer;

[0104] 3) Place in a 65°C water bath until the gel melts;

[0105] 4) If the target fragment is less than 500 bp, add isopropanol in an equal volume to the gel;

[0106] 5) Transfer the mixed solution to a spin column, centrifuge at 6000 g for 1 minute, and discard the liquid in the tube;

[0107] 6) Add 500 μL Extraction Buffer to the spin column, centrifuge at 12000 g for 1 minute, and discard the liquid in the tube;

[0108] 7) Add 500 μL Wash Buffer to the spin column, centrifuge at 12000 g for 1 minute, and discard the liquid in the tube;

[0109] 8) Repeat step 7) once;

[0110] 9) Centrifuge the empty column at 12000g for 1 minute;

[0111] 10) Transfer the Spin column to a new 1.5 mL centrifuge tube, add 30 μL Elution Buffer, let stand at room temperature for 2 minutes, and centrifuge at 12000 g for 1 minute; then connect the recovered target DNA fragment to the PMD19 vector. The reaction system is shown in Table 9 below:

[0112] Table 9. DNA fragment and PMD19 vector connection system

[0113]

[0114] Reaction conditions: 16°C, 8 hours. The obtained vector was then transformed into E. coli. The specific steps are as follows:

[0115] 1) Mix the prepared E. coli competent cells and the plasmid to be transfected in a ratio of 10:1 and place on ice for 30 minutes;

[0116] 2) Heat shock at 42°C for 90 seconds, immediately place on ice and let stand for 2 minutes;

[0117] 3) Add 800 μL of LB liquid medium without selection pressure, incubate at 37°C, 200 rpm, and recover for 1 hour;

[0118] 4) Centrifuge at 4000 g for 5 minutes;

[0119] 5) Discard the supernatant, keep about 100 μL of culture medium to resuspend the bacteria and apply to the corresponding resistance LB medium, and culture at 37°C. Then use PMD19 universal primers to perform colony PCR to screen positive colonies. The reaction conditions are as shown in Tables 10-11:

[0120] Table 10. PCR reaction system

[0121]

[0122] Table 11. PCR reaction conditions

[0123]

[0124] The selected positive clones were sequenced and sequence comparison was performed using DNAMAN to determine that L7 and L13 were positive transgenic lines. Figure 2 As shown. The tissue culture seedlings were sterile cultured and transplanted for subsequent experiments. The macroscopic phenotypic indicators of the PtoABAPT2 knockout transgenic strains and the wild-type white poplar in the control group were measured. The results showed that the transgenic strains showed obvious advantages in plant height, stem diameter and biomass compared with the wild-type white poplar. Figure 3 shown.

[0125] Example 4: Determination of xylem width and xylem cell layer number

[0126] The tenth internode of the transgenic strains and the wild-type white poplar in the control group was sliced ​​and observed by an oscillating slicer. The development of the xylem was observed by toluidine blue staining. The xylem width and the number of xylem cell layers of the corresponding strains were measured and counted to characterize the development of the xylem. The results showed that compared with the wild-type white poplar, the number of xylem cell layers and the xylem width of the transgenic strains were significantly increased. Figure 4 shown.

[0127] Example 5: Cell wall thickness detection

[0128] The xylem cells of the tenth internode of the transgenic plants and wild-type plants were observed by scanning electron microscopy after gold spraying. The statistical results of the cell wall thickness of the xylem fiber cells of each strain are as follows: Figure 5 As shown, statistics showed that the cell wall thickness of transgenic plants was thicker than that of wild-type plants.

[0129] The above results show that compared with the wild type, the transgenic poplars of the PtoABAPT2 mutant strain are significantly taller and thicker, and the number of xylem cell layers and cell wall thickness are significantly increased, indicating that the PtoABAPT2 gene is a key regulatory gene that regulates the formation of poplar wood, and has important application value in the fields of forest genetic engineering and asexual forestry.

[0130] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on 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 knocking out PtoABAPT2 gene in increasing poplar biomass, Features: The nucleotide sequence of the PtoABAPT2 gene is shown in SEQ ID NO.

1.

2. The use of the knockout PtoABAPT2 gene in increasing poplar biomass according to claim 1, Features: The knockout of PtoABAPT2 gene is used to improve the height of poplar trees.

3. The use of the knockout PtoABAPT2 gene in increasing poplar biomass according to claim 1, Features: The application of the knockout PtoABAPT2 gene in increasing the stem thickness of poplars.

4. The use of the knockout PtoABAPT2 gene in increasing poplar biomass according to claim 1, Features: The application of knocking out the PtoABAPT2 gene in increasing the number of xylem layers.

5. The use of the knockout PtoABAPT2 gene according to claim 1 in increasing poplar biomass, Features: The application of the knockout PtoABAPT2 gene in increasing the thickness of xylem cell walls.

6. The use of the knockout PtoABAPT2 gene according to claim 1 in increasing poplar biomass, Features: The method for knocking out the PtoABAPT2 gene is to use CRISPR / Cas9 technology.

7. The use of the knockout PtoABAPT2 gene according to claim 1 in increasing poplar biomass, Features: The method of knocking out the PtoABAPT2 gene is to transform the CRISPR / Cas9 gene editing vector of the PtoABAPT2 gene into poplar trees to obtain transgenic plants with edited mutations of the PtoABAPT2 gene, and the wood yield of the obtained transgenic plants is increased.

8. Methods for increasing the yield of poplar wood, Features: The specific method is to transform poplar with the CRISPR / Cas9 gene editing vector of the PtoABAPT2 gene to obtain transgenic plants with PtoABAPT2 gene editing mutations.

9. The method according to claim 5, Features: The method for transforming poplar is mediated by Agrobacterium.

10. The method according to claim 6, Features: The Agrobacterium is Agrobacterium GV3101.

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