Application of rice 9-cis-epoxy carotenoid dioxygenase gene OsNCED4
By overexpressing the OsNCED4 gene in rice and implementing controlled irrigation, the problem of inconsistent chemical phosphate fixation and alternating wet and dry irrigation was solved, thereby improving rice root growth and phosphorus absorption.
Patent Information
- Application Number
- CN202411719114.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-01-10
AI Technical Summary
In existing technologies, the excessive use of chemical phosphate fertilizers leads to soil fixation, which cannot effectively improve the absorption and utilization of residual phosphorus by crops. Furthermore, the effects of alternating wet and dry irrigation on rice yield are inconsistent, and there is a lack of effective water control irrigation methods to improve phosphorus absorption by rice roots.
By overexpressing the rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4, combined with controlled irrigation conditions (irrigating once every 2 days, with soil moisture content controlled at 30%), rice root growth and phosphorus absorption were promoted, and the phosphorus content of rice was increased under controlled irrigation conditions.
It significantly promotes rice root growth, increases root phosphorus content and total phosphorus content, and improves phosphorus absorption efficiency of rice under controlled irrigation conditions.
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Figure CN119286922A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of genetic engineering, and relates to the application of rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4. Background Art
[0002] Phosphorus (P), as a non-renewable resource, is one of the most important constant elements that restrict crop growth and development. Although the application of large amounts of chemical P fertilizers can alleviate soil P deficiency and increase crop yields, the input of large amounts of chemical fertilizers not only increases the economic burden on farmers, but also creates environmental risks. At the same time, since nearly 80% of chemical P fertilizers are fixed in the soil and cannot be absorbed by plants, the continuous high-intensity application of P fertilizers in the past few decades has resulted in soil P accumulation that is expected to be sufficient to sustain crop production needs for nearly a hundred years. Therefore, improving the absorption and utilization of soil residual P by crops has become an urgent issue to be addressed in current agriculture, and is of great significance to green agriculture and sustainable development.
[0003] Rice (Oryza sativa L.) is one of the most important food crops in the world, and more than half of the population relies on rice as their staple food. The traditional mid-season drainage irrigation method can not only inhibit ineffective tillering, but also improve root activity. The root system is the organ for water and nutrient absorption and transportation of rice, and its morphological and physiological characteristics have an important influence on the absorption of water and nutrients. Studies have shown that long-term flooding will lead to the accumulation of toxic reducing products in the soil, which is not conducive to the growth of rice roots. Alternating dry-wet irrigation treatment significantly improved the root morphology (root weight, root number, root length, root surface area, root volume and root aerenchyma area, etc.) and root oxidation capacity of rice, thereby improving the absorption of water and nutrients (P) by rice. However, previous studies have shown that the effects of alternating dry-wet irrigation on rice yield are inconsistent. Some studies believe that it increases yield, some studies believe that it reduces yield, and some studies believe that alternating dry-wet irrigation has no significant effect on rice yield, which may be related to the rice variety. Therefore, breeding rice varieties suitable for water-controlled irrigation and combining them with water-controlled irrigation methods are of great significance to achieving sustainable agricultural development.
[0004] Abscisic acid (ABA), as a stress-induced hormone, plays a key role in plant growth and development and adaptive responses to environmental stimuli. ABA biosynthesis is an important pathway affecting its content, and 9-cis-epoxycarotenoid dioxygenases (NCEDs) are considered to be key enzymes in ABA synthesis. Studies have shown that the OsNECD4 gene can regulate rice ABA levels and stress resistance under drought stress. However, there are few studies on how the OsNECD4 gene regulates rice roots and improves soil P utilization under controlled water irrigation. Summary of the invention
[0005] The purpose of the present invention is to provide application of rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4.
[0006] The technical solution adopted by the present invention is as follows: The first aspect of the present invention provides the use of a rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 in promoting the growth of rice under water-controlled irrigation conditions, wherein the nucleotide sequence of the rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; Furthermore, the conditions of the water control irrigation are: the water control period is the seedling stage, irrigation is once every 2 days, and the amount of water each time is to control the soil moisture content at 30%; Furthermore, the promoting the growth of rice under water-controlled irrigation conditions includes promoting the root growth of rice under water-controlled irrigation conditions and promoting the leaf growth of rice under water-controlled irrigation conditions; Furthermore, the promoting root growth of rice under water-controlled irrigation conditions includes increasing the root fresh weight of rice under water-controlled irrigation conditions, increasing the root dry weight of rice under water-controlled irrigation conditions, increasing the total root length of rice under water-controlled irrigation conditions, and increasing the root surface area of rice under water-controlled irrigation conditions; the promoting leaf growth of rice under water-controlled irrigation conditions includes increasing the leaf length of rice under water-controlled irrigation conditions.
