Knocking out the puki gene of a plant in a method of promoting plant growth and development and enhancing plant growth under nutrient deficiency conditions

Abstract

The application discloses a method for promoting plant development and enhancing plant growth under nutrient deficiency conditions by knocking out PUKI genes of plants. The application focuses on the regulation and application of the knockout of PUKI genes of plants to the development of aerial tissues and root systems under normal and nutrient deficiency conditions. After the PUKI genes in plants are knocked out, the development of aerial tissues and root systems of the plants is superior to that of wild types. The PUKI gene deletion mutant strains have more significant advantages in the development of aerial tissues and root systems under the culture conditions of the single deficiency of nitrogen, phosphorus and potassium and the simultaneous deficiency of the three nutrient elements. The application promotes the overall growth of plants by knocking out the PUKI genes in plants, and can be applied to land barren areas, and provides a new target gene and material for the cultivation of high-quality stress-resistant crop varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for promoting plant growth and development and enhancing plant growth under nutrient deficiency conditions by knocking out PUKI gene of plants, and belongs to the field of gene application. BACKGROUND

[0002] Pseudouridine is one of the most common RNA modifications in cells, which exists in various types of RNAs such as mRNA, tRNA, rRNA, snRNA and snoRNA. As a post-transcriptional modification mechanism, pseudouridine can affect the structure, function, location and half-life of RNA, and thus regulate gene expression. It has been reported that the expression regulation of some genes in plants may depend on pseudouridine modification, and affect the growth and development process of plants, such as root development, leaf morphogenesis, flowering and fruit ripening, etc. The metabolic pathway of pseudouridine is completed by pseudouridine kinase (PUKI) and pseudouridine nucleotide hydrolase (PUMY). Therefore, PUKI gene is particularly important for the regulation of plant growth and development, but such research is still relatively few.

[0003] Rice is one of the most important food crops in China, and its production development plays a crucial role in ensuring food security in China and even the world. Root development plays a decisive role in the effective absorption of water and various mineral nutrients such as nitrogen, phosphorus and potassium by rice, providing sufficient material basis for the normal growth and development of rice plants. The demand for nitrogen, phosphorus, potassium and other macronutrients is extremely large during the growth of rice, but in reality, not all lands can provide sufficient nitrogen, phosphorus and potassium nutrients. Under this background, it is of utmost importance to cultivate rice varieties with excellent adaptability. And further exploration of plant stress tolerance genes can provide an important idea to solve this nutrient supply problem. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for promoting plant growth and development and enhancing plant growth under nutrient deficiency conditions by knocking out PUKI gene of plants.

[0005] Technical scheme: In order to solve the above technical problem, the present application provides an application of knocking out PUKI gene of plants in promoting plant growth and development.

[0006] The plant includes Arabidopsis thaliana or rice.

[0007] The gene number of the PUKI gene is AT1G49350 (Arabidopsis thaliana) or LOC_Os05g09370 (rice).

[0008] The present application also provides an application of PUKI gene in regulating the growth and development of plants under nutrient element-deficient culture conditions.

[0009] The nutrient element comprises one or more of nitrogen, phosphorus or potassium.

[0010] The plant growth and development is promoted.

[0011] The plant growth and development is promoted by knocking out the PUKI gene of the plant.

[0012] The gene number of the PUKI gene is AT1G49350 or LOC_Os05g09370.

[0013] The application of knocking out the Arabidopsis PUKI gene in the regulation of root development under normal and nutrient deficiency culture conditions, the Atpuki mutant and the wild type plant are cultured under normal and nutrient deficiency culture conditions, the nucleotide sequence of the AtPUKI gene is shown as SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.

