Application of Osmads91 gene in regulating rice taste quality

Abstract

The present invention discloses the application of the Osmads91 gene in regulating the taste quality of rice, belonging to the fields of biotechnology and plant genetic engineering technology. The taste quality includes nutritional quality and cooking quality; the nutritional quality includes the content of amylose; the cooking quality includes rice hardness and rice viscosity; the nucleotide sequence of the Osmads91 gene is shown in SEQ ID NO.1. After the present invention uses CRISPR / Cas9 technology to knock out the Osmads91 gene, the content of amylose in rice is significantly reduced, the hardness of rice after cooking is reduced, the viscosity of rice after cooking is increased, and the taste quality of rice is significantly improved, but there is no significant effect on the plant height, tiller number, and 1000-grain weight of rice. The present invention provides a new gene resource for the high-quality breeding of cereal crops such as rice, and also provides technical support for the cloning of related genes in other crops.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology and plant genetic engineering, and in particular to the application of the Osmads91 gene in regulating the taste quality of rice. Background Art

[0002] Rice is a staple food crop. With rising consumer spending and quality of life, consumers' perception of rice consumption has shifted from "satisfying themselves" to "eating well and healthily." This shift has led to higher expectations for rice's flavor and nutritional qualities, and high-quality rice is increasingly popular. Taste quality directly influences consumer choice, making it a crucial indicator of rice quality. Despite the promulgation of the national standard "Method for Sensory Evaluation of Steamed and Cooked Rice" (GB / T15682.2008), manual tasting remains incapable of accurately assessing rice quality due to the inability to completely eliminate subjective factors.

[0003] Previous research has shown that amylose content significantly influences the taste quality of rice. Therefore, it is widely considered a crucial indicator of rice taste quality, along with apparent amylose content (AC), gel consistency, gelatinization temperature, and rice viscosity profile (RVA). High amylose content results in less sticky rice after cooking, resulting in a harder texture and a poorer taste. However, low amylose content is not recommended, as it can make the rice too sticky and less elastic, making it unpalatable.

[0004] Amylose synthesis in the rice endosperm is controlled by the Wx locus, which encodes the enzyme granule-bound starch synthase I (GBSSI), also known as the Wx protein. This locus is primarily found in storage tissues such as the endosperm and embryo sac. In glutinous rice, the Wx gene is not expressed, resulting in extremely low, almost no, amylose content. The non-glutinous gene Wx is dominant over the waxy gene wx, and there is a significant dosage effect. Research by Peng Jisong et al. suggests that soluble starch synthase plays a significant role in determining the ratio of amylose to amylopectin content. Research by Zhong Lianjin et al. also demonstrates that the activity of the granule-bound starch synthase GBSS is closely correlated with amylose content. While people generally prefer rice with a medium amylose content, many varieties do not meet this requirement. Therefore, fine-tuning amylose content is essential. To enhance rice competitiveness and meet market and consumer demands, improving rice taste quality has become a key focus of rice industry development. Although some genes related to taste quality have been cloned so far, they are still far from meeting the needs of improving rice taste quality through genetic engineering. Therefore, isolating and identifying genes that can regulate the expression of Wx genes is of great significance to improving rice taste quality. Summary of the Invention

[0005] The present invention aims to provide the application of the Osmads91 gene in regulating rice taste quality, thereby addressing the problems of the prior art described above. By utilizing CRISPR / Cas9 technology to knock out the Osmads91 gene, the present invention significantly reduces the amylose content in rice, reduces the hardness of rice after cooking, increases the viscosity of rice after cooking, and significantly improves the taste quality of rice, while having no significant effect on plant height, tiller number, or 1000-grain weight. This invention provides a new genetic resource for high-quality breeding of rice and other cereal crops, and also offers technical support for cloning related genes in other crops.

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

[0007] The present invention provides the use of the Osmads91 gene, the protein encoded by the Osmads91 gene, or a biological material with the Osmads91 gene knocked out in regulating the taste quality of rice, wherein the taste quality includes nutritional quality and cooking quality; the nutritional quality includes the content of amylose; the cooking quality includes the hardness and viscosity of rice;

[0008] The nucleotide sequence of the Osmads91 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the Osmads91 gene is shown in SEQ ID NO.2.

