Application of nucleoside phosphorylase genes PtNP10 / 11 / 12 in altering the high ammonium tolerance of Populus plants

Abstract

Application of nucleoside phosphorylase genes PtNP10 / 11 / 12 in altering the high ammonium tolerance of Populus plants, which relates to the field of genetic engineering, and particularly to an application of nucleoside phosphorylase genes PtNP10 / 11 / 12 in altering the high ammonium tolerance of Populus plants. The present invention provides a new use of nucleoside phosphorylase genes, laying a foundation for cultivating new germplasm materials of forest trees with efficient nitrogen utilization. Application of nucleoside phosphorylase genes PtNP10 / 11 / 12 in altering the high ammonium tolerance of Populus plants. Under high ammonium stress, the growth vigor, aboveground biomass and photosynthetic capacity of the nucleoside phosphorylase gene function-deficient mutants are stronger than those of wild-type plants. The present invention is used for wood improvement and cultivating new germplasm materials of forest trees.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly to the application of a nucleoside phosphorylase gene PtNP10 / 11 / 12 in changing the high ammonium tolerance of Populus plants. Background Art

[0002] The area of Populus plantations ranks first in the world, providing a huge amount of wood for the industry every year and playing an important ecological function in soil and water conservation, water conservation, etc. Affected by factors such as human agricultural production activities, climate change, altered precipitation patterns, and nitrogen deposition, nitrogen is overly enriched in the soil in some parts of the country and extends to the water ecological environment. Excessive nitrogen deposition changes the physical and chemical properties of the soil and often causes serious harm to the ecosystem imbalance, such as algal blooms. Ammonium nitrogen is a main form of nitrogen, and excessive ammonium nitrogen concentration in the soil inhibits the growth of forest trees, reduces their biomass and nitrogen use efficiency. Therefore, exploring the key functional genes of forest trees (Populus) with high ammonium tolerance and promoting the growth ability of forest trees in high ammonium habitats through genetic engineering technology helps to increase wood production and is also of great significance for ecological environment construction.

[0003] Nucleoside phosphorylases (NPs) are widely present in animals, plants and microorganisms and catalyze the enzymatic phosphorolysis reaction of nucleosides accompanied by the cleavage of N-glycosidic bonds. At present, NPs have been studied more in the medical field, while people know little about the physiological functions of NPs in plants. The purine nucleoside phosphorylase in potatoes can act as a negative regulator of cytokinin, and down-regulating the expression of this gene can shorten the dormancy time of tubers. Overexpressing the purine nucleoside phosphorylase gene of Rosa roxburghii can significantly improve the resistance of Poncirus trifoliata to salt, oxidative stress, drought and pests. However, so far, there is no report on the involvement of nucleoside phosphorylase genes in changing the growth ability of plants (including forest trees) with high ammonium tolerance. Summary of the Invention

[0004] The purpose of the present invention is to provide a new use of the nucleoside phosphorylase gene PtNP10 / 11 / 12, laying a foundation for cultivating new germplasm materials of forest trees with efficient nitrogen utilization.

[0005] The present invention provides the application of the nucleoside phosphorylase gene PtNP10 / 11 / 12 in changing the high ammonium tolerance of Populus plants.

[0006] Furthermore, under high ammonium stress, the growth potential of the functional deletion mutants of the nucleoside phosphorylase gene PtNP10 / 11 / 12 is stronger than that of wild-type plants.

[0007] The growth potential includes plant height, internode length and internode diameter.

[0008] Furthermore, under high ammonium stress, the above-ground biomass of the loss-of-function mutants of the nucleoside phosphorylase genes PtNP10 / 11 / 12 is higher than that of wild-type plants.

[0009] The above-ground biomass includes the fresh weight and dry weight of the above-ground part.

[0010] Furthermore, under high ammonium stress, the photosynthetic capacity of the loss-of-function mutants of the nucleoside phosphorylase genes PtNP10 / 11 / 12 is stronger than that of wild-type plants.

[0011] The parameters related to photosynthetic capacity include net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate.

[0012] Furthermore, under high ammonium stress, the growth vigor of the triple mutant np10np11np12 is better than that of the single mutant np12 and the double mutant np10np11.