[0014] SEQ ID No. 1 (wherein the capital letters are CDS sequences):

[0015]

[0016] SEQ ID No. 2:

[0017] MEPVIIGALILDVHAKPSTTPISGTTVPGQVLFAPGGVARNVADCIFKLGITPFMIGTLGLDGPANVLLKEWKLSMKGILRREDISTPIVSLVYDTNGEVAAGVAGVDAVENFLTPEWIQRFEYNISSARLLMVDANLSSLALEASCKLAAESSVPVWFEPVSVTKSQRIASIAKYVTIVSPNQDELIAMANALCAKNLFHPFRSDENKLSIEDMFRALKPAILVLLKNGVKVVIVTLGSNGALLCSKGNPKKALNIDRKFLRSGEVFKRVQSVCSPNRFSELGSNRSPSLFAMHFPTIPAKVKKLTGAGDCLVGGTVASLSDGLDLIQSLAVGIASAKAAVESDDNVPPEFKLDLISGDAELVYNGAKMLMVHQSML

[0018] The application relates to the application of knockout of rice PUKI gene in the regulation of the growth and development of the aerial part and / or root system of plants under normal and nutrient deficiency culture conditions. The specific method is as follows: knockout of the rice PUKI gene to obtain an Ospuki mutant, culture of the Ospuki mutant and wild-type plants under normal and nutrient deficiency culture conditions, and the nucleotide sequence of the OsPUKI gene is shown in SEQ ID No. 3, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 4.

[0019] SEQ ID No. 3 (wherein the capital letters represent the CDS sequence):

[0020]

[0021] SEQ ID No.4:

[0022] MAGATTTSSPLRRMESVCRHLLPASPPILYQNPLGAIRLESSPVIIGGMVLDIHAKPSMQPHPGTTVPGMVKYVSGGVARNIAECICKLETRPFMISVVGNDMAGDFLLKYWRSAGLCTDGILQIDDVTTPIVSNVFDGSGELIAGVASVGAVEKFLSPSWICQFRLHISTAPLLMLDANLSPDSLEAACKIAHESGVPVFFEPVSLAKGSRIAPIAKYITYTSPNEIELVAMANSLSPPEKYTFVKMEQSKNKAKAVEYLFEMLSPAMFFLLEKGIKFLLVTLGSNGVFVCCKESTSLMDQRKSEMMSFSTPLLQKLERCFPSNMLVDLPREGSSRTCVFHFPAVSASVVSLTGAGDCFVGGVISALCGGLGMMQSVAVGIAIAKSSVESEANIPDKFSAATIADDARRTLLSAKMMWCK

[0023] The promoting plant growth and development comprises promoting growth of aerial tissues and / or root systems of the plant.

[0024] Compared with the prior art, the PUKI gene knockout plant in the application has better performance in the growth and development process, especially under the conditions of nitrogen, phosphorus and potassium deficiency, and the development advantages of aerial tissues and root systems of the plant are more significant, and the plant can be applied to growth in land-poor areas. The application not only provides a new genetic resource for crop variety improvement, but also opens up a new way for the cultivation of stress-tolerant crops, and has great application value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure 1 is a sequence alignment diagram of plant-derived PUKI genes;

[0026] Figure 2 Figure 2 is a root length phenotype change of Arabidopsis thaliana PUKI gene function loss mutant and wild type plant under normal culture conditions; wherein a is a development phenotype comparison of the plant, and b is a fold line statistical diagram of root elongation; wherein WT is wild type, Atpuki-1 and Atpuki-2 are Arabidopsis thaliana PUKI gene function loss mutants; and asterisk indicates significant difference (variance analysis; p value is less than 0.05);

[0027] Figure 3 The root length phenotypic changes of Arabidopsis thaliana PUKI gene loss-of-function mutant and wild-type plants under phosphorus-deficient culture conditions are shown in Figure a. Figure a compares the developmental phenotypes of plant roots, and Figure b is a line graph of seedling root elongation. WT represents the wild-type, while Atpuki-1 and Atpuki-2 are Arabidopsis thaliana PUKI gene loss-of-function mutants. Asterisks indicate significant differences (ANOVA; *p < 0.05).

[0028] Figure 4 Comparison of root development phenotypes between wild-type and PUKI gene loss-of-function mutant rice; where a is a comparison of root development phenotypes, and b is a line graph of seedling root elongation; asterisks indicate significant differences (analysis of variance; *p value < 0.05);

[0029] Figure 5 The development of wild-type and PUKI gene loss-of-function mutant rice plants under phosphorus-deficient culture conditions is shown in Figure a. Plant development phenotype comparison, Figure b. Relative root elongation, and Figure c. Relative plant height growth; asterisks indicate significant differences (analysis of variance; p-value < 0.01).

