Gene for regulating tree xylem development and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a gene for regulating tree xylem development and application thereof. The nucleotide sequence of the gene for regulating and controlling tree xylem development is shown as SEQ ID NO. 1. By comparing the transgenic poplar overexpressing the MYB46 with a wild type, the phenotype that the xylem width of the transgenic poplar overexpressing the MYB46 is increased is found, so that the MYB46 gene is a key gene for regulating and controlling the development of the xylem of the poplar, and has important application value in the field of forest high-yield genetic engineering.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and in particular relates to a gene for regulating the development of tree xylem and an application thereof. Background Art

[0002] Poplar is the main afforestation tree species in my country. It not only plays an important role in ecological protection, but also has broad application prospects in urban greening and garden landscape design. In addition, poplar is also an important industrial material, providing raw materials for production activities such as papermaking and furniture manufacturing. The secondary xylem produced by the secondary growth of the stem is the main source of tree biomass and has great economic and ecological value in papermaking, construction, biofuels and the global carbon cycle. The development of the secondary cell wall is crucial to the formation of wood. Revealing the molecular regulatory mechanism of the formation of the secondary wall of forest trees will provide a theoretical basis for improving wood quality.

[0003] The V-myb avian myeloblastosis viral oncogene homolog (MYB) gene family is widely involved in regulating physiological processes such as plant growth and development, physiological metabolism, cell morphology and pattern building. Many MYB transcription factors are involved in regulating the biosynthesis of secondary walls. However, the molecular regulatory network and hierarchy of the MYB gene family regulating the secondary growth of poplars are still incomplete. Therefore, further research is needed on how the MYB gene family regulates the biosynthesis of secondary walls in poplars. Summary of the invention

[0004] The purpose of the present invention is to provide a gene for regulating the development of tree xylem and its application. The gene for regulating the development of tree xylem of the present invention can significantly increase the secondary growth of plant stems and promote xylem development.

[0005] The present invention provides a gene for regulating the development of tree xylem, and the nucleotide sequence of the gene for regulating the development of tree xylem is shown as SEQ ID NO.1.

[0006] The present invention also provides the CDS sequence of the gene regulating the development of tree xylem.

[0007] The present invention also provides application of the gene or the CDS sequence in regulating the development of tree xylem.

[0008] The present invention also provides a primer set for amplifying the gene or the CDS sequence, the primer set comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown as SEQ ID NO.3.

[0009] The present invention also provides a recombinant vector, which contains the gene or the CDS sequence.

[0010] As a preferred embodiment, the basic skeleton of the recombinant vector includes pBI121 vector.

[0011] The present invention also provides application of the recombinant vector in regulating the development of tree xylem.

[0012] The present invention also provides a method for enhancing the development of tree xylem, comprising the following steps: overexpressing the gene or the CDS sequence in the genome of the target tree.

[0013] The present invention also provides the application of the gene, the CDS sequence or the recombinant vector in creating seed resources with increased xylem width.

[0014] The present invention also provides a method for obtaining germplasm with increased xylem width, comprising the following steps: overexpressing the gene or CDS sequence as described above in the genome of a target tree.

[0015] Beneficial effects: The present invention provides a gene for regulating the development of tree xylem, and the nucleotide sequence of the gene for regulating the development of tree xylem is shown in SEQ ID NO. 1. By comparing the transgenic poplars overexpressing MYB46 with the wild type, the present invention found that the transgenic poplars overexpressing MYB46 showed a phenotype of increased xylem width, so the MYB46 gene is a key gene for regulating the development of poplar xylem, which can significantly increase the secondary growth of plant stems and promote xylem development, providing a new idea for the field of high-yield genetic engineering of forest trees, and has important application value in the field of high-yield genetic engineering of forest trees.