[0013] Furthermore, the Populus plant is Populus trichocarpa.

[0014] Advantages of the present invention:

[0015] In the present invention, target site sequences are designed according to the PtNP10 / 11 / 12 gene sequences, and the PtNP10 / 11 / 12 genes are artificially mutated by the CRISPR / Cas9 technology. Loss-of-function mutants of the PtNP12 gene (np12), loss-of-function mutants of the PtNP10 / 11 double genes (np10np11), and loss-of-function mutants of the PtNP10 / 11 / 12 triple genes (np10np11np12) are respectively screened out. Using 10 mM NH 4+ High ammonium stress treatment is carried out on three types of mutants and wild-type Populus trichocarpa saplings. It is found that the three types of mutant plants are stronger than the wild type in terms of plant height, stem node length, stem node width, above-ground tissue biomass, photosynthetic capacity, etc., and the growth vigor of the triple mutant np10np11np12 is better than that of the single mutant np12 and the double mutant np10np11. It shows that the PtNP10 / 11 / 12 genes are involved in changing the high ammonium tolerance ability of Populus trichocarpa and have functional redundancy.

[0016] The present invention discovers for the first time that the PtNP10 / 11 / 12 genes are involved in changing the high ammonium tolerance ability of Populus trichocarpa. Knocking out the PtNP10 / 11 / 12 genes significantly improves the high ammonium stress tolerance ability of Populus trichocarpa, and the plants show obvious ammonium tolerance. As candidate genes for forest tree improvement, PtNP10 / 11 / 12 will help relieve the pressure of forest trees facing eutrophic soil and enable forest trees to achieve high yields under environmental stress, which can not only meet the industrial and agricultural demands for wood, but also be of great significance for environmental improvement. Brief Description of the Drawings

[0017] Figure 1 shows the growth phenotypes of wild-type and mutant plants under normal ammonium salt conditions;

[0018] Figure 2 shows the growth phenotypes of wild-type and mutant plants under high ammonium stress conditions;

[0019] Figure 3 shows the plant height statistics of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0020] Figure 4 shows the statistics of the number of stem nodes of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0021] Figure 5 shows the statistics of the stem node length of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0022] Figure 6 shows the statistics of the stem node diameter of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0023] Figure 7 shows the analysis of the fresh weight of the above-ground parts of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0024] Figure 8 shows the analysis of the fresh weight of the roots of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0025] Figure 9 shows the analysis of the dry weight of the above-ground parts of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0026] Figure 10 shows the analysis of the dry weight of the roots of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0027] Figure 11 shows the analysis of the net photosynthetic rate of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0028] Figure 12 shows the analysis of the stomatal conductance of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0029] Figure 13 shows the analysis of the intercellular CO2 concentration of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions;

[0030] Figure 14 shows the analysis of the transpiration rate of wild-type and mutant plants under normal ammonium salt and high ammonium stress conditions. Detailed implementation manners

[0031] The following is a detailed description of the embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0032] Example 1: Method for obtaining knockout mutants of Populus trichocarpa nucleoside phosphorylase gene

[0033] 1. Construction of gene editing vector:

[0034] Manually screen out guide RNA sequences complementary to the DNA sequences of the target genes to be knocked out (the target genes to be knocked out are PtNP12 gene, PtNP10 / 11 gene, and PtNP10 / 11 / 12 gene respectively). The PHSE401 gene editing vector carries 2 guide RNA fragments. Since the protein similarity between PtNP10 and PtNP11 is as high as 97.8%, therefore, 2 guide RNA fragments targeting both PtNP10 and PtNP11 are screened out. This gene editing vector is used to obtain PtNP10 / 11 double gene knockout mutants. In addition, the protein similarity between PtNP12 and PtNP10, PtNP11 is as high as 92.4% and 91.5% respectively. Therefore, 1 guide RNA fragment targeting both PtNP10 and PtNP11 is screened out, and another guide RNA fragment targets PtNP12. This gene editing vector is used to obtain PtNP10 / 11 / 12 triple gene knockout mutants.

