Application of Rice OsUGPase1 Gene

Abstract

The present invention discloses the application of the rice OsUGPase1 gene, specifically the application of the rice OsUGPase1 gene and related biomaterials in regulating sucrose accumulation and phosphorus absorption and accumulation in rice. The method proposed by the present invention for increasing rice yield and improving rice phosphorus utilization efficiency by utilizing the OsUGPase1 gene is as follows: first, the expression of OsUGPase1 in rice was studied using specific primers. The research results showed that the gene was highly abundantly expressed, expressed both above and below ground, and that the expression of the aboveground part was induced by phosphorus deficiency stress; then, a transgenic material was constructed in which the promoter of the OsUGPase1 gene was fused to the GUS reporter gene. GUS staining experiments revealed that the gene was expressed in all vegetative rice tissues; finally, a transgenic rice material was constructed in which the OsUGPase1 gene was overexpressed. The research results showed that sucrose accumulation in the photosynthetic source organs of the overexpressing plants was significantly increased, which can be used to modify sugar distribution and thus improve yield.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to the application of rice OsUGPase1 gene. Background Art

[0002] UGPase was first discovered in yeast in 1957. It is an important enzyme in sugar metabolism and is widely present in plants, animals and bacteria. It is responsible for catalyzing the synthesis and degradation of UDPG and has high specificity for the substrate G1P:

[0003] Two types of UGPase (UGPase-A and UGPase-B) exist in plants, catalyzing the same reaction, yet their amino acid sequences share only 14% similarity. UGPase-A is widely found in both eukaryotes and prokaryotes, while UGPase-B has so far been found only in plant chloroplasts. The Arabidopsis genome contains two genes, AtUGP1 and AtUGP2, believed to encode UGPases. When grown under natural conditions, AtUGP1 mutants in Arabidopsis thaliana exhibit seed yields that are more than halved. Furthermore, AtUGP1 acts as a key factor in regulating programmed cell death. UGP1 can simultaneously participate in two or even more seemingly independent physiological processes, further highlighting its important biological functions.

[0004] Rice is one of my country's most important food crops. Current population growth and shrinking arable land areas are placing tremendous pressure on increasing rice production. Furthermore, rice is an important monocot model plant. Further tapping into rice's inherent yield potential to increase yield is the primary approach to ensuring sustainable rice production. From a biological perspective, there are two approaches to increasing rice yield: increasing biomass and improving the harvest index. The assimilates that contribute to rice yield are derived from photosynthesis in the current leaves and non-structural carbohydrates stored in the stem sheath prior to heading. Therefore, increasing the carbohydrate content of leaves and sheaths, and thereby increasing yield, is one of the key approaches to achieving high rice yields. Sucrose is the most important nutrient in plant growth and development and the primary form of assimilate transport within plants. Therefore, there is an urgent need for methods to increase the carbohydrate content of rice to improve rice yield. This paper identifies a UGPase-A rice gene, OsUGPase1, from rice and proposes methods for utilizing OsUGPase1 to increase rice yield and improve phosphorus utilization efficiency. Summary of the Invention

[0005] The present invention aims at the deficiencies in the prior art and provides application of rice OsUGPase1 gene.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides an application of the rice OsUGPase1 gene in increasing rice yield. The rice OsUGPase1 gene increases rice yield by regulating the accumulation of carbohydrates in rice.

[0008] On the other hand, the present invention provides an application of the rice OsUGPase1 gene in cultivating nutrient-efficient rice. The rice OsUGPase1 gene cultivates nutrient-efficient rice by regulating the absorption and accumulation of phosphorus in rice.

[0009] To optimize the above technical solutions, specific measures taken also include:

[0010] Furthermore, the above application is achieved by overexpressing the OsUGPase1 gene in rice.

[0011] Furthermore, the cDNA sequence of the OsUGPase1 gene is shown as SEQ ID No.9.

[0012] Furthermore, the OsUGPase1 gene also includes biological materials related thereto, and the biological materials include any one of an expression cassette, a recombinant vector, a recombinant bacterium, and a transgenic cell line encoding a protein of the OsUGPase1 gene or containing the OsUGPase1 gene.

[0013] Furthermore, the amino acid sequence of the protein encoded by the OsUGPase1 gene is shown as SEQ ID No.10.

[0014] Furthermore, the OsUGPase1 gene can also be used to increase the yield of monocotyledonous plants or to regulate the absorption and accumulation of phosphorus by monocotyledonous plants, wherein the monocotyledonous plants are selected from any one of corn and wheat.

