Populus salt stress resistance gene and application thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a poplar salt stress resistance gene and application thereof. The poplar salt stress resistance gene provided by the application is N-ALPHA-ACETYLTRANSFERASE 20 gene, and the nucleotide sequence is shown as SEQ ID No. 1. The expression amount of the gene is significantly positively correlated with the salt resistance of plants. According to the verification of the examples, the N-ALPHA-ACETYLTRANSFERASE 20 gene can significantly improve the salt resistance of the plants after overexpression in the poplar, and does not inhibit the growth characteristics of the plants, so that the gene can be used for cultivating new varieties with high yield, high quality and salt resistance, and provides a new molecular tool for genetic improvement of the salt resistance of forest trees.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a N-ALPHA-ACETYLTRANSFERASE 20 gene and application thereof. BACKGROUND

[0002] Soil salinization seriously restricts the production of agriculture and forestry, and is one of the key limiting factors for global agricultural and forestry production and sustainable development. High concentration of salt ions can cause ion toxicity to plant cells, leading to osmotic stress and oxidative stress, thereby limiting and inhibiting the growth and development of plants. As sessile organisms, plants have evolved highly complex signal transduction pathways to enable them to cope with the damage caused by salt stress.

[0003] The GCN5-related N-acetyltransferase (GNAT) gene family is an important family of protein acetyltransferases, which plays a key role in gene expression regulation, metabolic regulation and stress response biological processes. However, there is little research on the influence of GNAT family on the salt tolerance of forest trees. SUMMARY

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

[0005] The present application provides a poplar salt stress resistance gene, which is an N-ALPHA-ACETYLTRANSFERASE 20 gene, and the nucleotide sequence is shown in SEQ ID No. 1.

[0006] The present application also provides a recombinant expression vector, which comprises a basic vector and the N-ALPHA-ACETYLTRANSFERASE 20 gene of the above technical solution inserted into the basic vector.

[0007] The present application also provides the application of the N-ALPHA-ACETYLTRANSFERASE 20 gene of the above technical solution or the recombinant expression vector of the above technical solution in one or more of the following:

[0008] (1) regulating the salt tolerance of forest trees;

[0009] (2) screening new salt-resistant germplasm of forest trees;

[0010] (3) creating new salt-resistant germplasm of forest trees.

[0011] Preferably, the forest trees include poplar trees.

[0012] The present application also provides a method for improving the salt tolerance of forest trees, characterized in that it comprises the following steps:

[0013] Overexpressing the N-ALPHA-ACETYLTRANSFERASE 20 gene in a forest tree plant.

[0014] The application further provides a salt-resistant molecular marker of the N-ALPHA-ACETYLTRANSFERASE 20 gene, wherein a single nucleotide polymorphism site exists; the single nucleotide polymorphism site is located at the 21474335bp site on chromosome 5 of Populus tomentosa, and the polymorphism is C / A.

[0015] The application further provides a primer combination for detecting the salt-resistant molecular marker, wherein the primer combination comprises NNA20-Chr05-21474335T-F, NNA20-Chr05-21474335C-F and NNA20-R, the nucleotide sequence of the NNA20-Chr05-21474335T-F is shown in SEQ ID No. 3, the nucleotide sequence of the NNA20-Chr05-21474335C-F is shown in SEQ ID No. 4, and the nucleotide sequence of the NNA20-R is shown in SEQ ID No. 5.

[0016] The application further provides application of the molecular marker or the primer combination in screening and / or creating a salt-resistant new germplasm of a forest tree.

[0017] The application further provides a method for screening a salt-resistant new germplasm of a poplar, comprising the following steps:

[0018] The primer combination is used to amplify the genomic DNA of the poplar to be tested, and an amplification product is obtained;

[0019] If the NNA20-Chr05-21474335C-F and the NNA20-R amplify a 218bp target band, and the NNA20-Chr05-21474335T-F and the NNA20-R do not amplify a band, it is indicated that the poplar to be tested is a salt-resistant germplasm, and the genotype of the Chr05-21474335 SNP site is CC.

[0020] The application further provides a method for creating a salt-resistant new germplasm of a poplar, comprising the following steps:

[0021] The genotype of the N-ALPHA-ACETYLTRANSFERASE 20 gene Chr05-21474335 SNP site is CC, and the genotype of the other poplar germplasm is crossed to obtain hybrid offspring; the nucleotide sequence of the N-ALPHA-ACETYLTRANSFERASE 20 gene is shown in SEQ ID No. 1, and the CDS sequence is shown in SEQ ID No. 2; the genomic DNA of the hybrid offspring plant is amplified by using the primer combination of the above technical solution, and the amplification product is obtained;

[0022] If 218bp target band is amplified by using NNA20-Chr05-21474335C-F and NNA20-R, and no amplification band is obtained by using NNA20-Chr05-21474335T-F and NNA20-R primer combination, it represents that the hybrid offspring plant is a new forest salt-resistant germplasm.