[0035] The CDS sequence of the PtNP10 gene is shown as SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of its encoded protein is shown as SEQ ID NO: 2 in the sequence listing. The CDS sequence of the PtNP11 gene is shown as SEQ ID NO: 3 in the sequence listing, and the amino acid sequence of its encoded protein is shown as SEQ ID NO: 4 in the sequence listing. The CDS sequence of the PtNP12 gene is shown as SEQ ID NO: 5 in the sequence listing, and the amino acid sequence of its encoded protein is shown as SEQ ID NO: 6 in the sequence listing.

[0036] The RB to LB region of the PHSE401 vector carries guide RNA (the fragment that guides the editing of the purine nucleoside phosphorylase gene), cas gene, and hygromycin phosphotransferase gene.

[0037] The PHSE401 vector has been disclosed in the article "A CRISPR / Cas9 toolkit for multiplex genome editing in plants" (BMC Plant Biology 2014, 14:327) and was gifted by the article's author. The specific method for ligating the guide RNA in the PHSE401 vector refers to the article "A CRISPR / Cas9 toolkit for multiplex genome editing in plants".

[0038] 2. Obtaining the transformation receptor material:

[0039] Select aseptic seedlings of Populus trichocarpa that have grown well for 28 days, cut off the second, third, and fourth stem segments from top to bottom in morphology, and then cut them into small segments of 0.8 - 1.0 cm as the transformation receptor material.

[0040] 3. Activation of the Agrobacterium strain:

[0041] Thaw the Agrobacterium strain carrying the above plant expression vector stored at -80°C on ice. Dip a sterile toothpick into a small amount of the bacterial solution and streak it on a solid YEP medium plate. Seal the plate and place it upside down in an incubator at 28°C for 48 h; pick a single colony and inoculate it into 20 ml of liquid YEP medium, and culture it on a constant temperature shaker at 28°C and 200 rmp for 16 h. Then transfer the bacterial solution to 50 ml of liquid YEP medium at a ratio of 2%, and continue to culture it on a constant temperature shaker at 28°C and 200 rmp until the OD 600 value reaches 0.6. Centrifuge the bacterial solution at 2200 g for 10 min, carefully discard the supernatant, resuspend the bacterial cells in the suspension in a laminar flow hood, observe the concentration of the bacterial cell suspension, and measure the OD 600 value until the OD of the bacterial cell suspension 600 value reaches 0.4 for use.

[0042] Both the above-mentioned YEP solid and liquid media contain 50 mg / L rifampicin, 50 mg / L gentamicin, and 50 mg / L kanamycin.

[0043] 4. Genetic transformation process of Populus trichocarpa:

[0044] Put the prepared transformation receptor material into the bacterial cell suspension, add 80 μM acetosyringone, gently shake for 20 min, discard the suspension, place the receptor material on sterile filter paper to absorb the surface part of the bacterial solution, and horizontally inoculate it into a co-culture medium containing 0.04 mg / L 6-benzyladenine, 0.02 mg / L indolebutyric acid, 0.0008 mg / L thidiazuron, and 80 μM acetosyringone, and incubate it in the dark at 24 ± 1°C for 2 d.

[0045] The receptor materials after co-culture were subjected to bacterium removal treatment: first, rinsed once with 250 mg / L cefotaxime solution, and then rinsed three times with sterile water for 3 - 5 minutes each time. Then, the excess liquid on the surface of the materials was blotted dry with sterile filter paper, and they were horizontally inoculated into the initial adventitious bud screening medium containing 0.04 mg / L 6-benzyladenine, 0.02 mg / L indole-3-butyric acid, 0.0008 mg / L thidiazuron, 10 mg / L hygromycin, and 250 mg / L cefotaxime. Under the conditions of a photoperiod of 16 h / d and a light intensity of 50 μmol·m -2 ·s -1 After culturing for 20 days, they were transferred to the late screening medium containing 0.04 mg / L 6-benzyladenine, 0.02 mg / L indole-3-butyric acid, 0.0008 mg / L thidiazuron, 5 mg / L hygromycin, and 250 mg / L cefotaxime for further culturing, and resistant buds were obtained after 20 days.

