Application of Rice LOC_Os01g68930 Gene in Regulating Protein Content in Rice Grains

Abstract

The invention discloses rice LOC_Os01g68930 The application of genes in regulating the protein content in rice grains relates to the field of genetic engineering technology. LOC_Os01g68930 The CDS sequence of the gene 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. The present invention obtains OE‑LOC_Os01g68930 Overexpression plants. Experiments have shown that: PLACE_ Os01g68930 The total content of glutenin and alcohol-soluble proteins in the seeds of overexpressing transgenic rice reached 7.88% to 7.97%. In comparison, the protein content of wild-type rice grains was 6.97%, and the protein content of transgenic rice grains increased by 13.1%-14.3%. LOC_Os01g68930 It has been confirmed that this gene can effectively regulate the protein content of rice grains, laying a solid foundation for the subsequent breeding of high-protein rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology and relates to rice LOC_Os01g68930 Application of genes in regulating protein content in rice grains. Background Art

[0002] Protein plays a vital role in human physiological metabolism and is closely linked to all aspects of life activities. Plant protein is a source of high-quality protein. It is not only an important raw material in the food industry, but also widely used in many fields due to its excellent processing characteristics. Appropriate intake of plant protein also has many physiological effects such as lowering blood lipids, lowering blood pressure, anti-tumor, anti-microbial and anti-inflammatory.

[0003] Rice is a high-yield crop, and about one-third of the world's population relies on it as their staple food. Although the protein content in rice grains is relatively low, it is still one of the main sources of plant protein. Rice protein contains amino acids that are essential to the human body but cannot be synthesized by the human body. It is low in fat, cholesterol-free, and low in allergenicity, and has extremely high nutritional value. In addition, the lysine content is dominant among all types of grain proteins, and the amino acid composition ratio is reasonable, making it considered to be a relatively ideal plant protein.

[0004] As one of the most important food crops in the world, improving the nutritional content of rice has always been a research focus. In addition, protein is one of the important nutrients in rice seeds. Improving the protein content of rice helps to improve the nutritional value. With the growing demand for protein among consumers and the scarcity of resources, plant protein is gradually becoming a substitute for animal protein due to its high production efficiency, high sustainability and green manufacturing characteristics. Given the high yield of rice and the high nutritional value of protein, increasing the protein content of rice will also help meet the growing demand for protein. Summary of the invention

[0005] The present invention aims to dig a rice LOC_Os01g68930 The study identified the gene and studied its mechanism of action in regulating the protein content of rice grains, laying a solid foundation for the subsequent breeding of high-protein rice varieties.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a rice LOC_Os01g68930 Application of genes in regulating protein content in rice grains, the rice LOC_Os01g68930 The CDS sequence of the gene is shown in SEQ ID No. 1, and the rice LOC_ Os01g68930The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2; the application method is to overexpress the gene in rice. LOC_Os01g68930 Gene, increasing the protein content of grains of rice overexpressing plants.

[0008] In a specific embodiment, the present invention technicians constructed LOC_Os01g68930 The gene overexpression vector was successfully transformed into rice plants. The specific operations are as follows:

[0009] based on LOC_Os01g68930 Primers SfiI-LOC_Os01g68930-F (as shown in SEQ ID No.3) and SfiI-LOC_Os01g68930-R (as shown in SEQ ID No.4) were designed from the gene sequence. SfiI restriction sites were designed at both ends of the primers. RNA of NIP Nipponbare was extracted and reverse transcribed. The cDNA of NIP Nipponbare was used as a template for amplification. LOC_Os01g68930 The full length of the cDNA of the gene from the start codon to the stop codon (as shown in SEQ ID No.1).

[0010] Use Takara's T4 DNA Ligase to connect the target gene fragment cut with SfiI to the vector (see the plasmid map for details). Picture 1 ). Take out the competent cells of E. coli DH5α from the low temperature environment in advance, place them on ice to thaw slowly, and after the competent cells are completely thawed, add the above-mentioned ligation product to it, gently tap the wall of the reaction tube to ensure that the ligation product and the competent cells are fully mixed. The mixed system is placed on ice for 30 minutes, and then quickly placed in a 42°C water bath for heat shock treatment for 60 seconds. After the heat shock is over, it is immediately placed on ice to cool for 2 minutes. Add 650μL of LB culture medium without antibiotics to the cooled system, transfer it to a 37°C shaker, and shake and culture for 45 minutes. The cultured bacterial solution is centrifuged and the supernatant is discarded. The remaining bacteria are resuspended and evenly coated on the LB solid plate containing kanamycin resistance. The coated plate is inverted and placed in a 37°C incubator for 13 to 16 hours. After a single colony grows on the plate, the colony is identified and screened to determine the positive engineering bacteria and extract the plasmids in the positive engineering bacteria. The extracted plasmids are then sequenced and verified to ensure the successful construction of the overexpression vector.