[0023] Beneficial effects: the present application provides a N-ALPHA-ACETYLTRANSFERASE 20 gene, the nucleotide sequence of the N-ALPHA-ACETYLTRANSFERASE 20 gene is shown in SEQ ID No. 1; the expression amount of the gene is significantly positively correlated with the salt resistance of the plant, and the N-ALPHA-ACETYLTRANSFERASE 20 gene can significantly improve the salt resistance of the plant after overexpression in the poplar, and will not inhibit the growth characteristics of the plant, so it can be used for cultivating high-yield, high-quality, salt-resistant new varieties, and provides a new molecular tool for genetic improvement of forest salt resistance.

[0024] Further, the high salt-resistant characteristic molecular marker site and primer combination of the N-ALPHA-ACETYLTRANSFERASE 20 gene provided by the present application can effectively realize the genotyping of the poplar plant, and then the salt-resistant plant is screened. BRIEF DESCRIPTION OF DRAWINGS

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

[0026] Figure 1 It is a eukaryotic cell expression vector pBI121 vector map, in which LB represents the left border, and RB represents the right border;

[0027] Figure 2 It is the growth characteristics of wild type poplar plant and overexpression N-ALPHA-ACETYLTRANSFERASE 20 positive plant;

[0028] Figure 3Root development traits of Populus wild type plants, N-ALPHA-ACETYLTRANSFERASE 20 overexpression positive plants under 100 mM salt stress;

[0029] Figure 4 Figure for N-ALPHA-ACETYLTRANSFERASE 20 gene Chr05-21474335 SNP site genotyping results;

[0030] Figure 5 Peroxidase activity, malondialdehyde content, net photosynthetic rate and plant height of Populus wild type plants, N-ALPHA-ACETYLTRANSFERASE 20 overexpression positive plants. DETAILED DESCRIPTION

[0031]

[0032] The application further provides a recombinant expression vector comprising a basic vector and the N-ALPHA-ACETYLTRANSFERASE 20 gene according to the above technical solution inserted into the basic vector.

[0033] The application further provides the use of the N-ALPHA-ACETYLTRANSFERASE 20 gene according to the above technical solution or the recombinant vector according to the above technical solution in one or more of the following aspects:

[0034] (1) regulating the salt tolerance of forest trees;

[0035] (2) screening new salt-resistant germplasm of forest trees;

[0036] (3) creating new salt-resistant germplasm of forest trees.

[0037] In the application, the forest trees preferably include poplar trees, and more preferably include populus tomentosa.

[0038] The application further provides a method for improving the salt tolerance of forest trees, comprising the following steps:

[0039] overexpressing the N-ALPHA-ACETYLTRANSFERASE 20 gene according to the above technical solution in the forest tree plants.

[0040] In the application, the overexpression of the N-ALPHA-ACETYLTRANSFERASE 20 gene according to the above technical solution in the forest tree plants preferably comprises the following steps:

[0041] transforming the recombinant expression vector according to the above technical solution into the cells of the target plants to overexpress the N-ALPHA-ACETYLTRANSFERASE 20 gene.

[0042] The application further provides a salt-resistant molecular marker site of an N-ALPHA-ACETYLTRANSFERASE20 gene, wherein a single nucleotide polymorphism site exists; the single nucleotide polymorphism site is located at the 21474335bp site on chromosome 5 of a poplar, and the polymorphism is C / A.

[0043] The application further provides a primer combination for detecting the salt-resistant molecular marker, wherein the primer combination comprises NNA20-Chr05-21474335T-F, NNA20-Chr05-21474335C-F and NNA20-R, the nucleotide sequence of the NNA20-Chr05-21474335T-F is shown in SEQ ID No. 3, specifically 5'-GGTATGAATAATAGCGCAGAA-3'; the nucleotide sequence of the NNA20-Chr05-21474335C-F is shown in SEQ ID No. 4, specifically 5'-GGTATGAATAATAGCGCAGGA-3'; and the nucleotide sequence of the NNA20-R is shown in SEQ ID No. 5, specifically 5'-CACTCCTGATGAATTGGAGTATG-3'. Preferably, the penultimate nucleotide on the 3' end of the NNA20-Chr05-21474335T-F and the NNA20-Chr05-21474335C-F is modified by LNA.