[0009] Furthermore, the application is to reduce the content of amylose, reduce the hardness of rice, and increase the viscosity of rice by knocking out or inhibiting the expression of Osmads91 gene in rice.

[0010] Optionally, the biological material includes a recombinant vector capable of knocking out the Osmads91 gene or a recombinant microorganism capable of knocking out the Osmads91 gene.

[0011] The present invention also provides the use of the Osmads91 gene, the protein encoded by the Osmads91 gene, or a biological material with the Osmads91 gene knocked out in cultivating any of the following transgenic rice:

[0012] (1) Transgenic rice with low amylose content;

[0013] (2) Transgenic rice with low hardness;

[0014] (3) High viscosity transgenic rice;

[0015] The nucleotide sequence of the Osmads91 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the Osmads91 gene is shown in SEQ ID NO.2.

[0016] Furthermore, the application is to construct transgenic rice with low amylose content, low hardness or high viscosity by knocking out the Osmads91 gene in rice.

[0017] Optionally, the biological material includes a recombinant vector capable of knocking out the Osmads91 gene or a recombinant microorganism capable of knocking out the Osmads91 gene.

[0018] The present invention also provides an Osmads91 gene mutant, the nucleotide sequence of the Osmads91 gene mutant is shown as SEQ ID NO.8 or SEQ ID NO.10.

[0019] The present invention also provides a protein encoded by the above-mentioned Osmads91 gene mutant, the amino acid sequence of which is shown in SEQ ID NO.9 or SEQ ID NO.11.

[0020] The present invention also provides the use of the Osmads91 gene mutant or the protein in regulating the taste quality of rice. The taste quality includes nutritional quality and cooking quality; the nutritional quality includes the content of amylose; and the cooking quality includes rice hardness and rice viscosity.

[0021] The present invention also provides the use of the Osmads91 gene mutant or the protein in cultivating any of the following transgenic rice:

[0022] (1) Transgenic rice with low amylose content;

[0023] (2) Transgenic rice with low hardness;

[0024] (3) High viscosity genetically modified rice.

[0025] The present invention discloses the following technical effects:

[0026] This study identified a gene, Osmads91 (also known as MADS-box91), that controls rice flavor quality. Knocking out this gene using CRISPR / Cas9 technology significantly reduced the amylose content in rice, reduced rice hardness after cooking, and increased rice viscosity after cooking, significantly improving the rice's flavor quality. However, this study had no significant effect on plant height, tiller number, or 1000-grain weight. This study provides a new genetic resource for high-quality breeding of rice and other cereal crops, and also offers technical support for cloning related genes in other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 The results of temporal and spatial expression analysis of Osmads91 gene during the whole growth period of rice;

[0029] Figure 2 Schematic diagram of the structures of SK-gRNA vector and pC1300-Cas9 vector; A is a schematic diagram of the structure of SK-gRNA vector; B is a schematic diagram of the structure of pC1300-Cas9 vector;

[0030] Figure 3 It is the restriction site for the SK-gRNA intermediate vector and the pC1300-Cas9 vector;

[0031] Figure 4 Figure 2 is the growth status and agronomic trait test results of the Osmads91 gene homozygous mutant strain and the wild-type NIP; A is the growth status of the Osmads91 gene homozygous mutant strain and the wild-type NIP; B is the plant height of the Osmads91 gene homozygous mutant strain and the wild-type NIP; C is the tiller number of the Osmads91 gene homozygous mutant strain and the wild-type NIP; D is the 1000-grain weight of the Osmads91 gene homozygous mutant strain and the wild-type NIP;

[0032] Figure 5 Figure 2 is the detection result of the relative expression of Wx gene and GBSSI protein content in the Osmads91 gene homozygous mutant strain and wild-type NIP; A is the detection result of the relative expression of Wx gene in the Osmads91 gene homozygous mutant strain and wild-type NIP; B is the detection result of the GBSSI protein content in the Osmads91 gene homozygous mutant strain and wild-type NIP;

