Application of PtoESK1 gene in improving mechanical strength and wood yield of Populus tomentosa

Abstract

The present invention discloses the application of the PtoESK1 gene in improving the mechanical strength and wood yield of white poplar. The PtoESK1 gene is introduced into white poplar plants by genetic engineering methods to obtain white poplars with improved mechanical strength and wood yield. The mechanical strength of white poplars with the PtoESK1 gene is increased by more than 23%, and the fresh weight and dry weight yields of wood are increased by more than 76% and 110%. The present invention is of great significance for improving the strength and yield of poplar wood, provides new gene resources for plant genetic engineering, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of a PtoESK1 gene in improving the mechanical strength and wood yield of white poplar. Background Art

[0002] Poplar (Populus spp.) is the most widely planted fast-growing tree species in the world. It is an important raw material for papermaking and construction, and is also the main raw material for the bioenergy industry (Wang et al., 2012). However, in the development and utilization of poplars, although poplars grow rapidly and have a short growth cycle, their mechanical strength is low, which limits the use of poplar wood. In addition, there are still certain gaps in the utilization of wood, and the dependence on foreign wood exceeds 50%. If the output of poplars, a high-quality forest resource, can be increased, the current shortage of wood can be alleviated. Therefore, improving the mechanical strength and wood yield of poplars is of great help to the utilization of wood resources.

[0003] The mechanical strength of wood is closely related to the structure and function of plant cell walls. Plant cell walls are complex polymer networks composed of cellulose, hemicellulose, lignin, etc., which play an important role in the growth and development of plants (Somerville C et al., 2004). For plant growth, the cell wall needs to have a certain elasticity and hardness to resist the internal turgor pressure and the pressure of the external environment. The biomechanical properties of the cell wall are related to the supramolecular assembly between cellulose, hemicellulose and lignin (Jennie Berglund et al., 2020). Studies have found that hemicellulose, as a connecting bridge between cellulose and lignin, has different acetyl substitution patterns, and its acetyl modification is extremely critical to the formation of the advanced structure of the cell wall. The acetylation modification of hemicellulose polysaccharides requires the participation of many genes, including acetyltransferases and acetylesterases. Therefore, by studying the acetylation modification mechanism of poplar hemicellulose, revealing the functions of related genes, and improving the cell wall acetyl level through genetic manipulation to change the growth and development of plants. This provides genetic resources for improving and cultivating new high-quality germplasm with fast growth, high wood yield and high mechanical strength. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide an application of a PtoESK1 gene in improving the mechanical strength and / or wood yield of Populus tomentosa; a second object of the present invention is to provide a method for improving the mechanical strength and / or wood yield of Populus tomentosa.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. Application of the PtoESK1 gene in improving the mechanical strength and / or wood yield of Populus tomentosa, the sequence of the PtoESK1 gene being shown in SEQ ID NO.1.

[0007] 2. Use of a recombinant vector containing a PtoESK1 gene in improving the mechanical strength and / or wood yield of Populus tomentosa, wherein the sequence of the PtoESK1 gene is shown in SEQ ID NO.1.

[0008] 3. Application of an engineered bacterium containing the PtoESK1 gene in improving the mechanical strength and / or wood yield of Populus tomentosa, wherein the sequence of the PtoESK1 gene is shown in SEQ ID NO.1.

[0009] Preferably, the engineered bacteria is Agrobacterium GV3101.

[0010] 4. A method for improving the mechanical strength and / or wood yield of Populus tomentosa, comprising the following steps: introducing the PtoESK1 gene into Populus tomentosa plants by genetic engineering methods to obtain plants overexpressing the PtoESK1 gene, i.e., Populus tomentosa with improved mechanical strength and wood yield.

[0011] In the present invention, the method for introducing the PtoESK1 gene into Populus tomentosa plants is to introduce a recombinant vector containing the PtoESK1 gene into the Populus tomentosa plants through Agrobacterium-mediated introduction.

[0012] Preferably, in the present invention, the recombinant vector containing the PtoESK1 gene is obtained by ligating the PtoESK1 gene into the XCM I restriction site of the pCXSN vector through homologous recombination.