[0044] The application further provides an application of the molecular marker or the primer combination in screening and / or creating a new salt-resistant germplasm of a forest tree.

[0045] The application further provides a method for screening a salt-resistant new germplasm of a poplar, comprising the following steps:

[0046] The genomic DNA of the to-be-tested poplar germplasm is amplified by using the primer combination, and an amplification product is obtained;

[0047] If the 218bp target band is amplified by using the NNA20-Chr05-21474335C-F and the NNA20-R, and no amplification band is obtained by using the NNA20-Chr05-21474335T-F and the NNA20-R, it is represented that the to-be-tested poplar germplasm is a salt-resistant germplasm, and the genotype of the Chr05-21474335 SNP site is CC. The high salt-resistant molecular marker site and the primer combination of the N-ALPHA-ACETYLTRANSFERASE20 gene can effectively realize the genotyping of the poplar plant, and then a salt-resistant plant is screened.

[0048] The application further provides a method for creating a new salt-resistant poplar germplasm, comprising the following steps:

[0049] The genotype of the SNP site of the N-ALPHA-ACETYLTRANSFERASE20 gene Chr05-21474335 of the poplar germplasm is CC, and the poplar germplasm is crossed with other poplar germplasms to obtain hybrid offspring; the nucleotide sequence of the N-ALPHA-ACETYLTRANSFERASE20 gene is shown in SEQ ID No. 1, and the CDS sequence is shown in SEQ ID No. 2;

[0050] The genomic DNA of the hybrid offspring plant is amplified by using the primer combination in the above technical solution, and an amplification product is obtained;

[0051] If the 218bp target band is amplified by using the NNA20-Chr05-21474335C-F and NNA20-R primer combinations, and no amplification band is obtained by using the NNA20-Chr05-21474335T-F and NNA20-R primer combinations, it is represented that the hybrid offspring plant is a new salt-resistant forest tree germplasm.

[0052] In order to further illustrate the application, the poplar salt stress resistance gene and the application thereof provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0053] Example 1

[0054] 1. Cloning of N-ALPHA-ACETYLTRANSFERASE20 gene

[0055] In this example, the CDS (SEQ ID No. 2) of the N-ALPHA-ACETYLTRANSFERASE20 gene is amplified by using the cDNA of the poplar 1316 as a template by the method of RT-PCR. The primers used in the RT-PCR are as follows:

[0056] Forward primer F: 5'-ATGACGACTATTCGACGATTCAGCTGCAAC-3' (SEQ ID No. 6);

[0057] Reverse primer R: 5'-TTAATCATCTCCAATTCATCAGGAGTGAC-3' (SEQ ID No. 7).

[0058] The amplification system of RT-PCR is 25 μL, specifically: super-faith Taq enzyme 10.0 μL, forward primer 0.5 μL, reverse primer 0.5 μL, buffer 0.5 μL, cDNA template 2.0 μL, ddH2O 7.0 μL; the PCR amplification condition is: pre-denaturation 94℃ 3 min; denaturation 94℃ 10 s, annealing 62℃ 30 s, extension 72℃ 40 s, 40 cycles; the PCR product is identified in size by agarose gel electrophoresis, and then the gel is recovered. The recovered product and pBI121 vector are respectively double-enzymatically cut with Sac I and Xba I, and then recovered by gel; the T4 ligase is connected at 4℃ overnight, then the connection product is inactivated at 70℃ for 15 min, and then transferred to E. coli by heat shock method, positive clones are screened on LB medium containing 50 mg / L kanamycin, and identified by sequencing. The identification result of the sequence containing SEQ NO. 2 is the overexpression vector.

[0059] 2. Construction of N-ALPHA-ACETYLTRANSFERASE20 overexpression vector

[0060] (1) Enzymatic cutting: select BamH I as the enzyme cutting site to cut the overexpression vector pBI121. Incubate at 37℃ for several hours to ensure complete cutting of the cutting reaction. The linearized vector cut by BamH I is detected by electrophoresis, and the gel is recovered.

[0061] (2) Ligation: use the Seamless Cloning and Assembly Kit kit to connect the DNA fragment with the pBI121 vector (pBI121) which has been cut with BamH I. Figure 1 ) The detailed operation is described in the kit instruction manual.

[0062] (3) Transformation: transform the ligation product into E. coli DH5α competent cells. Use the heat shock freezing method for cell transformation. Uniformly spread the transformation product on LB agar plates containing an appropriate amount of antibiotic for culture.