[0015] The beneficial effects of the present invention are:

[0016] The present invention identifies a UGPase-A type rice gene, OsUGPase1, from rice for the first time and proposes a method for increasing rice yield and improving rice phosphorus utilization efficiency using OsUGPase1: (1) First, the expression of OsUGPase1 in rice was studied using specific primers. The results showed that the gene was highly abundant and expressed both above and below ground, and that its expression was induced above ground by phosphorus deficiency stress; (2) Then, a transgenic material was constructed in which the promoter of the OsUGPase1 gene was fused to the GUS reporter gene. GUS staining experiments showed that the gene was expressed in all vegetative rice tissues; (3) Finally, a transgenic rice material was constructed in which the OsUGPase1 gene was overexpressed. The results showed that sucrose accumulation in the photosynthetic source organs of the overexpressing plants was significantly increased, which can be used to change sugar distribution and improve yield. The present invention found that the OsUGPase1 gene was strongly upregulated at the transcriptional level by phosphorus deficiency stress. After overexpressing it in rice, it can significantly promote the accumulation of sucrose and inorganic phosphorus in leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 RT-qPCR analysis of the response of OsUGPase1 to various nutrient deficiency stresses in rice leaves (A) and roots (B) during the vegetative growth period in Experiment 1;

[0018] Figure 2 The results of GUS staining in Experiment 2: A is the main root, B is the lateral root, C is the leaf blade, and D is the leaf sheath.

[0019] Figure 3 Identification of the OsUGPase1 gene overexpressing rice created by the Over Expression technology in Experiment 3;

[0020] Figure 4 Figure 3 shows the phenotype (A), sucrose content (B), and inorganic phosphorus content (C) of rice plants overexpressing the OsUGPase1 gene under full nutrition (HP) and low phosphorus (LP) conditions in Experiment 3. Figure 4 B and Figure 4 Each series in C represents from left to right: WT, overexpression line Ox15, overexpression line Ox30, and overexpression line Ox36. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the accompanying drawings.

[0022] All reagents and drugs used in the following experiments, unless otherwise specified, are commercially available. Materials and methods not specifically described are based on the Molecular Cloning Manual (Sambrook and Russell, 2001).

[0023] 1. Study on the expression of OsUGPase1 gene

[0024] 1.1 Total RNA extraction and cDNA synthesis

[0025] Clean, plump, wild-type japonica rice seeds (Oryza sativa ssp. cv. Japonica) were selected and sterilized by soaking them in a 30% sodium hypochlorite solution for 20 minutes. The seeds were then rinsed with deionized water and incubated in a 37°C oven in the dark for approximately two days. Once the seeds appeared white, they were transferred to a climatic chamber (temperature: 28°C light / 22°C dark, photoperiod: 14 hours day / 10 hours night). When the rice seedlings had two leaves and one heart, seedlings of uniform height were selected for transplanting. Treatments with full nutrition, phosphorus, nitrogen, potassium, iron, and magnesium deficiency were initiated, with the nutrient solution changed every two days for up to 15 days. The full nutrient solution used was a modified Yoshida formula from International Rice. Leaf and root samples were collected, rapidly frozen in liquid nitrogen, and stored in a -80°C freezer. Tissue and cell lysis of the stored samples was performed in liquid nitrogen, and total RNA was extracted using TRIzol Reagent (Invitrogen, USA). The integrity and concentration of the extracted total RNA were detected by agarose gel electrophoresis and NanoDrop instrument. TM cDNA was synthesized using the RT reagent Kit.

[0026] 1.2 RT-PCR analysis of the response of OsUGPase1 gene to different nutrient deficiency stresses

[0027] The cDNA sequence of OsUGPase1 was obtained from the Rice Annotation Project database using the gene ID Os09g0553200. Specific primers were designed based on the sequence conservation and the 3' untranslated region (3'UTR) with high specificity for quantitative RT-qPCR analysis. The primer sequences are as follows:

[0028] QF (SEQ ID No. 1): GGCTGCTCACGGAAACCTT;

[0029] QR (SEQ ID No. 2): GCCGAATGCACACGACAAT.

[0030] The rice Actin (Os03g0718100) gene was used as an internal reference gene. Figure 1 As shown, OsUGPase1 gene is strongly induced by phosphorus deficiency in rice leaves during the vegetative growth period ( Figure 1A), which is weakly induced by phosphorus deficiency stress in rice roots ( Figure 1 B).