[0011] The successfully constructed overexpression vector was transformed into the callus tissue of NNIP (New Nipponbare) rice using the Agrobacterium-mediated method, and finally positive transformed seedlings were obtained.

[0012] The second aspect of the present invention provides the rice LOC_Os01g68930 Application of genes in high protein rice breeding.

[0013] The third aspect of the present invention provides an application of an overexpression vector to regulate the protein content in rice grains, wherein the overexpression vector contains rice LOC_Os01g68930 The gene is applied by transferring the overexpression vector into rice plants.

[0014] Beneficial effects of the present invention:

[0015] (1) Improving the protein content of rice grains: Creating overexpression through transgenic technology LOC_Os01g68930 The experimental data of the transgenic rice showed that the total content of glutenin and alcohol-soluble protein in its seeds reached 7.88%-7.97%, while that of wild-type rice was only 6.97%. The protein content of transgenic rice grains increased by 13.1%-14.3%. This greatly enhances the nutritional value of rice and meets people's demand for high-protein rice. For example, in daily diet, eating this high-protein rice can provide the human body with more abundant plant protein and help improve health.

[0016] (2) Clarifying gene function: LOC_Os01g68930 Genes can effectively regulate the protein content of rice grains, which provides key information for the research field of rice gene function. Based on this, researchers can further explore the molecular mechanism of gene regulation of rice protein synthesis, open up new directions for subsequent research, and promote the development of rice genetic engineering.

[0017] (3) Laying the foundation for variety breeding: It has laid a solid foundation for the breeding of high-protein rice varieties. With the help of this gene, breeders can use transgenic technology or traditional breeding methods to introduce it into different rice varieties, breed more new rice varieties with high protein content, and enrich rice germplasm resources.

[0018] (4) Optimizing carbon source allocation: Research has found that LOC_Os01g68930 In transgenic rice with overexpressed genes, carbon source distribution is adjusted, the synthesis of glutenin and alcohol-soluble proteins is enhanced, and starch synthesis is inhibited. This characteristic has potential application value in the food industry and other fields. For example, the proportion of rice ingredients can be adjusted according to different needs to produce rice products suitable for special groups of people (such as diabetic patients).

[0019] (5) Promote the development of plant protein: In the context of the growing global demand for protein and scarce resources, rice is an important food crop. Improving its protein content will help enhance the status of plant protein in the protein supply system. Plant protein has the characteristics of efficient production, sustainability and green manufacturing. The results of this study can further promote plant protein as a substitute for animal protein, alleviate protein supply pressure, and also conform to the concept of green development. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Picture 1 The overexpression vector pFOX-SfiI3-LOC_Os01g68930 provided by the present invention.

[0021] Picture 2 In the context of NNIP LOC_Os01g68930 Identification of expression levels in leaves of gene overexpressing plants: LOC_Os01g68930 -1. LOC_Os01g68930- 2 respectively represent the plant numbers of the two transgenic complementation events.

[0022] Picture 3 For overexpression LOC_Os01g68930 Comparison of genes and NNIP plants: from left to right: NNIP, LOC_Os01g68930 -1. LOC_Os01g68930- 2.

[0023] Picture 4 For NNIP, LOC_Os01g68930 -1. LOC_Os01g68930- 2Statistical charts of plant heading period, plant height and tiller number.

[0024] Picture 5 This is a statistical chart of seed length, width and thousand-grain weight: Picture 5 A in it is NNIP, LOC_Os01g68930 -1. LOC_ Os01g68930- 2. Statistical bar graphs of seed length, width and thousand-grain weight; Picture 5 The B in it is NNIP, LOC_Os01g68930 -1. LOC_ Os01g68930- 2. Statistical diagram of seed length and width phenotype.

[0025] Picture 6 for OE-LOC_Os01g68930 -1. OE-LOC_Os01g68930- 2. Comparison between seeds after husking and NNIP.

