Application of overexpressed OsNAC42 gene in improving low phosphorus stress resistance of rice

Abstract

The invention provides application of an overexpressed OsNAC42 gene in improving low-phosphorus stress resistance of rice, and belongs to the technical field of gene engineering. The nucleotide sequence of the OsNAC42 gene is shown as SEQ ID NO: 1, and a transgenic rice plant overexpressed by the OsNAC42 gene can be obtained by constructing an overexpression vector of the OsNAC42 gene and transforming rice. Under the condition of low-phosphorus stress, compared with wild type rice, a rice plant over-expressed with the OsNAC42 gene shows excellent characters such as improved root structure, enhanced phosphorus absorption capacity, remarkably improved biomass and phosphorus accumulation and the like, so that the adaptive capacity of rice to a low-phosphorus environment is effectively improved. The invention provides an important gene resource and a feasible technical approach for cultivating a new variety of low-phosphorus-resistant rice, and has important significance for saving phosphate fertilizer resources and promoting agricultural sustainable development.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of overexpression of the OsNAC42 gene in improving the resistance of rice to low phosphorus stress. Background Technology

[0002] Phosphorus is one of the essential macroelements for plant growth and development, participating in many key physiological and biochemical processes such as energy transfer, signal transduction, nucleic acid synthesis, and cell membrane construction. In agricultural production, the lack of available phosphorus in the soil is one of the main factors limiting crop yield. Especially in rice cultivation systems, phosphorus in the soil is easily fixed into forms that are difficult for plants to absorb and utilize, leading to problems such as slow growth, reduced tillering, purplish-red leaves, and a significant decrease in yield in rice under phosphorus-deficient conditions.

[0003] Currently, conventional approaches to improving crop tolerance to low phosphorus levels mainly include increasing phosphate fertilizer application, soil improvement, and breeding low-phosphorus tolerant varieties. However, phosphate fertilizer production relies on non-renewable phosphate rock resources, and long-term, large-scale application not only increases production costs but also leads to environmental pollution and ecological imbalance. While traditional breeding methods can screen for low-phosphorus tolerant germplasm, the breeding cycle is long and limited by existing genetic variations. Therefore, elucidating the genetic mechanisms of rice's response to low phosphorus stress at the molecular level and directionally improving rice's phosphorus efficiency through genetic engineering has become an important research direction for the sustainable development of modern agriculture.

[0004] Plants initiate a series of adaptive responses under low phosphorus stress, including changes in root architecture, activation of phosphorus transporters, secretion of organic acids, and redistribution of phosphorus. These processes are precisely regulated by a multi-level gene expression network. Transcription factors play a crucial role in this regulatory network, among which the NAC (NAM, ATAF1 / 2, CUC2) family, as a plant-specific class of transcription factors, has been shown to be widely involved in abiotic stress responses, growth and development, and hormone signal transduction. In recent years, studies have found that some NAC transcription factors are involved in regulating phosphorus deficiency responses. For example, ANAC042 in Arabidopsis has been reported to be associated with low phosphorus stress. However, the specific function and regulatory mechanism of the OsNAC42 gene in rice under low phosphorus stress remain unclear, and its application potential in the genetic improvement of low phosphorus tolerance in rice remains to be explored.

[0005] While several rice genes related to phosphorus uptake and utilization (such as phosphorus transporter genes OsPTs) have been identified in the current technology, genetic engineering research based on the regulation of multiple low-phosphorus tolerance traits by transcription factor systems remains relatively limited. As a member of the rice NAC family, whether OsNAC42 can enhance rice's adaptability to low-phosphorus environments by regulating the expression of a series of downstream phosphorus stress response genes has not yet been publicly reported or applied. Summary of the Invention

[0006] In view of this, the present invention provides the application of overexpression of the OsNAC42 gene in improving the resistance of rice to low phosphorus stress, providing a new gene resource and technical approach for solving the phosphorus nutrition problem in rice production.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of overexpression of the OsNAC42 gene in any one or more of the following:

[0009] (1) Its application in promoting rice growth;

[0010] (2) Application in improving phosphorus absorption in rice;

[0011] (3) Application in improving the low phosphorus tolerance of rice;

[0012] The nucleotide sequence of the OsNAC42 gene is shown in SEQ ID NO.1.

[0013] The present invention also provides an overexpression vector for expressing the OsNAC42 gene, comprising the OsNAC42 gene and a basic plasmid.

[0014] Preferably, the base plasmid is the pCAMBIA1300-UBI-HA-3*Flag-NOS vector.