[0063] (4) Screening: pick the resistant colonies from the LB agar plate, and perform PCR verification or sequencing to confirm the correctness and directionality of the inserted fragment.

[0064] Plasmid extraction and preservation: extract the plasmid from the verified positive colonies. Use the high-purity plasmid extraction kit (DP104) produced by Tiangen to extract the DNA plasmid, and store the reverse transcription reaction product at -20℃.

[0065] 3. Genetic transformation of N-ALPHA-ACETYLTRANSFERASE20 overexpression vector

[0066] ​Put competent Agrobacterium GV3101 on ice, add 1 g of plasmid DNA containing the 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 cells and spread on YEB plates containing 30 mg / L kanamycin and dry, and incubate at 28°C for 48 h to obtain Agrobacterium containing the overexpression vector.

[0067] Select fresh and disease-free leaves of healthy poplar tissue culture seedlings as leaf disc transformation materials. After surface cleaning and disinfection of the leaves, cut them into small pieces or blocks.

[0068] Incubate Agrobacterium containing the overexpression vector on LB medium at 28°C to the logarithmic growth phase.

[0069] Put the leaves into the resuspended Agrobacterium bacterial solution for co-culture, and then 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.

[0070] Transplant the new shoots from the differentiation medium to rooting medium to promote plant formation. Use the overexpression vector pBI121 with a selective marker gene to screen the transformed leaf discs to obtain transformed positive plants. Extract the DNA of the screened plants and detect by PCR to confirm the presence of the exogenous gene, thereby verifying the successful introduction of the overexpression vector.

[0071] 4. N-ALPHA-ACETYLTRANSFERASE20 overexpression poplar plants improve salt resistance

[0072] When the N-ALPHA-ACETYLTRANSFERASE20 overexpression poplar transgenic plants and wild type poplar plants (plants without introduction of exogenous genes) reached 4 weeks old, the tissue culture seedlings were taken out from the culture medium, transplanted into the prepared soil, placed in the poplar culture room, and subjected to 8 weeks of soil culture, as shown in Figure 2

[0073] As can be seen from Figure 2 , overexpression of the N-ALPHA-ACETYLTRANSFERASE20 gene has no significant inhibitory effect on the height of poplar plants.

[0074] ​The N-ALPHA-ACETYLTRANSFERASE 20 overexpression plants were subcultured, and the wild type (plants without introduction of foreign genes) and overexpression lines were treated with 0 mM, 50 mM, 85 mM and 100 mM salt stress for 7 days, respectively, as shown in Figure 3 .

[0075] It can be seen from Figure 3 that overexpression of N-ALPHA-ACETYLTRANSFERASE 20 gene promotes the development of poplar root system.

[0076] Example 2

[0077] 1. Salt-resistant genotyping of forest tree germplasm resource population

[0078] Using the DNA of the Populus tomentosa germplasm resource population as a template, primers were designed for the NNA20-Chr05-21474335 single nucleotide polymorphism site:

[0079] NNA20-Chr05-21474335T-F: 5'-GGTATGAATAATAGCGCAGAA-3' (SEQ ID No. 3)

[0080] NNA20-Chr05-21474335C-F: 5'-GGTATGAATAATAGCGCAGGA-3' (SEQ ID No. 4)

[0081] NNA20-R: 5'-CACTCCTGATGAATTGGAGTATG-3' (SEQ ID No. 5);

[0082] Among them, NNA20-Chr05-21474335 represents the 21474335 bp site on chromosome 5 of Populus.

[0083] The PCR amplification system was 25 μL, specifically: super-fidelity Taq enzyme 10.0 μL, forward primer 0.5 μL, reverse primer 0.5 μL, buffer 0.5 μL, cDNA template 2.0 μL, ddH2O 7.0 μL; the PCR amplification conditions were: pre-denaturation 94℃ for 3 min; denaturation 94℃ for 10 s, annealing 62℃ for 30 s, extension 72℃ for 40 s, 40 cycles; the PCR product was identified by electrophoresis, as shown in Figure 4As shown in the diagram. TF / R represents the primer combination of NNA20-Chr05-21474335T-F and NNA20-R; CF / R represents the primer combination of NNA20-Chr05-21474335C-F and NNA20-R. TT represents the genotype of the Chr05-21474335 SNP locus as TT, TC represents the genotype of the Chr05-21474335 SNP locus as TC, and CC represents the genotype of the Chr05-21474335 SNP locus as CC.