[0026] Picture 7 For NNIP, LOC_Os01g68930 -1. LOC_Os01g68930- 2 Statistical chart of protein, soluble sugar and starch content of seeds. DETAILED DESCRIPTION

[0027] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0028] Example 1 Construction LOC_Os01g68930 Gene overexpression vector

[0029] Refer to the National Rice Data Center (https: / / www.ricedata.cn / ) LOC_Os01g68930 Primers were designed based on the gene sequence, and SfiI restriction sites were designed at both ends of the primers. The cDNA of NIP was used as a template to amplify the full length of the cDNA of the target gene from the start codon to the stop codon (as shown in SEQ ID No.1), and the PCR product was recovered using the Zhongdao Life Purification Kit. The primer sequences are as follows:

[0030] SfiI-LOC_Os01g68930-F (as shown in SEQ ID No.3):gcattacggccATGGAATTCGGGGGCGCC;

[0031] SfiI-LOC_Os01g68930-R (as shown in SEQ ID No. 4): ggccgtaatggccTTAACCTCTAGTTCTTGTGATTGTGATACT.

[0032] Use Takara's T4 DNA Ligase to connect the fragment digested with SfiI to the vector (see the plasmid map for details). Picture 1 ). Take out the competent E. coli DH5ɑ cells in advance and thaw them on ice. Add the ligation product to the thawed competent cells and gently tap the tube wall to ensure that the two are fully mixed. After standing on ice for 30 mins, heat shock in a 42℃ water bath for 60 s. After the heat shock, immediately cool on ice for 2 min. Add 650μL of LB (Luria-Bertani) culture medium without antibiotics and shake at 37℃ for 45 mins. Centrifuge at 3700rpm for 2 min, carefully discard about 500 μL of supernatant in the clean bench, resuspend the remaining bacteria, spread on a kana resistance LB solid plate, and invert and culture in a 37℃ incubator for 13 to 16h. Identify and screen the single colonies that grow to identify positive engineering bacteria. The plasmids in these engineering bacteria were extracted using the Zhongdao Life Plasmid Miniprep Kit and sequenced to ensure that the pFOX-SfiI3-LOC_Os01g68930 overexpression vector was successfully constructed. The cultured cells were transformed into callus tissue of NNIP rice through Agrobacterium-mediated method to obtain positive transformed seedlings.

[0033] Example 2 LOC_Os01g68930 Quantitative expression analysis of genes in leaves of overexpressing plants

[0034] The cDNA was obtained by extracting and reverse-transcribing the sample RNA and then detected by qRT-PCR. LOC_Os01g68930The expression level of the gene is as follows:

[0035] Take rice NNIP and overexpression plants OE- LOC_Os01g68930 -1 and OE- LOC_Os01g68930 -2 Fresh leaf samples were used to extract RNA using the Trizol method. cDNA was synthesized using the reverse transcription kit Hifair® AdvanceFast1st Strand cDNA Synthesis Kit from Shanghai Yisheng Company, and random primers were used as primers. Using cDNA as a template, qRT-PCR was performed using the 2×SYBRGreen qPCR hot start premix 10 ul system from Zhongdao Life, with the specific system being: 1 μL cDNA, 5 μL SYBR Green Master Mix, 0.2 μL 10 μM Primer F (forward), 0.2 μL 10 μMPrimer R (reverse), and 3.6 μL ddH2O. The primer sequences are as follows:

[0036] Qrt-LOC_Os01g68930-F (as shown in SEQ ID No.5): CAATGCCCGCCTGAA

[0037] Qrt-LOC_Os01g68930-R (as shown in SEQ ID No.6): GTGTTGCCCTGCTCCC

[0038] The amplification program was: 95°C for 3 mins, 95°C for 10 s, 60°C for 30 s, 39 cycles, 65°C for 5 s, and 95°C for 0.5°C, in a fluorescence quantitative PCR instrument. The Ubiquitin (LOC_Os03g13170) gene was used as an internal reference, and the relative expression of the gene was calculated using 2-ΔΔCT.

[0039] The results are as follows Picture 2 As shown ( LOC_Os01g68930 -1 represents OE- LOC_Os01g68930 -1, LOC_Os01g68930 -2 represents OE- LOC_Os01g68930 -2), rice LOC_Os01g68930 The gene was successfully overexpressed.