[0031] 2. Analysis of tissue localization of promoter-fused GUS reporter gene materials

[0032] 2.1 Extraction of genomic DNA

[0033] A 2-cm-long section of Nipponbare rice leaf sample was cut and ground into a powder using liquid nitrogen. The sample was then transferred to a 2-mL centrifuge tube and 500 mL of TPS extractant (50 mL of 1 M Tris-HCl, pH 8.0; 10 mL of 0.5 M EDTA, pH 8.0; 37.25 g of KCl) was added and vortexed to mix thoroughly. The sample was placed in a 65°C water bath for 20 minutes, inverted two or three times. The sample was then centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was transferred to a 1.5-mL centrifuge tube, an equal volume of isopropanol was added, mixed thoroughly, and allowed to stand at room temperature for 10 minutes. The sample was centrifuged again at 4000 rpm for 5 minutes at 4°C. The supernatant was discarded, and 1 mL of 70% ethanol was added to wash the DNA precipitate. The sample was centrifuged at 4000 rpm for 2 minutes at room temperature, the supernatant discarded, and the sample was air-dried for 10 minutes before being dissolved in ddH2O.

[0034] 2.2 Creation of promoter-fused reporter gene materials

[0035] The 2475 bp preceding the ATG codon of the OsUGPase1 gene translation start codon were selected as candidate promoter sequences and analyzed for restriction enzyme distribution using Primer Premier 5.0 software. HindIII and KpnI were selected as the endonucleases for vector linearization. Amplification primers were designed as follows:

[0036] PF (SEQ ID No. 3): AAGCTT-AACATCCATCCAACCTCCC;

[0037] PR (SEQ ID No. 4): GGTACC-CCGGCTCGGCGATGCGATTC.

[0038] Primer synthesis was performed by Qingke. Using Nipponbare genomic DNA as a template, the target fragment was amplified using the high-fidelity enzyme KOD FX NEO. After gel running, a gel block of the target fragment was cut and recovered. Simultaneously, the expression vector pCAMBIA1300 was double-digested with HindIII and KpnI, and ligated using TaKaRa's T4 DNA Ligase to connect the target fragment to the expression vector. After sequencing, the recombinant plasmid was transformed into Agrobacterium tumefaciens via electroporation. Transgenic plants were then obtained using rice transgenic methods. Molecular identification confirmed that these plants were transgenic rice plants harboring a promoter-fused GUS reporter gene.

[0039] 2.3 GUS staining of promoter material

[0040] Refer to the seedling method in Experiment 1 and culture the promoter-fused GUS reporter gene material in full nutrition hydroponics for two weeks. Take different tissue parts and soak them in GUS dye solution and incubate them at 37℃ for 3 hours. Decolorize the aboveground parts repeatedly with 100% ethanol until the chlorophyll is completely washed away. Observe the GUS staining results with a stereoscope. Figure 2 As shown, OsUGPase1 gene is expressed in the main root of rice ( Figure 2 A) Lateral roots ( Figure 2 B) leaves ( Figure 2 C) and leaf sheaths ( Figure 2 D) Both are expressed.

[0041] 3. Phenotype and Inorganic Phosphorus Content Determination of OsUGPase1 Gene Overexpression Materials

[0042] 3.1 Construction and identification of overexpression materials

[0043] In this experiment, plants overexpressing the OsUGPase1 gene were constructed. The cDNA sequence of the OsUGPase1 gene was imported into Primer Premier 5.0 software to analyze the restriction endonuclease distribution, using SacI and BamHI as restriction endonucleases. The amplification primers were designed as follows:

[0044] Ox-F (SEQ ID No. 5): GAGCTC-ATGGCGGTCACCGCCGAC;

[0045] Ox-R (SEQ ID No. 6): GGATCC-TCAAAGATCCTCCGGAC.

[0046] Primer synthesis was performed by Qingke Co., Ltd. Using cDNA from wild-type Nipponbare seedlings as a template, amplification was performed using the high-fidelity enzyme KOD FXNEO. After electrophoresis, a fragment of the target fragment was excised and recovered, ligated to the pEASYBlune cloning vector. Positive clones were then verified by restriction enzyme digestion and sequenced. Sequencing-verified plasmids and the expression vector pCAMBIA1305-GUSPlus were simultaneously digested with SacI and BamHI, respectively. The target fragment and expression vector were recovered by gel electrophoresis and then ligated using T4 DNA Ligase, ligating the target fragment to the expression vector. After sequencing confirmed, the recombinant plasmid was transformed into Agrobacterium tumefaciens by electroporation. Transgenic plants were then obtained using Agrobacterium-mediated infection of rice callus tissue. Molecular identification confirmed that these plants were transgenic rice overexpressing the OsUGPase1 gene.

[0047] From the root tips of the transgenic seedlings obtained, approximately 2 mm was cut and immersed in GUS dye solution. The root tips were then placed in a 37°C incubator overnight. Transgenic-positive seedlings that stained blue were identified. Transgenic-positive seedlings that had been identified were transplanted into a turnover box and placed in an artificial climate chamber. They were cultured in rice nutrient solution for two weeks. Rice leaf samples were collected, RNA was extracted, and reverse transcribed into cDNA. Primers were designed to identify overexpressed OsUGPase1 genes. Amplification primers were designed as follows:

[0048] OQ-F (SEQ ID No. 7): GGAGCAGATCGAGTGGAGTAAGA;

[0049] OQ-R (SEQ ID No. 8): GAGCGTGTCGTAGGGAACCA.