[0046] When the stem length of the resistant buds was about 1 cm, they were transferred to the rooting screening medium containing 0.1 mg / L indole-3-butyric acid, 5 mg / L hygromycin, and 250 mg / L cefotaxime, and cultured under the conditions of a photoperiod of 16 h / d and a light intensity of 50 μmol·m -2 ·s -1 After 30 days, they were transplanted into nutrient soil for growth.

[0047] According to the above method, the PtNP12 gene function loss mutant lines were obtained and named np12-1, np12-2, and np12-3 in sequence; the PtNP10 / 11 double gene function loss mutant lines were obtained and named np10np11-2, np10np11-5, and np10np11-7 in sequence; the PtNP10 / 11 / 12 triple gene function loss mutant lines were obtained and named np10np11np12-3, np10np11np12-7, and np10np11np12-9 in sequence.

[0048] Example 2: Phenotypic characteristics and biomass analysis of mutant lines under high ammonium stress

[0049] The wild-type Populus trichocarpa plants were used as a control and named WT in this example. Four-week-old tissue culture seedlings of WT, np10np11, np12, and np10np11np12 mutant plants were selected for homogenization culture. The specific method was as follows: Two fully expanded leaves at the morphological upper end were retained, and they were transferred to a hydroponic box containing 1 / 2 nitrogen-free Hoagland nutrient solution with 0.5 mM NH4NO3. Five plants were planted in each hydroponic box, and the freshly prepared nutrient solution was replaced every 7 days.

[0050] After 25 days of uniform cultivation, WT, np10np11, np12, and np10np11np12 mutant saplings with consistent growth states were selected and treated with normal ammonium salts (1 mM NH 4+ ) and high ammonium (10 mM NH 4+ ), respectively. The specific method is as follows: Retain the 4 fully expanded leaves at the morphological upper end, and transfer them to hydroponic boxes containing 1 / 2 nitrogen-free Hoagland nutrient solution with normal ammonium salts and high ammonium, respectively. The height of the above-ground saplings is uniformly 13 cm, and marks are made at 13 cm from the morphological upper end downwards. Replace the freshly prepared nutrient solution every 7 days. Each strain was subjected to 3 biological replicates under the same treatment, and each biological replicate included 5 saplings. After continuous treatment for 25 days, phenotypic changes were observed. The plant height, number of stem nodes, length of stem nodes, diameter of stem nodes, fresh weight of above-ground parts, fresh weight of roots, dry weight of above-ground parts, and dry weight of roots were measured. The culture conditions for the plant materials were: 23 - 25 °C, 80 - 100 μmol·m -2 s -1 and 16 h light / 8 h dark.

[0051] Under normal ammonium salt conditions, there were no significant differences in the growth of WT, np10np11, np12, and np10np11np12 mutant saplings (as Figure 1 ). Under high ammonium conditions, the growth of WT and the three types of mutant saplings was inhibited to varying degrees. Among them, the growth inhibition of WT was the most significant, and the growth of np10np11np12 mutant saplings was hardly affected (as Figure 2 ).

[0052] Further statistical analysis data showed that under normal ammonium salt conditions, there were no obvious changes in the plant height, number of stem nodes, length of stem nodes, and diameter of stem nodes of WT and mutant saplings (as Figures 3 to 6 ). Under high ammonium conditions, the three types of mutant saplings were significantly higher than WT in terms of plant height, length of stem nodes, and diameter of stem nodes (as Figure 3 , Figure 5 , Figure 6 ), and the plant height of np10np11np12 mutant saplings reached a highly significant level compared with WT and was slightly higher than that of np10np11 and np12 mutant saplings (as Figure 3 ); the number of stem nodes of the three types of mutant saplings was slightly higher than that of WT, but did not reach a significant difference level (as Figure 4 ).

[0053] The results of biomass statistical analysis showed that under normal ammonium salt conditions, there were no significant differences in the fresh weight, dry weight of above-ground parts, fresh weight, and dry weight of roots between WT and the three types of mutant saplings (as Figures 7 to 10) Under high ammonium conditions, the fresh and dry weights of the above-ground parts of the three types of mutant saplings were significantly higher than those of the WT, and np10np11np12 was higher than np10np11 and np12 (as shown in Figure 7 and Figure 9 ); there were no significant differences in the fresh and dry weights of the roots of the three types of mutant saplings compared with the WT (as shown in Figure 8 and Figure 10 ).