[0040] Example 3 LOC_Os01g68930 Phenotypic observation and agronomic trait statistics of overexpression plants

[0041] The OE- LOC_Os01g68930 The overexpressing plants were planted in 3×6 lines in the experimental field of Fuyang Base of China National Rice Research Institute. All field experiments were uniformly managed in accordance with conventional field production methods.

[0042] Yield trait investigation: After the seeds mature, select NNIP and OE -LOC_Os01g68930 The middle plants within the strain were selected as the objects for investigation and analysis of agronomic traits.

[0043] The results showed that compared with NNIP, OE- LOC_Os01g68930 -1 and OE- LOC_Os01g68930 -2 delayed the heading date by 2-4 days, and the plant height was relatively short at maturity, but the number of tillers increased significantly ( Picture 4 ); however, in terms of grain morphology, overexpressed OE- LOC_Os01g68930 -1 and OE- LOC_Os01g68930 -2 Compared with NNIP, grain length, grain width and thousand-grain weight decreased ( Picture 5 ).

[0044] Example 4 LOC_Os01g68930 Determination of grain quality traits in overexpressed rice

[0045] The T2 generation harvested in 2024 at the experimental field of the Fuyang base of the China National Rice Research Institute OE-LOC_Os01g68930 The seeds of the wild type were shelled, ground, sieved, and dried in an oven at 42 degrees Celsius to constant weight for subsequent protein, starch, and soluble sugar determination.

[0046] Determination of glutenin and alcohol-soluble protein content in brown rice:

[0047] 1) Extraction of glutenin and alcohol-soluble protein from grains

[0048] Accurately weigh 0.1g of brown rice flour to be tested, put it into a 2ml centrifuge tube, add steel balls and 1.6ml 0.1mol / LNaOH, put it into a sampler at 50hz for 60s, and then place it for 30mins. Shake it several times during the placement process. Centrifuge at 3500 rpm for 15min, transfer the supernatant into a new 2ml centrifuge tube, add distilled water to 2ml, shake it well, and get gluten.

[0049] The above precipitate was repeatedly extracted with 1 ml of 70% ethanol, and the operation steps were the same as above to obtain alcohol-soluble protein (three replicates per group)

[0050] 2) Determination of protein content by Coomassie Brilliant Blue G250

[0051] Prepare Coomassie Brilliant Blue G250 solution: dissolve 10 mg of Coomassie Brilliant Blue G250 in 5 ml of 90% ethanol, add 10 ml of 85% (w / v) phosphoric acid, dilute to 100 ml with distilled water and filter.

[0052] 3) Drawing of standard curve and sample determination

[0053] Weigh 40 mg of bovine serum albumin and dissolve it in 10 ml of distilled water to prepare a standard protein solution. In a 200 uL microcentrifuge tube, add 20 ul of distilled water and 20 uL of 4000ug / mL BSA to obtain a 2000ug / mL BSA standard solution; repeat this process, taking 20 microliters of the current concentration of BSA solution each time for serial dilution, and finally 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625 ug / mL BSA standard solutions. -LOC_Os01g68930 Take 3uL of each of the wild-type gluten and alcohol-soluble protein solutions and each dilution standard solution, mix them evenly with 297uL of Coomassie Brilliant Blue G-250 staining working solution, place them at room temperature for 2 minutes, and place them in a microplate reader to measure their absorbance (OD595 value) at a wavelength of 595 nanometers. Draw a standard curve with the standard protein concentration (ug / mL) as the horizontal axis and the corresponding OD595 absorbance value as the vertical axis. Finally, according to the OD595 values ​​of the extracted OE-LOC_Os01g68930 transgenic samples and wild-type samples, refer to the standard curve to find out the gluten and alcohol-soluble protein concentrations of unknown samples.

[0054] Determination of soluble sugar content in brown rice:

[0055] 1) Extraction of soluble sugar: Weigh 0.1g of brown rice flour into a 15 mL centrifuge tube, add 10 mL of 80% ethanol, heat the centrifuge tube in an 80°C water bath for 30 minutes, then take it out and centrifuge it at 2000 rpm for 5 minutes after cooling, and transfer the obtained supernatant to a 50 mL centrifuge tube; for the residue left after centrifugation, repeat the above steps of adding ethanol, heating, centrifugation and collecting supernatant twice. The supernatant collected three times can be used to measure the soluble sugar content. The residue can be dried in an oven at 80°C overnight for the determination of starch content.