[0033] Figure 6 The figure is a comparison of the brown rice phenotypes of the homozygous mutant strain of the Osmads91 gene and the wild-type NIP;

[0034] Figure 7Figure 5 is the test results of rice taste quality of the homozygous mutant strain of Osmads91 gene and wild-type NIP; A is the chalkiness of rice of the homozygous mutant strain of Osmads91 gene and wild-type NIP; B is the amylose content of rice of the homozygous mutant strain of Osmads91 gene and wild-type NIP; C is the hardness of rice of the homozygous mutant strain of Osmads91 gene and wild-type NIP; D is the viscosity of rice of the homozygous mutant strain of Osmads91 gene and wild-type NIP. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1 Spatiotemporal expression analysis of Osmads91 gene

[0041] 1. Rice material: The original wild type material was the japonica rice variety Nipponbare (NIP).

[0042] 2. Osmads91 gene: The RAP-DB website (https: / / rapdb.dna.affrc.go.jp) was used to input the RAP_Locus number Os01g0213300 of the Osmads91 gene to obtain the nucleotide sequence of the Osmads91 gene (SEQ ID NO. 1).

[0043] The CDS sequence of the Osmads91 gene is shown in SEQ ID NO.1:

[0044]

[0045] The protein sequence encoded by the Osmads91 gene is shown in SEQ ID NO.2:

[0046] *.

[0047] 3. Spatiotemporal expression analysis: The spatiotemporal expression profile of Osmads91 gene was analyzed by detecting the relative expression of Osmads91 gene in different tissue parts (root, stem, leaf, sheath) and grains at different growth stages (3d, 5d, 10d, and 15d of grain filling). Figure 1 , it can be seen that the expression level of Osmads91 gene in grains is high.

[0048] Example 2 Construction of Osmads91 gene knockout rice strain

[0049] 1. Construction of knockout plasmid

[0050] (1) Target sequence selection and primer design:

[0051] A. Find the PAM sequence (NGG sequence) in the Osmads91 gene of NIP. The first three bases of the PAM sequence are the intended cleavage mutation site for the CAS9 protein. The selected target sequence is: CACCGGCGCTAGCGTCGCCGTGG (SEQ ID NO. 3), with a PAM sequence of TGG.

[0052] B. Design two complementary DNA sequences based on the selected target sequence: by adding GGCA before the forward target sequence and AAAC before the reverse complementary target sequence. The sequence information of the two complementary DNA sequences is as follows:

[0053] g++: GGCA-CACCGGCGCTAGCGTCGCCG (SEQ ID NO.4);

[0054] g--: AAAC-CGGCGACGCTAGCGCCGGTG (SEQ ID NO. 5).

[0055] (2) Construction of intermediate vector:

[0056] A. The SK-gRNA vector was digested with AarI (Ferment) to form a vector with sticky ends; the structural diagram of the SK-gRNA vector is shown in Figure 2 A;

[0057] B. After mixing g++ and g-- primers, they are denatured and annealed to form fragments with sticky ends;

[0058] C. Ligate the vector with sticky ends and the fragment with sticky ends at a molar ratio of 1:(3-10). After ligation, transform the vector into E. coli DH5α. Perform colony PCR using primers T3 and g-- for positive results.

[0059] D. Use public primers T7 or T3 for sequencing verification to obtain the SK-gRNA intermediate vector.

[0060] (3) Final vector construction:

[0061] The pC1300-Cas9 vector was digested with KpnI and BamHI, and the SK-gRNA intermediate vector was also digested with KpnI and BglII to recover the target fragment. The schematic diagram of the pC1300-Cas9 vector is shown in Figure 2 B. Schematic diagram of the structure of the restriction sites of the SK-gRNA intermediate vector and the pC1300-Cas9 vector is shown in Figure 3 The target fragment was ligated with the pC1300-Cas9 vector after enzyme digestion and then transformed into Escherichia coli DH5α.

[0062] The transformed E. coli was cultured and colony PCR positive detection was performed using primers T3 and g--. Sequencing verification was performed using common primers T7 or T3 to extract and identify the correct positive knockout plasmid.