[0007] The second aspect of the present invention provides the use of rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 in improving the phosphorus content of rice under water-controlled irrigation conditions, wherein the nucleotide sequence of the rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; Furthermore, the conditions of the water control irrigation are: the water control period is the seedling stage, irrigation is once every 2 days, and the amount of water each time is to control the soil moisture content at 30%; Furthermore, the method of increasing the phosphorus content of rice under water-controlled irrigation conditions includes increasing the phosphorus content of the rice roots under water-controlled irrigation conditions and increasing the total phosphorus content of the rice plants under water-controlled irrigation conditions.
[0008] The third aspect of the present invention provides the use of rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 in improving phosphorus absorption of rice under water-controlled irrigation conditions, wherein the nucleotide sequence of the rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; Furthermore, the conditions for water-controlled irrigation are as follows: the water-controlled period is the seedling stage, irrigation is performed once every 2 days, and the amount of water for each irrigation is to control the soil moisture content at 30%.
[0009] The significant advantages of the present invention are: The present invention has found that overexpression of the rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 can promote rice growth under water-controlled irrigation conditions, increase the phosphorus content of rice under water-controlled irrigation conditions, and increase phosphorus absorption of rice under water-controlled irrigation conditions. The present invention has great application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 :Identification of OsNCED4 overexpressing strains. Identification of expression levels of OsNCED4 overexpressing strains.
[0011] Figure 2 :Overexpression of OsNCED4 promotes rice root growth under controlled irrigation conditions. A: Rice phenotype; B: Rice root fresh weight; C: Rice root dry weight; D: Rice total root length; E: Rice root surface area.
[0012] Figure 3 :Overexpression of OsNCED4 increases phosphorus content in rice under controlled irrigation conditions. A: leaf phosphorus content; B: root phosphorus content; C: total phosphorus content in the plant.
[0013] Note: WT is the wild-type rice Nipponbare (NIP), OE-1 and OE-2 are OsNCED4 overexpressing rice. DETAILED DESCRIPTION
[0014] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.
[0015] The nucleotide sequence of the rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 of the present invention is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO.2.
[0016] Embodiment 1: 1. Creation of OsNCED4 overexpressing plants Construction of OsNCED4 overexpression material using ubi promoter transgenic technology: The nucleotide sequence of rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 was searched in MSU-DB database, and the Primer software was used to generate the nucleotide sequence of rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4. 5.0 Design amplification primers: NCED4(+): 5'-cagtGGTCTCacaacatggcgctcctccgcccttc-3', NCED4(-): 5'-cagtGGTCTCataactaagcttgtcgtcgagct-3'; Using rice genomic DNA as a template, PCR amplification of rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 was performed using primers NCED4(+) and NCED4(-), and the target fragment was obtained by 1% agarose electrophoresis detection and gel recovery and purification; the target fragment and pBWA(V)HU-gus vector containing ubi promoter (purchased from Wuhan Boyuan Biotechnology Co., Ltd., project number MOLE_2018_1032) were double-digested with restriction endonucleases BsaI and Eco31I, and the T4 The ligation product was connected by DNA ligase, and the ligation product was transformed into Escherichia coli DH5a competent cells, spread on LB solid culture medium containing kanamycin and cultured at 37°C for 12 hours, and positive clones were picked for plaque PCR identification to obtain ubi::OsNCED4 overexpression vector; the ubi::OsNCED4 overexpression vector was transformed into Agrobacterium tumefaciens EHA105 competent cells to obtain Agrobacterium transformed with the ubi::OsNCED4 overexpression vector, and the Agrobacterium was infected with callus tissue of wild-type rice Nipponbare, and ubi::OsNCED4 overexpression T0 generation transgenic rice material was obtained through selective culture, differentiation, rooting and seedling hardening.
[0017] II. Identification of OsNCED4 overexpressing plants The leaves of T0 transgenic rice seedlings were collected, and the rice genomic DNA was extracted. The ubi::OsNCED4 overexpression vector fragment was amplified by PCR. The PCR amplification product was sequenced and compared with the sequence of wild-type rice Nipponbare to select positive plants. In addition, real-time fluorescence quantitative PCR was performed on wild-type rice Nipponbare and OsNCED4 overexpression positive plants. The steps are as follows: (1) Extract total RNA from rice leaves (Trizol, commercially available); (2) Total RNA was reverse transcribed into cDNA as a template using a reverse transcription kit (commercially available), and real-time fluorescence quantitative PCR was performed using primers (Forward Primer: 5'-TCCATCTCCTTCTCCCTCCTCCCA-3', Reverse Primer: 5'-CCTCGCACCCTGCTTGATCTTGCC-3') to detect the relative expression level of the OsNCED4 gene.
[0018] Based on the relative expression of OsNCED4 gene, two single-copy strains were selected and named OE-1 and OE-2 ( Figure 1 ), the relative expression levels of OsNCED4 genes in the two single-copy lines were upregulated by more than 20 times compared with that in wild-type rice.