[0016] The embodiment of the present invention shows that after overexpressing the MYB46 gene, the width of the poplar xylem increases significantly, the average xylem width of the wild type poplar is 0.86 cm, and the average xylem width of the transgenic poplar overexpressing MYB46 is 1.26 cm, indicating that overexpressing MYB46 promotes the development of the poplar xylem. By overexpressing the MYB46 gene in plants, high-yield, high-quality new varieties can be cultivated, which has great application value in the field of high-yield genetic engineering research of forest trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 is the position where the CDS sequence of the MYB46 gene is connected to the expression vector pBI121, wherein LB indicates the left border and RB indicates the right border;

[0019] Figure 2 Schematic diagram of gRNA connected to the vector;

[0020] Figure 3 This is a real-time quantitative detection diagram of the transcription level of transgenic poplar overexpressing MYB46;

[0021] Figure 4 The cross-section of the stem of wild-type poplar and MYB46-overexpressing transgenic positive plants;

[0022] Figure 5 The scanning electron micrographs of cross sections of stems of wild-type poplar and MYB46-overexpressing transgenic positive plants;

[0023] Figure 6 This is a statistical diagram of stem cross-sectional area of ​​wild-type poplar and MYB46-overexpressing transgenic positive plants;

[0024] Figure 7 The poplar phenotypes of wild-type, MYB46 knockout and MYB46 overexpressing plants are shown in Figure 1.

[0025] Figure 8 The statistical results of poplar plant height are shown for wild-type poplar, MYB46 knockout plants, and MYB46 overexpressing plants. DETAILED DESCRIPTION

[0026]

[0027]

[0028] The present invention also provides application of the gene or the CDS sequence in regulating the development of tree xylem.

[0029] As a specific implementation method, overexpressing the MYB46 gene in poplar can significantly increase the secondary growth of poplar stems and promote xylem development, providing new ideas for the field of high-yield genetic engineering of forest trees.

[0030] The present invention also provides a primer set for amplifying the gene or the CDS sequence, the primer set comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown as SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown as SEQ ID NO.3.

[0031] The sequence of the primer set of the present invention is as follows:

[0032] Forward primer (SEQ ID NO. 2): 5'-ATGAGGAAGCCAGAGGCCTC-3',

[0033] Reverse primer (SEQ ID NO. 3): 5'-TTGGAAATCAAGGAATGGAAAGA-3'.

[0034] The present invention also provides a recombinant vector, which contains the gene or the CDS sequence. After the recombinant vector is transferred into a tree, the MYB46 gene can be overexpressed in the tree, thereby enhancing the xylem development of the tree.

[0035] The basic framework of the recombinant vector of the present invention includes the pBI121 vector.

[0036] The present invention also provides the use of the recombinant vector in regulating the development of tree xylem. As a specific embodiment, after overexpressing the MYB46 gene, the width of the poplar xylem increased significantly, the average xylem width of the wild-type poplar was 0.86 cm, and the average xylem width of the transgenic poplar overexpressing the MYB46 gene was 1.26 cm, indicating that overexpressing MYB46 promoted the development of the poplar xylem.

[0037] The present invention also provides a method for enhancing the development of tree xylem, comprising the following steps: overexpressing the gene or the CDS sequence in the target tree genome. As a specific embodiment, the recombinant vector can be transformed into cells of the target plant to overexpress the MYB46 gene. The transformation method can be Agrobacterium transformation; the Agrobacterium can be GV3101. By overexpressing the MYB46 gene in plants, high-yield and high-quality new varieties can be cultivated, which has great application value in the field of high-yield genetic engineering research of forest trees.

[0038] The present invention also provides application of the gene, the CDS sequence or the recombinant vector in creating germplasm resources with increased xylem width.

[0039] The present invention also provides a method for obtaining germplasm with increased xylem width, comprising the following steps: overexpressing the gene or CDS sequence as described above in the genome of a target tree.

[0040] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products known to those skilled in the art can be used.