[0015] The present invention also provides a method for improving the resistance of rice to low phosphorus stress, comprising the following steps:

[0016] (1) Using rice genomic cDNA as a template, PCR amplification was performed using OsNAC42-OE-F and OsNAC42-OE-R as primers to obtain the OsNAC42 gene sequence;

[0017] (2) The pCAMBIA1300-UBI-HA-3*Flag-NOS vector was digested with BamHI and ligated with the OsNAC42 gene sequence to obtain an overexpression vector;

[0018] (3) The overexpression vector from step (2) is introduced into rice callus tissue and cultured into transgenic rice.

[0019] Preferably, the sequences of OsNAC42-OE-F and OsNAC42-OE-R are as shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0020] Preferably, the PCR amplification reaction system is 50 μL, including 1 μL of cDNA template, 2 μL each of upstream and downstream primers, 25 μL of Biorun Pfu PCR Mix, and 20 μL of Nuclease-free Water.

[0021] Preferably, the PCR amplification reaction program is as follows: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 50 °C annealing for 45 s, 72 °C extension for 72 s, cycle number 30; 72 °C extension for 10 min.

[0022] By adopting the above technical solution, the present invention has the following beneficial effects: The nucleotide sequence of the OsNAC42 gene described in this invention is shown in SEQ ID NO:1. By constructing an overexpression vector of the OsNAC42 gene and transforming it into rice, transgenic rice plants overexpressing the OsNAC42 gene can be obtained. Under low phosphorus stress conditions, compared with wild-type rice, rice plants overexpressing the OsNAC42 gene exhibit superior traits such as improved root structure, enhanced phosphorus absorption capacity, and significantly increased biomass and phosphorus accumulation, thereby effectively improving the adaptability of rice to low phosphorus environments. This invention provides important gene resources and feasible technical approaches for breeding new low-phosphorus tolerant rice varieties, and is of great significance for saving phosphorus fertilizer resources and promoting sustainable agricultural development. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the OsNAC42 gene overexpression vector constructed in this invention.

[0024] Figure 2 The relative expression level of the OsNAC42 gene in rice overexpression lines is shown.

[0025] Figure 3 This is a comparison of the aboveground dry weight of OsNAC42-overexpressing plants and wild-type plants.

[0026] Figure 4 This is a comparison of root dry weight between OsNAC42 overexpressing plants and wild-type plants.

[0027] Figure 5 This is a comparison of phosphorus concentration in the aboveground parts of OsNAC42-overexpressing plants and wild-type plants.

[0028] Figure 6 This is a comparison of root phosphorus concentration between OsNAC42-overexpressing plants and wild-type plants. Detailed Implementation

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] Constructing primer sequence information for overexpression vectors:

[0032] OsNAC42-OE-F sequence:

[0033] 5'-ttccagattacgctggatccATGAAGAGGGGTTGTGAAGATGAA-3' (SEQ ID NO. 2);

[0034] OsNAC42-OE-R sequence:

[0035] 5'-tggtctttgtagtcggatccAGCATATATATGATCATAGAACAA-3' (SEQ ID NO. 3).

[0036] The pCAMBIA1300-UBI-HA-3*Flag-NOS vector was preserved by our research group, and its plasmid map is shown below. Figure 1 As shown.

[0037] OsNAC42 gene coding sequence:

[0038]

[0039] Using Nipponbare rice genomic cDNA as a template, and OsNAC42-OE-F and OsNAC42-OE-R as primers, the OsNAC42 gene (SEQ ID NO.1) was amplified by PCR. The PCR amplification reaction system was 50 μL, including: 20 μL of nuclease-free water, 2 μL each of forward and reverse primers, 25 μL of Biorun Pfu PCR Mix, and 1 μL of cDNA template. The PCR amplification reaction conditions were: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 50 ℃ annealing for 45 s, 72 ℃ extension for 72 s, 30 cycles; 72 ℃ extension for 10 min. The PCR amplification product was purified and recovered, and the recovered fragment was stored at -20 ℃.

[0040] The pCAMBIA1300-UBI-HA-3*Flag-NOS vector was digested with BamHI to obtain a linearized vector. The PCR amplification product was ligated between the HA tag and the 3*Flag of the pCAMBIA1300-UBI-HA-3*Flag-NOS linearized vector using a universal homologous recombination seamless cloning kit (purchased from Beijing TransGen Biotech Co., Ltd.). The resulting recombinant vector was transformed into E. coli, and positive clones were selected for detection.