[0063] 2. Construction of rice knockout lines

[0064] The correctly identified positive knockout plasmid was transformed into Agrobacterium EHA105, and then transformed into NIP using the Agrobacterium-mediated rice genetic transformation method to obtain T0 generation transgenic plants.

[0065] DNA was extracted from leaves of the transgenic T0 generation plants and amplified by PCR using primers Osmads91-F (SEQ ID NO. 6) and Osmads91-R (SEQ ID NO. 7). The PCR products were sequenced. Sequencing confirmed the presence of two homozygous mutant lines, both of which terminated prematurely at 82 aa. They were named osmads91-1 and osmads91-2, respectively.

[0066] The primer pairs used are as follows:

[0067] Osmads91-F: AAACCTCCATCGATCCCCTC (SEQ ID NO.6);

[0068] Osmads91-R: CGGAGCTTGCTGTGATTCAA (SEQ ID NO. 7).

[0069] The nucleotide sequences of the Osmads91 gene mutants and the amino acid sequences of their encoded proteins in the two homozygous mutant lines are as follows:

[0070] The nucleotide sequence of the osmads91-1 gene mutant is shown in SEQ ID NO.8 (one base A is inserted, and a stop codon TGA appears after amino acid position 82):

[0071]

[0072] The amino acid sequence of the protein encoded by the osmads91-1 gene mutant is shown in SEQ ID NO.9:

[0073] MPRRARRTGAAYVDDERERDITFFKRRNGLFKCASDLSILTGASVDRGHRGPEPQQVPRGRDADGAGRRRRRPLKRRGGGGG*.

[0074] The nucleotide sequence of the osmads91-2 gene mutant is shown in SEQ ID NO. 10 (a base C is inserted, and a stop codon TGA appears after amino acid position 82):

[0075]

[0076] The amino acid sequence of the protein encoded by the osmads91-2 gene mutant is shown in SEQ ID NO.11:

[0077] MPRRARRTGAAYVDDERERDITFFKRRNGLFKCASDLSILTGASVARGHRGPEPQQVPRGRDADGAGRRRRRPLKRRGGGGG*.

[0078] Example 3 Detection of agronomic traits of Osmads91 gene homozygous mutant strains

[0079] The homozygous mutant lines osmads91-1 and osmads91-2 and wild-type NIP were cultivated under the same environment. The growth status and agronomic performance of the three lines were shown in Figure 2. Figure 4 As can be seen, there are no significant differences in plant height, tiller number, and 1000-grain weight between the homozygous mutant and wild-type NIP lines, indicating that the Osmads91 gene does not affect the agronomic traits of rice.

[0080] Example 4 Detection of related genes in the homozygous mutant strain of Osmads91 gene

[0081] Leaf DNA was extracted from homozygous mutant lines osmads91-1 and osmads91-2, as well as wild-type NIP. PCR amplification was performed using primer pairs Wx-F and Wx-R, with Ubiquitin as the internal reference gene. The relative expression of the Wx gene was calculated. Western blotting was used to measure GBSSI protein levels in the three lines. The primer pairs used are as follows:

[0082] Wx-F: AACGTGGCTGCTCCTTGAA (SEQ ID NO. 12);

[0083] Wx-R: TTGGCAATAAGCCACACACA (SEQ ID NO. 13).

[0084] See the results Figure 5 As shown in Figure 3, compared with wild-type NIP, knockout of the Osmads91 gene significantly reduced the expression of Wx genes and the content of GBSSI protein in rice. This suggests that the Osmads91 gene can regulate the expression of Wx genes and is related to the abundance of GBSSI protein.

[0085] Example 5 Detection of Taste Quality of Rice Grains of Osmads91 Gene Homozygous Mutant Lines

[0086] Homozygous mutant lines osmads91-1 and osmads91-2 and wild-type NIP were cultivated under the same environment until harvest, and mature rice grains of the three lines were collected. The brown rice phenotypes of the homozygous mutant line osmads91-1 and wild-type NIP were shown in Figure 2. Figure 6 .