[0084] Depend on Figure 4 It can be seen that the primer combination of NNA20-Chr05-21474335T-F and NNA20-R amplified a 218bp target band, while the primer combination of NNA20-Chr05-21474335C-F and NNA20-R did not amplify the target band, indicating that the genotype of the Chr05-21474335SNP site is TT;

[0085] The primer combination NNA20-Chr05-21474335T-F and NNA20-R did not amplify the target band, while the primer combination NNA20-Chr05-21474335C-F and NNA20-R amplified a 218bp target band, indicating that the genotype of the Chr05-21474335SNP site is CC.

[0086] The NNA20-Chr05-21474335T-F primer combination with NNA20-R amplified a 218bp target band, and the NNA20-Chr05-21474335C-F primer combination with NNA20-R amplified a 218bp target band, indicating that the genotype of the Chr05-21474335SNP locus is TC.

[0087] 2. Hybridization of salt-tolerant forest tree germplasm

[0088] Individuals with the homozygous CC genotype at the NNA20-Chr05-21474335 single nucleotide polymorphism site were selected as the parents for the mating combination, with the male parent being Populus tomentosa'2-12' and the female parent being Populus tomentosa'5017'. In this example, both the male and female parent plants were obtained from the National Excellent Populus tomentosa Germplasm Resource Bank (Guanxian County, Shandong Province). Hybrid breeding was carried out to create the F1 generation hybrid population.

[0089] 3. Screening of new salt-tolerant forest tree germplasm

[0090] The DNA of the F1 hybrid population of Populus tomentosa was used as a template, and NNA20-Chr05-21474335T-F and NNA20-R, NNA20-Chr05-21474335C-F and NNA20-R primer combinations were used to screen the salt-resistant new germplasm of Populus.

[0091] The NNA20-Chr05-21474335T-F and NNA20-R primer combination did not amplify the 218 bp target band, and the NNA20-Chr05-21474335C-F and NNA20-R primer combination amplified the 218 bp target band, representing the CC genotype of the salt-resistant new germplasm of Populus.

[0092] 4. Improvement of salt resistance of new salt-resistant germplasm of forest trees

[0093] When the screened salt-resistant new germplasm of Populus reached 4 weeks old, the tissue culture seedlings were removed from the culture medium and transplanted into the prepared soil in the Populus culture room for 8 weeks of soil culture. Subsequently, the CC three genotype offspring of the NNA20-Chr05-21474335 single nucleotide polymorphism site and the TT and TC genotype individuals of the natural population were treated with 100 mM NaCl for 7 days.

[0094] The peroxidase activity, malondialdehyde content and net photosynthetic rate of Populus 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.

[0095] It can be seen from Figure 5 that compared with the other two genotypes, the CC genotype of the salt-resistant new germplasm of Populus increased the average peroxidase activity to 5157 μmol (guaincol) mg -1 (Protein)min -1 under 100 mM salt stress Figure 5 (A), the average malondialdehyde content decreased to 11.8 μmol g -1 FW Figure 5 (C), and the average net photosynthetic rate was maintained at 14.2 μmol m -2 s -1 under 100 mM salt stress Figure 5Medium (B)), the plant height trait was not significantly inhibited Figure 5 Medium (D)).

[0096] As can be seen from the above examples, the overexpression of the N-ALPHA-ACETYLTRANSFERASE 20 gene in poplar can significantly improve the salt resistance of the plant, and will not inhibit the growth characteristics of the plant. Using the high salt resistance molecular marker site and primer combination of the N-ALPHA-ACETYLTRANSFERASE 20 gene provided by the present application, the genotyping of the poplar plant can be effectively realized, and then the salt-resistant plants are screened out.

[0097] 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. Application of a Populus salt stress resistance gene or a recombinant expression vector in regulating salt tolerance of Populus: the Populus salt stress resistance gene is an N-ALPHA-ACETYLTRANSFERASE20 gene, and a CDS sequence of the N-ALPHA-ACETYLTRANSFERASE20 gene is shown as SEQ ID No. 2; the recombinant expression vector comprises a base vector and an N-ALPHA-ACETYLTRANSFERASE20 gene inserted into the base vector; and the regulation is that overexpression of the N-ALPHA-ACETYLTRANSFERASE20 gene improves salt resistance characteristics of Populus. The method comprises the following steps: overexpressing the N-ALPHA-ACETYLTRANSFERASE20 gene in a forest plant; 2. A method for improving salt tolerance of a forest tree, characterized by, the forest plant is Populus. ​ ​

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