Poplar anti-osmotic stress gene and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a poplar anti-osmotic stress gene and application thereof. The poplar anti-osmotic stress gene is a NATB CATALYTIC SUBUNIT gene, the nucleotide sequence of which is shown as SEQ ID No. 1. Inhibition of the expression of the NATB CATALYTIC SUBUNIT gene in a plant body can effectively improve the net photosynthetic rate of the plant under osmotic stress conditions and promote root system development. Therefore, the NATB CATALYTIC SUBUNIT gene can be used for cultivating new varieties with high yield, high quality and anti-osmosis, and has great application value for genetic improvement of comprehensive stress resistance of forest trees and provides a new idea for creating new poplar anti-osmosis germplasm.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a poplar anti-osmotic stress gene and application thereof. BACKGROUND

[0002] Soil osmotic stress can cause an increase in osmotic stress ions in the soil, making it difficult for trees to absorb water, causing water deficiency in the plant body, and thus inhibiting the growth and development of trees. Secondly, osmotic stress can also damage the cell membrane structure of trees, affect the normal physiological functions of cells, and lead to metabolic disorders. In addition, soil osmotic stress can also affect the photosynthesis and respiration of trees, reduce their photosynthetic efficiency and net photosynthetic productivity, and further limit their growth potential. Due to the intensification of climate change, extreme drought weather occurs frequently, and the genetic improvement of forest trees against osmotic stress has become the primary task of forest tree genetic breeders.

[0003] The GCN5-related N-acetyltransferase gene family plays a key role in gene expression regulation, metabolic regulation, and stress response biological processes. GNAT family member NAA15, as one of the components of the N-terminal acetyltransferase complex NatA, can trigger ABA-mediated drought stress response transcriptional regulatory networks. The loss of function or abnormality of the NatA complex can lead to accelerated protein degradation, thereby affecting the normal physiological processes of cells. Acetyltransferases play an important role in maintaining plant cell wall homeostasis and enhancing plant resistance to abiotic stress. However, there is still little research on the influence of acetyltransferase-related genes on the osmotic resistance of forest trees. SUMMARY

[0004] The purpose of the present application is to provide a poplar anti-osmotic stress gene to effectively improve the osmotic resistance of forest trees.

[0005] The present application provides a poplar anti-osmotic stress gene, which is a NATB CATALYTIC SUBUNIT gene, and the nucleotide sequence is shown in SEQ ID No. 1.

[0006] The present application also provides the application of the NATB CATALYTIC SUBUNIT gene in one or more of the following:

[0007] (1) regulating the ability of forest trees to resist mannitol osmotic stress;

[0008] (2) screening new germplasm of forest trees resistant to mannitol osmotic stress;

[0009] (3) creating new germplasm of forest trees resistant to mannitol osmotic stress,

[0010] The application further provides a gRNA of the NATB CATALYTIC SUBUNIT gene, and a nucleotide sequence of the gRNA is shown in SEQ ID No. 2.

[0011] The application further provides a silencing vector of the NATB CATALYTIC SUBUNIT gene, and the silencing vector comprises a basic vector and the gRNA of claim 3 inserted into the basic vector.

[0012] The application further provides a method for improving the anti-permeability of forest trees, comprising the following steps.

[0013] The NATB CATALYTIC SUBUNIT gene in the forest tree plant is knocked out, and a nucleotide sequence of the NATB CATALYTIC SUBUNIT gene is shown in SEQ ID No. 1.

[0014] The application further provides an anti-permeability characteristic molecular marker of the NATB CATALYTIC SUBUNIT gene, and a single nucleotide polymorphism site exists in the molecular marker.

[0015] The application further provides a primer combination for detecting the high anti-permeability characteristic molecular marker, and the primer combination comprises NCS-Chr05-21472971G-F, NCS-Chr05-21472971A-F and NCS-R.

[0016] The application further provides a method for screening new germplasm of poplar with anti-permeability, comprising the following steps.

[0017] The DNA of the to-be-tested poplar germplasm is amplified by using the primer combination, and then the amplified product is genotyped.

[0018] The primer combination of NCS-Chr05-21472971G-F and NCS-R does not amplify the target band, and the primer combination of NCS-Chr05-21472971A-F and NCS-R amplifies the target band of 296 bp, which represents that the to-be-tested poplar germplasm is excellent germplasm of poplar with anti-permeability stress, and the genotype is AA.