[0041] In order to further illustrate the present invention, a gene for regulating the development of tree xylem and its application provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example

[0043] 1. MYB46 gene cloning

[0044] TIANGEN's RNA prep Pure plant total RNA extraction kit (DP432) was used to extract total RNA from Populus tomentosa "Yi Yang No. 3" (Ma Huandi. Cloning and genetic transformation of members of the sucrose synthase PtSS gene family of Populus tomentosa and analysis of wood properties differences of hybrid asexual lines of Populus tomentosa [D]. Beijing Forestry University, 2015.). For detailed operation steps, please refer to the kit manual. The extracted RNA was subjected to agarose gel electrophoresis, and the results showed clear and obvious bands, indicating that the quality of the extracted RNA met the standards for further testing. The concentration and quality (OD260 / 280 ratio) of RNA were also tested, and the quality (OD260 / 280 ratio) was 1.9, indicating that the sample can be used for subsequent experiments. The TAKARA cDNA synthesis kit (PrimeScript TM IV 1ststrand cDNA Synthesis Mix) was used to synthesize cDNA using plant total RNA as template. The specific operation steps are shown in the product manual. The cDNA was stored at -20℃ for future use.

[0045] The 2× Gold high-fidelity gene cloning kit was used. High Fidelity (HiFi) PCR SuperMix II (-dye) (AS131)) was used for gene amplification. In this example, the cDNA of Populus tomentosa "Yi Yang No. 3" was used as a template, and the CDS fragment of the MYB46 gene (SEQ ID NO. 4) was amplified by RT-PCR. The primers used for RT-PCR were:

[0046] Forward primer (SEQ ID NO. 2): 5'-ATGAGGAAGCCAGAGGCCTC-3',

[0047] Reverse primer (SEQ ID NO. 3): 5'-TTGGAAATCAAGGAATGGAAAGA-3'.

[0048] The amplification system of RT-PCR is 25 μL, specifically: 10.0 μL of ultra-fidelity Taq enzyme, 0.5 μL of forward primer, 0.5 μL of reverse primer, 0.5 μL of buffer, 2.0 μL of cDNA template, and ddH2O to 25 μL; the PCR amplification conditions are: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 10 s, annealing at 59°C for 60 s, extension at 72°C for 40 s, and 40 cycles; the amplified target fragment is detected by agarose gel electrophoresis, and the target fragment is excised and recovered using Tiangen's ordinary agarose gel DNA recovery kit. For specific operating instructions, please refer to the kit manual.

[0049] The CDS fragment of the MYB46 gene recovered from the gel was connected to the pEASY-T1 vector to obtain the T1-MYB46 vector. For specific operation steps, see the full-form gold pEASY-T1 ( -Basic Seamless Cloning and Assembly Kit (CU201-02)) Kit Instructions. The ligated vector was transformed into DH5α E. coli competent cells (Shanghai Weidi Biotechnology Co., Ltd. (DL1001M)), and the transformation method was carried out according to the instructions of DH5α E. coli competent cells.

[0050] 2. Construction of MYB46 overexpression vector

[0051] (1) Enzyme digestion: Select BamH I as the restriction site to digest the overexpression vector pBI121 (Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants. DOI: 10.1023 / A: 1023475710642) and the T1-MYB46 vector containing the target gene. Incubate at 37°C for 3 hours to ensure that the cleavage reaction is completely cleaved. The pBI121 vector and T1-MYB46 vector cut by BamH I were detected by electrophoresis and recovered by gel cutting to obtain the linearized pBI121 vector and the CDS fragment of the MYB46 gene; the enzyme digestion system and method refer to the instructions of BamH I restriction endonuclease (NEWENGLAND Biolabs BamH I restriction endonuclease (R0136S)).

[0052] (2) Connection: Use -Basic Seamless Cloning and Assembly Kit kit to cleave the CDS fragment of MYB46 gene to the linearized pBI121 vector (see the position of CDS ligation to the vector for details). Figure 1 ) to obtain a ligation product. For detailed operations, refer to the instructions of the kit.

[0053] (3) Transformation: The ligation product was transformed into E. coli DH5α competent cells. The cells were transformed using the freeze-thaw method (Study on transformation efficiency of E. coli and Agrobacterium by different methods. Journal of Huaihai Institute of Technology (Natural Science Edition), 2007, (02): 55-58.). The transformation product was evenly spread on LB agar plates containing 50 mg / L kanamycin for culture.