[0013] The beneficial effects of the present invention are as follows: the present invention discovered a gene PtoESK1 that improves mechanical strength and wood yield in white poplar. By using genetic engineering methods, the PtoESK1 gene was introduced into white poplar plants to obtain white poplars with improved mechanical strength and wood yield. The mechanical strength of white poplars with the PtoESK1 gene was increased by more than 23%, and the fresh weight and dry weight yields of wood were increased by more than 76% and 110%. The present invention is of great significance for improving the strength and yield of poplar wood, provides new gene resources for plant genetic engineering, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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:

[0015] Figure 1 . Quantitative detection of PtoESK1 transgenic plants;

[0016] Figure 2.Phonotype observation of PtoESK1 transgenic plants;

[0017] Figure 3 .The mechanical strength measurement diagram of the stems of transgenic plants;

[0018] Figure 4 .Determination of fresh and dry weight yield of wood in transgenic plants. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1: Construction of a recombinant plant overexpression vector containing the PtoESK1 gene

[0021] The amplified PtoESK1 gene (its DNA sequence is shown in SEQ ID NO.1) was connected to the pCXSN vector by homologous recombination enzyme. The pCXSN vector was digested with XCMI, and the PtoESK1 gene fragment and the pCXSN vector skeleton fragment were recovered. The homologous recombination was connected and transformed into Escherichia coli DH5α, and positive clones were screened. Then the plasmid was extracted for PCR detection and enzyme digestion verification to obtain a recombinant plant overexpression vector containing the PtoESK1 gene, named pCXSN-PtoESK1 vector. The correctness of the gene was confirmed by sequencing by Shenzhen BGI Genomics Technology Co., Ltd.

[0022] The pCXSN-PtoESK1 vector was transformed into Agrobacterium tumefaciens (GV3101), and positive clones were screened to obtain engineered bacteria containing the pCXSN-PtoESK1 vector.

[0023] Example 2: Genetic transformation of Populus tomentosa

[0024] 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. The specific steps are as follows:

[0025] 1) Cultivation of Agrobacterium

[0026] Agrobacterium GV3101 containing the recombinant plasmid was transformed into YEP solid medium (containing 20 mg / ml Rif and 50 mg / ml Kan), and inverted at 28°C for culture; a single clone was selected and inoculated into YEP liquid medium containing the corresponding antibiotics, and cultured at 28°C with shaking until the logarithmic growth OD600 was 0.6-0.8; then the activated Agrobacterium was inoculated into the same 50 ml YEP liquid medium at a ratio of 1:100-1:50, and cultured at 28°C until OD 600The pH value is 0.6-0.8, add the diactive Agrobacterium liquid into a 50ml centrifuge tube, centrifuge at 4°C, 4000rpm for 10min, collect the bacterial precipitate, add 10ml WPM resuspension (WPM powder + 2.14g + 30g sucrose + 100μmol AS), mix with a pipette, add 20-30ml WPM resuspension, transfer into a round-mouth bottle, and culture at 28°C, 200rpm for 40min-1h.

[0027] 2) Agrobacterium-mediated leaf disc infection

[0028] 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.

[0029] 3) Co-cultivation of Populus tomentosa

[0030] The infected leaf disc was picked out with sterilized tweezers, placed on sterilized filter paper, the bacterial solution was absorbed, and the leaf disc was spread flat on WPM co-cultivation medium (WPM powder + 2.14g + 30g sucrose + 100μmol AS + 1.0mg NAA + 2.0mg ZT), and cultured in the dark at 25℃ for 2 days.

[0031] 4) Selective cultivation of Populus tomentosa

[0032] After 2 days of co-cultivation, the transformed explants were transferred to a callus-inducing selection medium (WPM powder + 2.14 g + 30 g sucrose + 9 mg Hyg + 1.0 mg NAA + 2.0 mg ZT + 400 mg Cef)) and cultured at 25°C in the dark for 3 to 5 weeks, during which time the selection medium was replaced every 5 days.

[0033] 5) Populus tomentosa bud induction culture

[0034] When white loose callus tissue appears around the leaf edge, move the callus tissue to WPM budding medium (WPM powder + 2.14g + 30g sucrose + 9mg Hyg + 0.1mg NAA + 2.0mg ZT + 400mg Cef) on the clean bench and culture at 25℃ under light for about 4 to 5 weeks, replacing new WPM budding medium every 10 days.

[0035] 6) Rooting culture of Populus tomentosa

[0036] When the adventitious buds grew to about 5 cm, they were transferred to WPM rooting medium (WPM+30 g sucrose+9 mg Hyg+0.1 mg NAA+400 mg Cef) containing corresponding antibiotics to induce rooting.

[0037] 7) Transplantation of Populus tomentosa

[0038] 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 warm and moist. Remove the film after one week of growth.

[0039] 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.

[0040] 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.

[0041] Example 3: Identification of transgenic positive seedlings

[0042] In order to screen the overexpressed transgenic strains, specific quantitative primers were designed, and RNA extracted from transgenic materials was reverse transcribed and amplified using the reverse transcribed cDNA template. The results are shown in the attached figure. Figure 1 As shown, strains 2, 4, 5 and 12 all overexpressed.