[0019] The application also provides a method for creating a new poplar osmosis-resistant germplasm, comprising the following steps:

[0020] The poplar osmosis-resistant excellent germplasm with the genotype AA of the NATB CATALYTIC SUBUNIT gene Chr05:21472971 SNP site is used as a male parent / female parent to perform hybridization to obtain hybrid offspring; the genomic DNA of the hybrid offspring plant is amplified by using the primer combination in the above technical solution to obtain an amplification product;

[0021] If the NCS-Chr05-21472971G-F and NCS-R primer combination does not amplify the target band, and the NCS-Chr05-21472971A-F and NCS-R primer combination amplifies a 296bp target band, it represents that the hybrid offspring plant is a new poplar osmosis-resistant germplasm.

[0022] Beneficial effects:

[0023] The application provides a NATB CATALYTIC SUBUNIT gene, the nucleotide sequence of the NATB CATALYTIC SUBUNIT gene is shown in SEQ ID No. 1; inhibiting the expression of the NATB CATALYTIC SUBUNIT gene in a plant body can effectively improve the net photosynthetic rate of the plant under osmotic stress conditions and promote root development; therefore, the NATB CATALYTIC SUBUNIT gene can be used for cultivating high-yield, high-quality, osmosis-resistant new varieties, has great application value for genetic improvement of comprehensive stress resistance of forest trees, and provides a new idea for creating a new poplar osmosis-resistant germplasm.

[0024] Further, the gRNA for knocking out the NATB CATALYTIC SUBUNIT gene is introduced into a plant plant by using an exogenous gene expression vector, the NATB CATALYTIC SUBUNIT gene can be effectively knocked out, and the knockout plant is verified by the examples to have normal root development under 3% mannitol osmotic stress. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0026] Figure 1 The figure is a pSpCas9 vector map; wherein, LB represents a left border, and RB represents a right border;

[0027] Figure 2Root development traits of wild type plant of poplar, NATB CATALYTIC SUBUNIT gene knockout positive plant under 3% mannitol osmotic stress;

[0028] Figure 3 Growth traits of wild type plant of poplar, NATB CATALYTIC SUBUNIT gene knockout positive plant under 3% mannitol osmotic stress;

[0029] Figure 4 NATB CATALYTIC SUBUNIT gene Chr05-21472971 SNP site genotyping result diagram of poplar;

[0030] Figure 5 Peroxidase activity, malondialdehyde content, net photosynthetic rate and root length of poplar plant. DETAILED DESCRIPTION

[0031]

[0032] The application also provides the application of the NATB CATALYTIC SUBUNIT gene in one or more of the following aspects:

[0033] (1) regulating the ability of trees to resist mannitol penetration;

[0034] (2) screening new germplasm of trees that resist mannitol penetration;

[0035] (3) creating new germplasm of trees that resist mannitol penetration. In the application, the trees preferably include poplar, and more preferably include populus tomentosa.

[0036] The application also provides a gRNA of the NATB CATALYTIC SUBUNIT gene, and the nucleotide sequence of the gRNA is shown in SEQ ID No. 2, and specifically is 5'-CTCG GCTTCTTTCTTAGCTCGGG-3'. The gRNA provided by the application can knock out the NATB CATALYTIC SUBUNIT gene in a plant body, and thus improve the anti-mannitol penetration property of the plant.

[0037] The application also provides a silencing vector of the NATB CATALYTIC SUBUNIT gene, and the silencing vector includes a basic vector and the gRNA of the above technical solution inserted into the basic vector. In the application, the basic vector preferably includes a CRISPR / Cas9 vector pSpCas9 suitable for plant cells.

[0038] The application also provides a method for improving the anti-penetration property of trees, including the following steps:

[0039] knocking out the NATB CATALYTIC SUBUNIT gene in a tree plant, and the nucleotide sequence of the NATB CATALYTIC SUBUNIT gene is shown in SEQ ID No. 1.

[0040] In the application, the step of knocking out the NATB CATALYTIC SUBUNIT gene in the tree plant preferably includes the following steps:

[0041] The recombinant expression vector in the technical scheme is transformed into cells of a target plant to knock out a NATB CATALYTIC SUBUNIT gene.

[0042] The application further provides an anti-osmotic property molecular marker of the NATB CATALYTIC SUBUNIT gene, wherein the molecular marker has a single nucleotide polymorphism site; the single nucleotide polymorphism site is located at the 21472971bp site on the 5th chromosome of a poplar, and the polymorphism is A / G.