[0050] The expression abundance of OsUGPase1 gene in transgenic materials was detected using cDNA as a template, and strains with good overexpression effects were selected for subsequent use.

[0051] Three of these lines were selected for this experiment and named Ox15, Ox30, and Ox36. The effects of overexpression of the OsUGPase1 gene in these three transgenic rice lines were identified as follows: Figure 3 As shown, the expression level of OsUGPase1 gene in these three lines increased by about 3 times compared with that in wild-type rice WT.

[0052] 3.2 Phenotypic analysis of mutant materials and determination of sucrose and inorganic phosphorus content

[0053] Rice seed germination and pretreatment were the same as in Experiment 1, with cultivation under full nutrition (HP) and low phosphorus (LP) conditions for three weeks. Transgenic rice lines were selected as described above: Ox15, Ox30, and Ox36, which exhibit significant overexpression, and wild-type rice (WT). After cultivation, rice plants were photographed, and both the aerial and root parts were sampled and weighed. Dried plant samples were crushed and incubated at 80°C for 30 minutes before sucrose content was determined. Inorganic phosphorus concentration was determined using perchloric acid extraction followed by molybdenum blue colorimetry.

[0054] The results are as follows Figure 4 As shown in Figure 2, overexpression of the OsUGPase1 gene did not affect the growth of rice during the vegetative growth period ( Figure 4 A), the biomass of the overexpressing plants in the shoots and roots showed no significant difference compared with the wild-type materials under HP and LP treatment conditions. It is worth noting that the sucrose content in the overexpressing plants in the shoots and roots was significantly higher than that in the wild-type rice under HP, that is, under phosphorus-sufficient conditions; but there was no significant difference under low phosphorus conditions ( Figure 4 B). Similarly, when phosphorus was adequately supplied, the inorganic phosphorus content in the shoots of overexpressing rice was significantly higher than that of wild-type rice; however, under low-phosphorus conditions, the inorganic phosphorus content of overexpressing plants was not significantly different from that of wild-type plants ( Figure 4 C).

[0055] The above experiments show that the synthesis of sucrose and the absorption and accumulation of phosphorus in rice can be promoted by positively regulating the OsUGPase1 gene of the present invention.

[0056] The present invention screened a gene related to sugar metabolism, OsUGPase1, from the monocotyledonous plant Oryza sativa. Its cDNA sequence is shown in SEQ ID No. 9, and the amino acid sequence of the protein encoded by the OsUGPase1 gene is shown in SEQ ID No. 10. RT-qPCR results showed that the expression of this gene was induced in the aboveground part of rice by low phosphorus stress. Overexpression of the OsUGPase1 gene did not affect the growth of rice during the vegetative growth period, but it promoted the accumulation of sucrose in the aboveground part and roots of rice, as well as the accumulation of inorganic phosphorus in rice leaves. The present invention provides a guarantee for the cultivation of high-yield, high-quality rice varieties that efficiently utilize phosphorus.

[0057] SEQ ID No.9:

[0058]

[0059] MAVTADVKLEGLRAATDKLDQISENEKSGFISLVSRYLSGEAEQIEWSKIQTPTDEVVV PYDTLSAAPEDLNETKKLLDKLVVLKLNGGLGTTMGCTGPKSVIEVRNGFTFLDLIVIQIESLNKKYGCNVPLLLMNSFNTHDDTQKIVEKYSNSNIEIHTF NQSQYPRIVTEDFLPLPSKGKTGKDGWYPPGHGDVFPSLNNSGKLDTLLAQGKEYVFVANSDNLGAIVDIKILNHLIHNQNEYCMEVTPKTLADVKGGTLISY EGRVQLLEIAQVPDEHVNEFKSIEKFKIFNTNNLWVNLKAIKRLVEAEALKMEIIPNPKEVDGVKVLQLETAAGAAIRFFEKAIGINVPRSRFLPVKATSDLL LVQSDLYTLVDGFVIRNPARTNPSNPSIELGPEFKKVANFLARFKSIPSIVELDTLKVSGDVWFGSGVTLKGKVTITAKSGKLEIPDGAVLENKDINGPEDL.

[0060] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the principles of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Rice OsUGPase1 The application of the gene in rice cultivation is characterized in that: When phosphorus is adequately supplied, the OsUGPase1 The rice is cultivated by increasing the absorption and accumulation of phosphorus in rice; OsUGPase1 The cDNA sequence of the gene is shown in SEQ ID No.

9. OsUGPase1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.10.

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