[0056] 2) Preparation of glucose standard solution (1 mg / mL): Weigh 50 mg of anhydrous glucose and dissolve it in water, dilute to 40 mL to obtain 1 mg / mL glucose standard solution, and dilute 1 mg / mL 7 times in a row.

[0057] 3) Preparation of anthrone reagent: Dissolve 0.5 g of anthrone in 500 mL of 80% H2SO4.

[0058] 4) Drawing of standard curve and sample determination: Take 100 μL of a series of glucose standard solutions and test solutions, add them to test tubes containing 500 μL of anthrone reagent, and heat them in a 100°C water bath for 10 min. After cooling to room temperature, take out 300 μL of solution, add it to the ELISA plate, and then place it in an ELISA reader to measure its absorbance at a wavelength of 620 nanometers (OD620 value). Calculate the concentration and content of soluble sugar in the sample.

[0059] Determination of starch content in brown rice:

[0060] 1) Starch extraction: After the soluble sugars are extracted, the residue remaining is dried to constant weight, and 2 mL of distilled water is added to the residue, and the mixture is treated in a water bath at 100 °C for 20 min, and then removed and cooled. 2 mL of 9.2 N HClO4 is added, and then the mixture is treated in a water bath at 100 °C for 30 min, removed and cooled, and 6 mL of distilled water is added, and the mixture is centrifuged at 2500 rpm for 10 min, and the supernatant is collected in a new centrifuge tube. 2 mL of 4.6 N HClO4 is added to the residue again, and the mixture is treated in a water bath at 100 °C for 30 min, removed and cooled, and 6 mL of distilled water is added, and the mixture is centrifuged at 2500 rpm for 10 min, and the supernatant is collected in the same new centrifuge tube, and the volume is fixed to 40 mL with distilled water.

[0061] 2) Sample determination: 100 μL of the extract was taken out from the fixed solution for determination of starch content using the same method as previously used for determination of soluble sugars.

[0062] The results are as follows Picture 7 As shown, LOC_Os01g68930 The total content of glutenin and alcohol-soluble proteins in the seeds of the same strain of overexpressed transgenic rice showed an upward trend with the increase of gene expression. The total content of glutenin and alcohol-soluble proteins in the seeds reached 7.88% to 7.97%. In comparison, the content of wild-type rice was 6.97%, and the protein content of transgenic rice grains increased by 13.1%-14.3%. Correspondingly, the starch content of transgenic rice seeds was significantly reduced, from 85% to 71%, and the soluble sugar content did not change significantly, indicating that the starch content of transgenic rice seeds was significantly reduced, from 85% to 71%, and the soluble sugar content did not change significantly. LOC_Os01g68930 In transgenic rice with gene overexpression, the distribution of carbon sources was adjusted, in which the synthesis of glutenin and alcohol-soluble proteins was enhanced, while the synthesis of starch was inhibited.

[0063] The present invention is described in detail above. Definitions of Terms Related to the Present Invention Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

[0064] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0065] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0066] For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application, and the changes made with conventional techniques known in the art.

Claims

1. A kind of rice LOC_Os01g68930 The application of the gene in increasing the total content of glutelin and alcohol-soluble protein in rice grains is characterized in that: The rice LOC_Os01g68930 The CDS sequence of the gene is shown in SEQ ID No. 1, and the rice LOC_ Os01g68930 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2; the application is to overexpress the gene in rice LOC_Os01g68930 Gene, increasing the total content of glutelin and alcohol-soluble proteins in the grains of rice overexpressing plants.

2. Rice LOC_Os01g68930 The application of the gene in the breeding of rice with high glutenin and total alcohol-soluble protein content is characterized in that: The rice LOC_Os01g68930 The CDS sequence of the gene is shown in SEQ ID No. 1, and the rice LOC_ Os01g68930 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

2.

3. An application of an overexpression vector in increasing the total content of glutelin and alcohol-soluble protein in rice grains, characterized in that: The overexpression vector contains rice LOC_Os01g68930 Gene, the rice LOC_Os01g68930 The CDS sequence of the gene is shown in SEQ ID No. 1, and the rice LOC_Os01g68930 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No.

2.

4. The use according to claim 3, characterized in that: The application is to transfer the overexpression vector into rice plants.

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