[0043] The application further provides a primer combination for detecting the anti-osmotic property molecular marker in the technical scheme, wherein the primer combination comprises NCS-Chr05-21472971G-F, NCS-Chr05-21472971A-F and NCS-R, the nucleotide sequence of the NCS-Chr05-21472971G-F is shown in SEQ ID No. 3, and specifically 5'-GGTAGTATTTGATGCTCTCACA-3'; the nucleotide sequence of the NCS-Chr05-21472971A-F is shown in SEQ ID No. 4, and specifically 5'-GGTAGTATTTGATGCTCTCATA-3'; and the nucleotide sequence of the NCS-R is shown in SEQ ID No. 5, and specifically 5'-GTTGCTGGACTTCTGTTCCAACA-3'. Preferably, the last second nucleotide at the 3' end of the NCS-Chr05-21472971G-F and the NCS-Chr05-21472971A-F is modified by LNA.

[0044] The application further provides a method for screening a new poplar germplasm with anti-osmotic property, comprising the following steps:

[0045] The DNA of the to-be-tested poplar germplasm is amplified by using the primer combination in the technical scheme, and then the amplified product is genotyped;

[0046] NCS-Chr05-21472971G-F and NCS-R primer combination does not amplify the target band, and NCS-Chr05-21472971A-F and NCS-R primer combination amplifies the target band of 296bp, which represents that the tested poplar germplasm is an excellent poplar germplasm resistant to osmotic stress, and the genotype of Chr05:21472971 SNP site of the NATB CATALYTIC SUBUNIT gene is AA.

[0047] The application also provides a method for creating a new poplar germplasm resistant to osmotic stress, comprising the following steps:

[0048] The excellent poplar germplasm resistant to osmotic stress with the genotype AA of the Chr05:21472971 SNP site of the NATB CATALYTIC SUBUNIT gene is used as a male parent / female parent to perform hybridization to obtain hybrid offspring; the primer combination of claim 7 is used to amplify the genomic DNA of the hybrid offspring to obtain an amplification product;

[0049] If NCS-Chr05-21472971G-F and NCS-R primer combination does not amplify the target band, and NCS-Chr05-21472971A-F and NCS-R primer combination amplifies the target band of 296bp, which represents that the hybrid offspring is a new poplar germplasm resistant to osmotic stress.

[0050] In order to further illustrate the application, the application of the NATB CATALYTIC SUBUNIT gene is described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the application.

[0051] The experimental methods used in the embodiments of the application are all conventional methods if no special instructions are given, and the reagents and materials used are all commercially available products if no special instructions are given.

[0052] Example 1

[0053] 1. Constructing a NATB CATALYTIC SUBUNIT gene editing vector

[0054] (1) gRNA design: using the online tool CRISPR-P2.0, input the sequence of the NATB CATALYTIC SUBUNIT gene (measured according to the method described in the literature https: / / onlinelibrary.wiley.com / doi / 10.1111 / pbi.14108) to design gRNA for the gene. The first exon is selected as the target site to improve the knockout efficiency. The finally designed gRNA sequence is: 5'-CTCGGCTTCTTTCTT AGCTCGGG-3'(SEQ ID No. 2).

[0055] (2) Vector construction: select CRISPR / Cas9 vector pSpCas9( Figure 1 ) suitable for plant cells, connect gRNA into the vector, insert site: Target-Sense: 5'-TTG-gRNA sense; Target-Anti: 5'-AAC-gRNA anti. Get knockout vector.

[0056] 2. Genetic transformation of NATB CATALYTIC SUBUNIT gene editing vector

[0057] Put competent Agrobacterium GV3101 on ice, add 1 g of plasmid DNA containing knockout vector, mix thoroughly, 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, and wait for it to melt; add 1 mL of YEB liquid culture without antibiotics, and incubate at 28°C, 230 r / min for 2-4 h to resuspend the bacterial body and spread on YEB plates containing 30 mg / L kanamycin and dry blow, and incubate at 28°C for 48 h to obtain Agrobacterium containing knockout vector.

[0058] Select fresh and disease-free leaves of healthy poplar tissue culture seedlings as leaf disc transformation material. After surface cleaning and disinfection of the leaves, cut them into small pieces or blocks. Incubate Agrobacterium containing knockout vector on LB medium to logarithmic growth phase.