[0054] (4) Screening: Resistant colonies were picked from the LB agar plate and PCR verification or sequencing was performed to confirm the correctness and direction of the inserted fragment.

[0055] Plasmid extraction and storage: Extract plasmids from verified positive colonies. Use the high-purity plasmid extraction kit (DP104) produced by Tiangen Company to extract the plasmid of the MYB46 overexpression vector, and store the plasmid of the MYB46 overexpression vector at -20°C.

[0056] 3. MYB46 overexpression vector genetic transformation

[0057] Place the competent Agrobacterium GV3101 (AC1001M) on ice, add 1 g of plasmid containing the MYB46 overexpression vector, mix well, and place on ice for 30 min; quickly cool in liquid nitrogen for about 1 min, then quickly transfer to a 37°C water bath to melt; add 1 mL of antibiotic-free YEB liquid culture medium at 28°C, 230 r / min for 3 h, resuspend the bacteria and spread them on a YEB plate containing 30 mg / L kanamycin, blow dry, and culture at 28°C for 48 h to obtain Agrobacterium containing the overexpression vector.

[0058] Fresh and disease-free leaves of healthy poplar tissue culture seedlings were selected as leaf disc transformation materials. After the leaves were surface cleaned and disinfected, they were cut into small pieces or pieces.

[0059] Agrobacterium containing the overexpression vector was cultured in LB medium at 28°C until the logarithmic growth phase.

[0060] The leaves were placed in the resuspended Agrobacterium solution for co-cultivation, and then the transformed leaf discs were transferred to differentiation medium (MS + 1.0 mg / L 6-BA and 0.1 mg / L NAA, pH adjusted to 5.8, agar 8g / L), the light cycle was set to 16h light / 8h dark, and the temperature was maintained at 25 ° C to promote stable transformation and expression of DNA.

[0061] Transplant new shoots from the differentiation medium to the rooting medium to promote plant formation. Use the overexpression vector pBI121 with a selective marker gene (resistant to kanamycin) (the pBI121 vector has kanamycin resistance and can grow normally on a medium containing kanamycin. Leaves that have not been successfully transformed do not contain the vector and therefore cannot grow on a kanamycin-resistant medium, ultimately achieving the purpose of screening positive plants.) Screen the transformed leaf discs to obtain transformed positive plants. Extract the DNA of the screened plants and perform PCR testing to confirm the presence of the exogenous gene, thereby verifying the successful introduction of the overexpression vector.

[0062] 4. Construction of MYB46 gene editing vector

[0063] (1) gRNA design: Use the online tool CRISPR-EG (http: / / skl.scau.edu.cn / targetdesign / ) to input the sequence of the MYB46 gene to design a gRNA targeting the gene. Select the first exon as the target site to improve the knockout efficiency. The final designed gRNA sequence is: 5'-TAAACAATGGACAAGGTTGC-3' (SEQ ID NO.5).

[0064] (2) Vector construction: Select the CRISPR / Cas9 vector p2gR-TRI (ACRISPR / Cas9toolkit for multiplex genome editing in plants. https: / / doi.org / 10.1186 / s12870-014-0327-y) suitable for plant cells. Figure 2 As shown, gRNA is connected to the vector, and the insertion sites are: Target-Sense: 5'-TTG-gRNAsense; Target-Anti: 5'-AAC-gRNA anti. The knockout vector is obtained. The method for constructing the knockout vector is the same as the method in "2. Constructing the MYB46 overexpression vector".

[0065] 5. Genetic transformation of MYB46 gene editing vector

[0066] The genetic transformation method of MYB46 gene editing vector is the same as the method of “3. Genetic transformation of MYB46 overexpression vector”.

[0067] 6. Application of MYB46 overexpressing poplar plants

[0068] Four-week-old tissue culture seedlings of MYB46-overexpressing poplar transgenic plants and wild-type poplar plants (plants without exogenous genes) were used as materials to extract RNA and perform reverse transcription. The expression level of the MYB46 gene was detected using the SYBR Premix Ex Taq TM Kit (TaKaRa) and the ACTIN gene as an internal reference, and the Light Cycler480 (Roche) real-time quantitative instrument. Figure 3 As shown in the figure, the bar graph shows the expression levels of MYB46 gene in non-transgenic poplar (ck) and transgenic poplar (1, 2, 3) (three strains of transgenic poplar overexpressing MYB46); Figure 3 It can be seen that the expression level of MYB46 in transgenic poplar (3) was significantly increased compared with that in non-transgenic poplar.