[0043] QESK1-F: 5'-GAAGGGTCCACCGAGTATGA-3' (SEQ ID NO. 2);

[0044] QESK1-R: 5'-CGGGTTCTCCCAGTCCAA-3' (SEQ ID NO.3);

[0045] The RNA extraction methods involved in this study are as follows:

[0046] The Biospin polysaccharide and polyphenol plant total RNA extraction kit was used, and the specific steps were as follows:

[0047] (1) Add 490 μL BGLysis and 10 μL β-mercaptoethanol to a 1.5 mL RNase-Free EP tube to a final concentration of 2% and mix well.

[0048] (2) Liquid nitrogen grinding: Grind an appropriate amount of tissue into powder in liquid nitrogen, weigh 50-100 mg of the fine powder and put it into the above-mentioned 1.5 mL centrifuge tube containing BGLysis and β-mercaptoethanol, and immediately shake vigorously until there are no obvious particles.

[0049] (3) Centrifuge the above samples at 12,000 pm for 5 minutes and carefully pipette the supernatant into a new RNase-Free 1.5 mL centrifuge tube.

[0050] (4) More accurately estimate the volume of the supernatant of the lysate and add 0.5 times the volume of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Shake and mix immediately.

[0051] (5) Pipette the entire mixture into the spin column and centrifuge at 12,000 rpm for 1 minute. Discard the liquid in the collecting tube.

[0052] (6) Add 600 μL PG Bufer to the spin column, centrifuge at 12,000 rpm for 30 seconds, and discard the liquid in the collecting tube.

[0053] (7) Add 500 μL Wash Buffer to the spin column, centrifuge at 12,000 pm for 30 seconds, discard the liquid in the collection tube, and then add 250 uL Wash Buffer and repeat the wash.

[0054] (8) The empty column was then centrifuged at 12,000 rpm for 1 min to remove as much wash buffer as possible to prevent residual ethanol in the wash buffer from inhibiting downstream reactions.

[0055] (9) Move the spin column into a new 1.5 mL RNase-Free centrifuge tube, add 25 μL of RElution Buffer to the center of the membrane, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, add the first eluate back to the spin column membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to obtain total RNA.

[0056] (10) The total RNA obtained is reverse transcribed to obtain cDNA.

[0057] Example 4: Phenotypic observation of transgenic plants and determination of wood yield

[0058] The phenotype of the three-month-old soil-grown seedlings after transplanting was observed (see Appendix Figure 2 ), determine plant height, diameter and wood yield (fresh weight, dry weight), such as the attached Figure 3 The results showed that the plant height, diameter, fresh weight and dry weight of the overexpression strain were higher than those of the wild type, indicating that overexpression of the PtoESK1 gene can increase poplar yield.

[0059] Example 5: Determination of mechanical strength of transgenic plant stems

[0060] The stem strength (puncture force) of the three-month-old soil-grown seedlings after transplanting was measured using a stem strength meter. The results are shown in the attached figure. Figure 4 As shown, the stem strength of the overexpression strain was significantly increased compared with the wild type, indicating that overexpression of the PtoESK1 gene can improve the mechanical strength of poplar.

[0061] 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 overexpression of PtoESK1 gene in increasing wood yield of Populus tomentosa, characterized by: The sequence of the PtoESK1 gene is shown in SEQ ID NO.1; the wood yield is plant height, diameter, fresh weight and dry weight.

2. Application of a recombinant vector containing the PtoESK1 gene in increasing the yield of Populus tomentosa wood, characterized in that: The sequence of the PtoESK1 gene is shown in SEQ ID NO.

1.

3. The application of an engineered bacterium containing the PtoESK1 gene in increasing the wood yield of Populus tomentosa, characterized in that: The sequence of the PtoESK1 gene is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that: The engineered bacteria is Agrobacterium GV3101.

5. A method for increasing the yield of Populus tomentosa wood, characterized in that: The method comprises the following steps: introducing the PtoESK1 gene into white poplar plants by genetic engineering to obtain plants over-expressing the PtoESK1 gene, namely white poplar plants with improved mechanical strength and wood yield; the sequence of the PtoESK1 gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that: The method for introducing the PtoESK1 gene into the Populus tomentosa plant is to introduce the recombinant vector containing the PtoESK1 gene into the Populus tomentosa plant through Agrobacterium-mediated introduction.

7. The method according to claim 6, characterized in that: The recombinant vector containing the PtoESK1 gene is obtained by connecting the PtoESK1 gene to the XCMI restriction site of the pCXSN vector through homologous recombination.

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