[0054] Example 3: Analysis of the photosynthetic capacity of mutant lines under high ammonium stress

[0055] Select the 4th - 6th mature leaves of WT, np10np11, np12, and np10np11np12 mutant saplings grown for 25 days under normal ammonium salt and high ammonium conditions. Between 9 am and 11 am, use a LI6400 portable photosynthesis system to measure the net photosynthetic rate (μmol / m 2 / s), stomatal conductance (mol / m 2 / s), intercellular CO2 concentration (mol / m 2 / s), and transpiration rate (mmol / m 2 / s) of the leaves, and measure 10 plants for each line.

[0056] The results showed that under normal ammonium salt conditions, there were no significant differences in the photosynthetic parameters of WT and the three types of mutant saplings (as shown in Figures 11 to 14 ). Compared with normal ammonium salt, the photosynthetic parameters of WT were significantly inhibited under high ammonium conditions, while the photosynthetic parameters of the three types of mutant saplings were significantly higher than those of the WT under high ammonium (as shown in Figures 11 to 14 ), and the net photosynthetic rate and intercellular CO2 concentration were higher than those of the plants treated with normal ammonium salt (as shown in Figure 11 and Figure 13 ). Under the two ammonium salt concentrations, there were no significant differences in the photosynthetic parameters among the three types of mutants (as shown in Figures 11 to 14 ).

[0057] From the results of the above examples, it can be seen that under high ammonium stress conditions, the growth potential, above-ground biomass, and photosynthetic capacity of the PtNP10 / 11 / 12 gene function - deleted mutant plants are stronger than those of the wild type, indicating that knocking out the PtNP10 / 11 / 12 gene can improve the high - ammonium tolerance of Populus trichocarpa plants. Therefore, as an important forest tree gene adapting to high - ammonium habitats, the PtNP10 / 11 / 12 gene can lay a foundation for cultivating new germplasm materials of forest trees with efficient nitrogen utilization.

Claims

1. Application of nucleoside phosphorylase gene PtNP10 / 11 / 12 in improving the high ammonium tolerance of Populus plants; the Populus plant is Populus trichocarpa; the application is to obtain a nucleoside phosphorylase gene through gene knockout PtNP10 / 11 / 12 function - defective mutant; the nucleoside phosphorylase gene PtNP10 / 11 / 12 function - defective mutant is PtNP12 a gene function - defective mutant, PtNP10 / 11 a double - gene function - defective mutant or PtNP10 / 11 / 12 a triple - gene function - defective mutant; Among them PtNP10 The CDS sequence of the gene is shown as SEQ ID NO: 1 in the sequence listing, PtNP11 The CDS sequence of the gene is shown as SEQ ID NO: 3 in the sequence listing, PtNP12 The CDS sequence of the gene is shown as SEQ ID NO: 5 in the sequence listing.

2. The application according to claim 1, wherein: Under high ammonium stress, the growth potential of the nucleoside phosphorylase gene PtNP10 / 11 / 12 loss-of-function mutants is stronger than that of wild-type plants.

3. The application according to claim 2, characterized in that: The growth potential includes plant height, internode length, and internode diameter.

4. The application according to claim 1, wherein: Under high ammonium stress, the shoot biomass of the nucleoside phosphorylase gene PtNP10 / 11 / 12 loss-of-function mutants is higher than that of wild-type plants.

5. The application according to claim 4, characterized in that: The aboveground biomass includes aboveground fresh weight and aboveground dry weight.

6. The application according to claim 1, characterized in that: Under high ammonium stress, the nucleoside phosphorylase gene PtNP10 / 11 / 12 The photosynthetic capacity of the loss-of-function mutant is stronger than that of the wild-type plants.

7. The application according to claim 6, characterized in that: The parameters related to photosynthetic capacity include net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate.

8. The application according to claim 1, wherein: Under high ammonium stress, the triple mutant np10np11np12 showed better growth vigor than the single mutant np12 and the double mutant np10np11 .

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