[0019] Embodiment 2: Seeds of wild-type rice Nipponbare (NIP) and OsNCED4 overexpressing rice materials with full grains were selected and germinated in a 28°C incubator. After the seeds turned white, they were planted in a milk tea cup filled with 400 g of paddy field soil with a water content of 30% (the volume of the milk tea cup was 1500 mL, the top diameter was 12 cm, the bottom diameter was 8 cm, the cup height was 20 cm, the cup wall was wrapped and covered with opaque tape, and there were water holes at the bottom). They were cultured in a monocot greenhouse in the dark for 1 to 2 days, and 10 mL of water was poured in the morning and evening every day. After the buds grew out of the soil, the seedlings were placed under a light intensity of 300 μmol·m -2 ·s -1 The seedlings were allowed to grow for 7 days at 28℃, 70% relative humidity and 28℃ temperature. After the seedlings were grown, the seedlings were irrigated with controlled water. The method was to weigh and water them once every 2 days. The amount of water each time was to control the soil moisture content at 30%. A total of 6 controlled water irrigations were performed. Four biological replicates were performed for each strain. After the controlled water irrigation treatment, the roots of the rice seedlings were scanned using EPSON Expression 11000XL root scanner. After the scanning, the scanned images were loaded into WinRHIZO Pro 2016a analysis software. The root scanned images were analyzed, and then the total root length and root surface area were counted. At the same time, after the controlled water irrigation treatment, the rice seedlings were dried in an oven at 45℃ for 72h and then weighed. The fresh weight and dry weight of the roots of the plants before and after drying were recorded. The phosphorus content of the leaves, the phosphorus content of the roots and the total phosphorus content of the plants were detected using the molybdenum antimony colorimetric method (national standard NY_T 2421-2013).
[0020] like Figure 2As shown, compared with wild-type rice, OsNCED4 overexpression lines (OE-1 and OE-2) grew more robustly, had longer leaves and more developed root systems under water-controlled irrigation conditions; quantitative analysis results showed that the root fresh weight, root dry weight, total root length and root surface area of OsNCED4 overexpression lines (OE-1 and OE-2) under water-controlled irrigation conditions were significantly higher than those of wild-type rice (p<0.01).
[0021] like Figure 3 As shown, there was no significant difference in leaf phosphorus content between wild-type rice and OsNCED4 overexpressing lines (OE-1 and OE-2) under water-controlled irrigation conditions (p>0.05); the root phosphorus content and plant total phosphorus content of OsNCED4 overexpressing lines (OE-1 and OE-2) under water-controlled irrigation conditions were significantly higher than those of wild-type rice (p<0.05 or p<0.01).
[0022] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. Rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 Application in promoting the growth of rice under water-controlled irrigation conditions, characterized in that: The rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.
2.
2. The use according to claim 1, characterized in that: The conditions of the water-controlled irrigation are as follows: the water-controlled period is the seedling stage, irrigation is performed once every 2 days, and the amount of water for each irrigation is such that the soil moisture content is controlled at 30%.
3. The use according to claim 1, characterized in that: The promoting the growth of rice under water-controlled irrigation conditions includes promoting the growth of the root system of rice under water-controlled irrigation conditions and promoting the growth of the leaves of rice under water-controlled irrigation conditions.
4. The use according to claim 3, characterized in that: The promoting of root growth of rice under water-controlled irrigation conditions comprises increasing root fresh weight of rice under water-controlled irrigation conditions, increasing root dry weight of rice under water-controlled irrigation conditions, increasing total root length of rice under water-controlled irrigation conditions, and increasing root surface area of rice under water-controlled irrigation conditions; the promoting of leaf growth of rice under water-controlled irrigation conditions comprises increasing leaf length of rice under water-controlled irrigation conditions.
5. Rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 The application of the invention in increasing the phosphorus content of rice under water-controlled irrigation conditions is characterized by: The rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.
2.
6. The use according to claim 5, characterized in that: The conditions of the water-controlled irrigation are as follows: the water-controlled period is the seedling stage, irrigation is performed once every 2 days, and the amount of water for each irrigation is such that the soil moisture content is controlled at 30%.
7. The use according to claim 1, characterized in that: The method of increasing the phosphorus content of rice under water-controlled irrigation conditions includes increasing the phosphorus content of the rice root system under water-controlled irrigation conditions and increasing the total phosphorus content of the rice plant under water-controlled irrigation conditions.
8. Rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 Application in improving phosphorus absorption of rice under water-controlled irrigation conditions, characterized in that: The rice 9-cis-epoxycarotenoid dioxygenase gene OsNCED4 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.
2.
9. The use according to claim 8, characterized in that: The conditions of the water-controlled irrigation are as follows: the water-controlled period is the seedling stage, irrigation is performed once every 2 days, and the amount of water for each irrigation is such that the soil moisture content is controlled at 30%.