[0087] After the harvested rice grains are processed, polished rice is obtained. The taste quality of polished rice is determined according to the following method:

[0088] (1) Determination of amylose content:

[0089] Weigh 0.0500g of rice flour into a 50mL volumetric flask. Gently shake the flask to remove any flour adhering to the flask wall. First, add 500μL of anhydrous ethanol and mix gently. Then, add 4.5mL of a 1 mol / L NaOH solution along the neck of the flask and let it stand at room temperature for 23 hours. Then, dilute to 50mL with deionized water and shake evenly. Pipette 0.5mL of the sample solution and 5mL of water into a test tube. Add 100μL of a 1 mol / L acetic acid solution, 200μL of KI-I2, and 4.2mL of H2O to the test tube. Mix vigorously on an oscillator and let it stand for 20 minutes. Draw a standard curve using standard samples with amylose contents of 1.5%, 10.4%, 16.2%, and 26.5%, respectively. Use a blank solution to adjust the zero point of the spectrophotometer at a wavelength of 620nm and measure the absorbance of the colored sample solution. The standard curve was then used to calculate the amylose content in the samples.

[0090] (2) Determination of chalkiness:

[0091] The harvested seeds were investigated for their chalkiness using the Wanshen SC-M Seed Appearance Quality Analyzer.

[0092] (3) Determination of hardness and viscosity:

[0093] Wash the polished rice twice, soak it in water for 30 minutes, seal it and steam it at normal pressure for 30 minutes, then stir it and simmer it for 10 minutes. After simmering, cool it in a cooling box for 20 minutes, and finally cool it at room temperature for 90 minutes.

[0094] The measurements were performed using a TA.XTplus texture analyzer (Stable MicroSystems, UK). The test conditions were as follows: probe: P / 36R; induction force: 20.0 g; pre-test speed: 1.0 mm / s, test speed: 1.0 mm / s, and post-test speed: 1.0 mm / s. A two-compression test was used, with the first compression ratio being 40%, the second compression ratio being 75%, and the interval between the two compressions being 5 seconds. During each test, three whole rice grains were randomly selected from the center of the rice sample and placed in a ring directly below the probe on the texture analyzer stage. Each sample was measured six times, and the maximum and minimum values ​​were removed, and the average values ​​were calculated to obtain the hardness and viscosity values.

[0095] The results of the eating quality of the three rice strains are shown in Figure 7 It can be seen that compared with wild-type NIP, although knocking out the Osmads91 gene increased the chalkiness of rice, it significantly reduced the amylose content and hardness of rice, increased the adhesion of rice, and made rice more delicious, indicating that knocking out the Osmads91 gene can improve the taste quality of rice. In addition, Figure 4 The results showed that knockout of the Osmads91 gene had no significant effect on plant height, tiller number and 1000-grain weight of rice, indicating that this gene has great application prospects in improving the taste quality of rice.

[0096] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of a biomaterial with knockout Osmads91 gene in regulating rice taste quality, characterized in that: The taste quality includes nutritional quality and cooking quality; the nutritional quality includes the content of amylose; the cooking quality includes rice hardness and rice viscosity; The nucleotide sequence of the Osmads91 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the Osmads91 gene is shown in SEQ ID NO.2; The application is to reduce the content of amylose, reduce the hardness of rice, and increase the viscosity of rice by knocking out or inhibiting the expression of Osmads91 gene in rice.

2. The use according to claim 1, characterized in that The biological material includes a recombinant vector capable of knocking out the Osmads91 gene or a recombinant microorganism capable of knocking out the Osmads91 gene.

3. Use of a biological material with Osmads91 gene knockout in cultivating any of the following transgenic rice: (1) Transgenic rice with low amylose content; (2) Transgenic rice with low hardness; (3) High viscosity transgenic rice; The nucleotide sequence of the Osmads91 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the Osmads91 gene is shown in SEQ ID NO.

2.

4. The use according to claim 3, characterized in that The biological material includes a recombinant vector capable of knocking out the Osmads91 gene or a recombinant microorganism capable of knocking out the Osmads91 gene.

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