[0030] Figure 6 The development of wild-type and PUKI gene loss-of-function mutant rice plants under nitrogen-deficient culture conditions is shown in Figure a. Plant development phenotype comparison, Figure b. Relative root elongation, and Figure c. Relative plant height growth; asterisks indicate significant differences (analysis of variance; p-value < 0.01).

[0031] Figure 7 The development of wild-type and PUKI gene loss-of-function mutant rice plants under potassium-deficient culture conditions is shown in Figure a. Plant development phenotype comparison, Figure b. Relative root elongation, and Figure c. Relative plant height growth; asterisks indicate significant differences (analysis of variance; p-value < 0.01).

[0032] Figure 8 The development of wild-type and PUKI gene loss-of-function mutant rice plants under nitrogen, phosphorus, and potassium deficiency culture conditions is shown in Figure a. Plant development phenotype comparison, Figure b. Relative root elongation, and Figure c. Relative plant height growth. Asterisks indicate significant differences (analysis of variance; p-value < 0.01). Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0034] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0035] The PUKI gene deletion mutant includes an Arabidopsis PUKI gene deletion mutant and a rice PUKI gene deletion mutant.

[0036] The Arabidopsis Atpuki mutant SALK_006361 (Atpuki-1) and SAIL_647C12 (Atpuki-2) used in the present application are from the Nottingham Arabidopsis Stock Centre, and the rice PUKI gene knockout target sequence is SEQ ID NO. 5: ACAGACTCCATCCTCCGCAGGGG. The rice PUKI gene deletion mutant (Ospuki) is ordered from BGI-GENOMICS (Jiangsu) CO., LTD., and the germplasm number is BG110392C07.

[0037] The present application uses sodium hypochlorite for seed disinfection.

[0038] The present application uses different plant nutrient solutions for rice plant cultivation. The normal nutrient solution formula is shown in Table 1. The composition of the nutrient solution with nutrient deficiency: in the normal nutrient solution, the KH2PO4 in the culture medium formula is deleted to obtain a nutrient solution lacking P element; the KNO3 in the normal culture medium formula is replaced with NH4NO3 to obtain a nutrient solution lacking K element; the KNO3 in the normal nutrient solution is deleted to obtain a nutrient solution lacking N element; the KH2PO4 and KNO3 in the normal nutrient solution are deleted to obtain a nutrient solution lacking all of K, P and N elements. The solid culture medium used for Arabidopsis culture needs to add 8% agar in the above-mentioned nutrient solution, and is used after sterilization at 121℃.

[0039] Table 1

[0040]

[0041]

[0042] The present application uses SPSS software for variance analysis of the root system and plant height growth of rice and Arabidopsis.

[0043] Example 1: Alignment analysis of PUKI gene sequences among different plants

[0044] The Arabidopsis thaliana AtPUKI gene used in the application is obtained from the Tair website (https: / / www.arabidopsis.org / ), and the gene number is: AT1G49350. The protein sequence of AtPUKI is used as bait to perform blast on the Phytozome website (https: / / phytozome-next.jgi.doe), select plant source search results and download sequences, perform sequence alignment using an online sequence alignment website (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo), and use Jalview software to display the alignment results, as shown in Figure 1 .

[0045] Example 2: Application of Arabidopsis thaliana (variety: Columbia (Col)) PUKI gene deletion in regulating plant growth and development under normal culture conditions

[0046] The application uses 10% sodium hypochlorite to surface disinfect Arabidopsis thaliana seeds of wild type (WT) and PUKI gene deletion mutants (Atpuki-1, Atpuki-2), respectively. After disinfection for 10 minutes, rinse 3-5 times with sterile water to remove the disinfectant. The seeds are planted in solid culture medium, 4°C vernalization for two days, and then placed in a light incubator for light culture (light / dark is 16 / 8 hours). After 5 days, plants with uniform root length are transferred to new solid culture medium for continuous culture to record root elongation. The results show that the root elongation of PUKI gene deletion mutants is significantly higher than that of wild type ( Figure 2 ), indicating that PUKI gene deletion promotes the growth and development of Arabidopsis thaliana roots.