[0059] Put the leaves in resuspended OD 600 value of 0.2-0.5 Agrobacterium bacterial solution for 3 min, remove the excess bacterial solution with sterile filter paper, and continue to place in MS medium, dark culture at 25°C, for a total of 2-4 days to obtain transformed leaf discs. Transfer the transformed leaf discs to differentiation medium (MS + 1.0 mg / L 6-BA and 0.1 mg / L NAA), set the photoperiod to 16 h light / 8 h dark, and maintain the temperature at 25°C to promote stable transformation and expression of DNA.

[0060] After 3-4 weeks of culture at 25°C on differentiation medium with 16 h light / 8 h dark, obtain the selected plants. Extract the DNA of the selected plants, and detect the editing type of the target site by PCR to verify the successful introduction of the gene editing vector. The plants containing the gene editing vector are NATB CATALYTIC SUBUNIT knockout plants.

[0061] 3. NATB CATALYTIC SUBUNIT knockout plants with improved osmotic resistance

[0062] The NATB CATALYTIC SUBUNIT knockout plants were subcultured. Wild type and knockout lines were treated with 0%, 1%, 3%, and 5% mannitol osmotic stress, respectively, for 7 days. The results are shown in Table 1. Figure 2 As can be seen from Table 1, the NATB CATALYTIC SUBUNIT knockout plants promoted root growth under osmotic stress. Figure 2

[0063] The NATB CATALYTIC SUBUNIT knockout plants and wild type poplar plants (plants without knockout) were cultured to 4 weeks old, and the tissue culture seedlings were removed from the culture medium, transplanted into the prepared soil (the mass ratio of perlite: vermiculite: grass carbon = 1:1:1), and placed in a poplar culture room for 8 weeks of soil culture. The conditions for soil culture were: temperature 25±1°C, relative humidity 50%, light time 16h light / 8h dark, and light intensity 3000-5000LUX.

[0064] Subsequently, the knockout lines and wild type lines were compared in terms of phenotypic traits, and the results are shown in Table 2. Figure 3 As can be seen from Table 2, the NATB CATALYTIC SUBUNIT knockout plants were significantly inhibited in height. Figure 3

[0065] Example 2

[0066] 1. Osmotic resistance genotyping of forest tree germplasm resource population

[0067] Using the DNA of the P. tomentosa germplasm resource population as a template, primers were designed for the NCS-Chr05-21472971 single nucleotide variation site, as follows:

[0068] NCS-Chr05-21472971G-F: 5'-GGTAGTATTTGATGCTCTCACA-3' (SEQ ID No. 3);

[0069] NCS-Chr05-21472971A-F: 5'-GGTAGTATTTGATGCTCTCATA-3' (SEQ ID No. 4);

[0070] NCS-R: 5'-GTTGCTGGACTTCTGTTCCAACA-3' (SEQ ID No. 5).

[0071] ​​The PCR amplification system was 25 μL, specifically: 10.0 μL of ultra-fidelity Taq enzyme, 0.5 μL of forward primer, 0.5 μL of backward primer, 0.5 μL of buffer, 2.0 μL of cDNA template, and 7.0 μL of ddH2O. The PCR amplification conditions were: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 10 s, annealing at 62℃ for 30 s, and extension at 72℃ for 40 s, for 40 cycles. The melting curve of the PCR product was obtained within the 72℃-95℃ range, as shown below. Figure 4 As shown in the diagram. Here, GF / R represents the primer combination of NCS-Chr05-21472971G-F and NCS-R; AF / R represents the primer combination of NCS-Chr05-21472971A-F and NCS-R. GG represents the genotype of the Chr05:21472971 SNP locus is GG, GA represents the genotype of the Chr05:21472971 SNP locus is GA, and AA represents the genotype of the Chr05:21472971 SNP locus is AA.

[0072] Depend on Figure 4 It can be seen that the NCS-Chr05-21472971G-F primer combination with NCS-R primers amplified the target band of 296bp, while the NCS-Chr05-21472971A-F primer combination with NCS-R primers did not amplify the target band, indicating that the genotype of the Chr05:21472971 SNP site is GG;

[0073] The NCS-Chr05-21472971G-F primer combination with NCS-R primers did not amplify the target band, while the NCS-Chr05-21472971A-F primer combination with NCS-R primers amplified a 296bp target band, indicating that the genotype of the Chr05:21472971 SNP site is AA.