[0069] Poplar seedlings were removed from the culture medium, transplanted into pre-prepared soil, placed in a poplar culture room for hardening, and then grown outdoors. The cross-sectional areas of the stems of wild-type and transgenic plants were compared. The results are as follows: Figure 4 shown. Figure 4 This is a cross-sectional comparison of the stems of wild-type and transgenic poplar plants grown outdoors for 15 months. By taking pictures with a camera, it can be seen that compared with the wild-type poplar, the stems of the transgenic poplar are significantly thicker, indicating that the MYB46 gene affects the growth and development of poplar.

[0070] Stem tissue sections of 5-month-old MYB46-overexpressing poplar plants were freeze-dried and placed under a scanning electron microscope for observation and photography. Figure 5 As shown. Figure 5 It can be seen that the xylem width of poplars overexpressing the MYB46 gene increased significantly. The average xylem width of wild-type poplars at 5 months old was 0.86 cm, and the average xylem width of transgenic plants was 1.26 cm, indicating that overexpression of MYB46 promoted the development of poplar xylem.

[0071] like Figure 6 As shown in the figure, the thickness of the stem of the 15-month-old transgenic poplar was measured. By calculating the cross-sectional area of ​​the stem of wild-type and transgenic poplar plants, it was found that the cross-sectional area of ​​the poplar overexpressing MYB46 was 1.25 cm on average. 2 The average cross-sectional area of ​​wild-type poplars is 0.61 cm 2 The stems of transgenic poplars were significantly thickened, further confirming that overexpression of MYB46 promoted the development of poplar xylem.

[0072] During the growth test of MYB46 knockout poplars, it was also found that after MYB46 was knocked out, the leaves of the poplars wilted and became smaller, and the plants were short. Figure 7 As shown in the figure, under the same growth environment and conditions during the same period, the MYB46 knockout poplars had an obvious growth retardation phenotype. Figure 8 The figure shows the plant height statistics, which confirms the importance of MYB46 to the growth and development of poplar from another perspective.

[0073] It can be seen that by comparing the transgenic poplars overexpressing and knocking out MYB46 with the wild type, the transgenic poplars overexpressing MYB46 showed a phenotype of increased xylem width. Therefore, the MYB46 gene is a key gene regulating the development of poplar xylem and has important application value in the field of high-yield genetic engineering of forest trees.

[0074] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A gene that regulates the development of tree xylem, characterized in that: The nucleotide sequence of the gene regulating the development of tree xylem is shown in SEQ ID NO.

1.

2. The CDS sequence of the gene regulating the xylem development of trees as claimed in claim 1.

3. Use of the gene according to claim 1 or the CDS sequence according to claim 2 in regulating the development of xylem in trees.

4. A primer set for amplifying the gene according to claim 1 or the CDS sequence according to claim 2, characterized in that: The primer set includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

3.

5. A recombinant vector, characterized in that: The recombinant vector comprises the gene according to claim 1 or the CDS sequence according to claim 2.

6. The recombinant vector according to claim 5, characterized in that The basic framework of the recombinant vector includes pBI121 vector.

7. Use of the recombinant vector according to claim 5 or 6 in regulating the development of xylem of trees.

8. A method for enhancing xylem development in trees, characterized in that: The following steps are involved: Overexpressing the gene described in claim 1 or the CDS sequence described in claim 2 in the genome of a target tree.

9. Use of the gene according to claim 1, the CDS sequence according to claim 2 or the recombinant vector according to claim 5 in creating a seed resource with increased xylem width.

10. A method for obtaining germplasm with increased xylem width, characterized in that The method comprises the following steps: overexpressing the gene described in claim 1 or the CDS sequence described in claim 2 in the genome of a target tree.

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