[0041] After correct sequencing, the vector ( Figure 1 The samples were sent to Wuhan Boyuan Biotechnology Co., Ltd., where they were transformed into Agrobacterium and then introduced into rice callus tissue using Nipponbare rice as a background for genetic transformation. The resulting transgenic rice was identified using hygromycin, followed by quantitative PCR. The primer sequences for identification were:

[0042] qOsNAC42-F: 5'-GATTTCTACTACAAGGATGGATGCA-3' (SEQ ID NO. 4);

[0043] qOsNAC42-R: 5'-TATGATCATAGAACAAGCTCGGATC-3' (SEQ ID NO. 5);

[0044] Finally, two homozygous OsNAC42 overexpression lines were obtained (OsNAC42 OE5-6 and OsNAC42 OE7-3). Compared with wild-type (WT) rice, the relative expression levels of the OsNAC42 gene in the two OsNAC42 overexpression lines increased by approximately 126 and 124 times, respectively. Figure 2 ).

[0045] Example 2

[0046] Wild-type rice varieties Nipponbare (WT) and OsNAC42 overexpression lines were cultured and grown for 30 days under normal phosphorus (300 μM) and low phosphorus (2 μM) conditions, respectively. Samples were then taken, and the aboveground dry weight, root dry weight, aboveground phosphorus concentration, and root phosphorus concentration were measured. The results are as follows: Figures 3 to 6 As shown.

[0047] The results showed that under normal phosphorus conditions, the aboveground dry weight, root dry weight, aboveground phosphorus concentration, and root phosphorus concentration of the OsNAC42 overexpression lines were not significantly different from those of the WT line; however, under low phosphorus conditions, all four indicators of the OsNAC42 overexpression lines were significantly higher than those of the WT line. Specifically, under low phosphorus conditions, the aboveground dry weights of OE5-6 and OE7-3 were 2.04 g and 2.16 g, respectively, representing increases of 61.9% and 71.8% compared to the WT dry weight of 1.26 g; the root dry weights of OE5-6 and OE7-3 were 1.02 g and 1.11 g, respectively, representing increases of 44.1% and 56.5% compared to the WT dry weight of 0.71 g; the aboveground phosphorus concentrations of OE5-6 and OE7-3 were 11.52 mg / g and 12.52 mg / g, respectively, representing increases of 45.5% and 58.1% compared to the WT dry weight of 7.92 mg / g; and the root phosphorus concentrations of OE5-6 and OE7-3 were 5.44 mg / g and 5.10 mg / g, respectively, representing increases of 95.1% and 82.5% compared to the WT dry weight of 2.79 mg / g.

[0048] As can be seen from the above embodiments, the present invention provides the application of overexpression of the OsNAC42 gene in improving the resistance of rice to low phosphorus stress. Overexpression of the OsNAC42 gene in rice plants can improve the resistance of rice to low phosphorus stress.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression OsNAC42 The application of genes in one or more of the following: (1) Its application in promoting rice growth; (2) Application in improving phosphorus absorption in rice; (3) Application in improving the resistance of rice to low phosphorus stress; The OsNAC42 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. An expression OsNAC42 Gene overexpression vectors, characterized in that, include OsNAC42 Genes and basic plasmids.

3. The overexpression vector according to claim 2, characterized in that, The basic plasmid is the pCAMBIA1300-UBI-HA-3*Flag-NOS vector.

4. A method for improving the resistance of rice to low phosphorus stress, characterized in that, Includes the following steps: (1) Using rice genomic cDNA as a template, PCR amplification was performed using OsNAC42-OE-F and OsNAC42-OE-R primers to obtain OsNAC42 Gene sequence; (2) The pCAMBIA1300-UBI-HA-3*Flag-NOS vector was digested with BamHI and then... OsNAC42 Gene sequences are linked to obtain overexpression vectors; (3) The overexpression vector from step (2) is introduced into rice callus tissue and cultured into transgenic rice.

5. The method according to claim 4, characterized in that, The sequences of OsNAC42-OE-F and OsNAC42-OE-R are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

6. The method according to claim 4, characterized in that, The PCR amplification reaction system is 50 μL, including 1 μL of cDNA template, 2 μL each of upstream and downstream primers, 25 μL of Biorun Pfu PCR Mix, and 20 μL of Nuclease-free Water.

7. The method according to claim 4, characterized in that, The PCR amplification reaction program was as follows: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 50 °C annealing for 45 s, 72 °C extension for 72 s, cycle number 30; 72 °C extension for 10 min.

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