[0074] The combination of NCS-Chr05-21472971G-F and NCS-R primers amplified a 296bp target band, and the combination of NCS-Chr05-21472971A-F and NCS-R primers amplified a 296bp target band, indicating that the genotype of the Chr05:21472971 SNP site is GA.

[0075] 2. Hybridization of forest tree germplasm with resistant permeability

[0076] The DNA of the Populus tomentosa resource population was amplified using the primer combination designed in step 1. Individuals with the genotype AA at the NCS-Ch r05-21472971 single nucleotide polymorphism site were selected as the parents for crossbreeding to create the Populus tomentosa F1 hybrid population.

[0077] 3. Identification of new forest tree germplasm with resistance to osmosis

[0078] The DNA of the F1 hybrid population of P. tomentosa was amplified by using the primer combination in step 1, and the new germplasm of poplar resistant to osmotic stress was identified.

[0079] The NCS-Chr05-21472971G-F and NCS-R primer combination did not amplify the target band, and the NCS-Chr05-21472971A-F and NCS-R primer combination amplified a 296 bp target band, representing the AA genotype of the new germplasm of poplar resistant to osmotic stress.

[0080] 4. Improvement of salt resistance of new germplasm of forest trees

[0081] When the identified F1 hybrid population of P. tomentosa reached 4 weeks old, the tissue culture seedlings were removed from the culture medium and transplanted into the prepared soil in the poplar culture room for 8 weeks of soil culture. Subsequently, the AA genotype of the NCS-Chr05-21472971 single nucleotide polymorphism site of the offspring strain and the GG and GA genotypes of the Chr05: 21472971 SNP site in the natural germplasm population were treated with 3% mannitol osmotic stress for 7 days.

[0082] The peroxidase activity, malondialdehyde content and net photosynthetic rate of poplar plants were determined by the method described in the literature Effects of high temperature on photosynthesis and related gene expression in poplar. BMC Plant Biol. (Song Y, Chen Q, Ci D, Shao X, Zhang D. 2014 Apr 28; 14: 111.), and the results are shown in Figure 5 , where different letters represent significant differences.

[0083] It can be seen from Figure 5 that compared with the other two genotypes, the average peroxidase activity of the AA genotype of the new germplasm of poplar resistant to osmotic stress increased to 5025 μmol (guaincol)·mg -1 (Protein)·min -1 under 3% mannitol osmotic stress Figure 5 (A), the average malondialdehyde content decreased to 12.1 μmol·g -1 FW Figure 5 (C), and the average net photosynthetic rate maintained at 13.5 μmol·m -2 ·s -1 Figure 5 ​The peroxidase activity of the AA genotype poplar under 3% mannitol osmotic stress was significantly higher than that of the other two genotypes (B), and the malondialdehyde content was significantly lower, and the photosynthesis was not significantly inhibited.

[0084] As can be seen from the above examples, knocking out the NATB CATALYTIC SUBUNIT gene in poplar plants can still improve the photosynthesis of the plants under osmotic stress conditions, and the root growth is not inhibited. Figure 5 (D), which provides a new idea for breeding new osmotic-resistant poplar varieties.

[0085] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. Use of a poplar gene for osmotic stress resistance in modulating the ability of poplar to resist mannitol osmotic stress, characterized in that, The poplar anti-osmotic stress gene is a NATB CATALYTIC SUBUNIT gene, and the nucleotide sequence is shown as SEQ ID No.

1. The regulation is that the NATB CATALYTIC SUBUNIT gene is knocked out to improve the anti-mannitol osmotic ability of the poplar.

2. A method of improving the resistance to permeability of a forest tree, characterized in that, The method comprises the following steps: The NATB CATALYTIC SUBUNIT gene in the forest plant is knocked out, and the nucleotide sequence of the NATB CATALYTIC SUBUNIT gene is shown as SEQ ID No.

1. The forest plant is a poplar. The anti-osmotic ability is the anti-mannitol osmotic ability.

3. The method of claim 2, wherein, The NATB CATALYTIC SUBUNIT gene in the forest plant is knocked out The method comprises the following steps: transforming a recombinant expression vector into cells of a target plant to knock out the NATB CATALYTIC SUBUNIT gene, wherein the recombinant expression vector comprises a basic vector and a gRNA inserted into the basic vector, and the nucleotide sequence of the gRNA is shown as SEQ ID No. 2.

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