[0047] Example 3: Application of Arabidopsis thaliana PUKI gene deletion in regulating plant growth and development under phosphorus deficiency culture conditions

[0048] Arabidopsis thaliana seedlings of wild type (WT) and PUKI gene deletion mutants (Atpuki-1, Atpuki-2) with uniform growth are transferred to solid culture medium lacking P element (reduce KH2PO4 in the normal nutrient solution in Table 1 and add 8% agar). The results show that under phosphorus deficiency culture, the root elongation of PUKI gene deletion mutants is significantly higher than that of wild type ( Figure 3 ), indicating that PUKI gene deletion promotes the growth and development of Arabidopsis thaliana plants under low-phosphorus culture conditions.

[0049] Example 4: Application of PUKI gene deletion in regulating the growth and development of rice (variety: Zhonghua 11, ZH11)

[0050] Selecting the wild type (Zhonghua 11) and PUKI gene deletion mutant (Ospuki) of rice seeds, using 30% sodium hypochlorite solution for disinfection, and using sterile water repeatedly for 4-5 times after washing, placing the culture dish in a humidified culture dish, placing the culture dish in a light culture room for germination, and using deionized water to keep the culture dish clean and humid. After the bud germination, the culture dish was moved to the culture box, and 1L of normal nutrient solution of Table 1 was added, and the culture solution was replaced every 3 to 4 days. The elongation of the root system was recorded daily. The results show that under normal culture conditions, the root length of Ospuki rice is significantly higher than that of wild type rice (Ospuki) (Ospuki), indicating that PUKI gene deletion also plays an important role in the growth and development of rice plants. Figure 4

[0051] Example 5: Application of PUKI gene deletion to the regulation of rice growth and development under phosphorus deficiency conditions

[0052] The experimental method is the same as that of Example 4, and the KH2PO4 in the normal nutrient solution of Table 1 is not added. The results show that under the condition of phosphorus deficiency culture, the root length and plant height of Ospuki rice are significantly higher than those of wild type rice (Ospuki) (Ospuki), indicating that PUKI gene deletion plays an important role in the regulation of rice plant growth and development under phosphorus deficiency conditions. Figure 5

[0053] Example 6: Application of PUKI gene deletion to the regulation of rice growth and development under potassium deficiency conditions

[0054] The experimental method is the same as that of Example 4, and KNO3 in the normal nutrient solution of Table 1 is replaced by NH4NO3. The results show that under the condition of potassium deficiency culture, the root length and plant height of Ospuki rice are significantly higher than those of wild type rice (Ospuki) (Ospuki), indicating that PUKI gene deletion plays an important role in the regulation of rice plant growth and development under potassium deficiency conditions. Figure 6

[0055] Example 7: Application of PUKI gene deletion to the regulation of rice growth and development under nitrogen deficiency conditions

[0056] The experimental method is the same as that of Example 4, and KNO3 in the normal nutrient solution of Table 1 is not added. The results show that under the condition of nitrogen deficiency culture, the root length and plant height of Ospuki rice are significantly higher than those of wild type rice (Ospuki) (Ospuki), indicating that PUKI gene deletion plays an important role in the regulation of rice plant growth and development under nitrogen deficiency conditions. Figure 7

[0057] ​​​​Example 8: Application of PUKI gene deletion in the regulation of rice growth and development under the conditions of potassium, nitrogen and phosphorus deficiency

[0058] The experimental method is the same as that in Example 4, except that KH2PO4 and KNO3 are not added in the normal nutrient solution in Table 1. The results show that under the culture conditions of simultaneous deficiency of potassium, nitrogen and phosphorus, the root length and plant height of Ospuki rice are still significantly higher than those of wild-type rice Figure 8 ), indicating that PUKI gene deletion plays an important regulatory role in the growth and development of rice plants under the culture conditions of simultaneous deficiency of potassium, nitrogen and phosphorus.

Claims

1. Knock out the plant PUKI The application of genes in promoting plant growth and development under normal conditions is characterized by, The plant is Arabidopsis thaliana or rice; PUKI The nucleotide sequence of the gene is shown in SEQ ID No. 1; the growth and development refers to root elongation or plant height growth.

2. Knock out the plants PUKI The application of genes in promoting plant growth and development under nutrient-deficient culture conditions is characterized by, The plant is Arabidopsis thaliana or rice; the nutrients include one or more of nitrogen, phosphorus, or potassium; PUKI The nucleotide sequence of the gene is shown in SEQ ID No. 1; the growth and development refers to root elongation or plant height growth.

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