Application of Osmads91 gene in regulation and control of rice taste quality

Abstract

The invention discloses application of an Osmads91 gene in regulation and control of rice taste quality, and belongs to the technical field of biotechnology and plant genetic engineering. The taste quality comprises nutritional quality and cooking quality; the nutritional quality comprises the content of amylose; the cooking quality comprises rice hardness and rice viscosity; the nucleotide sequence of the Osmads91 gene is as shown in SEQ ID NO. 1 (sequence identifier number 1). After the Osmads91 gene is knocked out by utilizing a CRISPR / Cas9 technology, the content of amylose in the rice is remarkably reduced, the hardness of the cooked rice is reduced, the viscosity of the cooked rice is enhanced, the eating quality of the rice is remarkably improved, but the plant height, the tiller number and the thousand seed weight of the rice are not remarkably influenced. A new gene resource is provided for high-quality breeding of cereal crops such as rice, and a technical support is also provided for cloning related genes in other crops.

Description

Technical Field

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

[0002] Rice is a major food crop. With the improvement of people's consumption level and quality of life, consumers' rice consumption concept has changed from "eating enough" to "eating well and eating healthily", and higher requirements are put forward for the taste quality and nutritional quality of rice. Rice with good quality is becoming more and more popular among consumers. Taste quality is a direct factor affecting consumer choice, and thus has become an extremely important evaluation indicator of rice quality. Although the national standard "Sensory Evaluation Method for Steamed Rice Edible Quality" (GB / T15682.2008) has been issued, it is still impossible to accurately identify the quality of rice by manual tasting because it is impossible to completely eliminate the influence of subjective factors.

[0003] Previous research results show that the level of amylose content has an important influence on the taste quality of rice. Therefore, it is regarded as an important indicator for measuring the taste quality of rice both at home and abroad, including apparent amylose content (AC), gelatin consistency, gelatinization temperature and rice starch viscosity spectrum (RVA). If the amylose content is high, the rice will be less sticky after steaming, the texture will be hard and the taste will be poor. However, the amylose content cannot be too low, otherwise the rice will be too sticky, have poor elasticity and be unpopular.

[0004] The synthesis of amylose in rice endosperm is controlled by the Wx locus, which encodes starch granule-bound starch synthase I (GBSSI), which is responsible for the synthesis of amylose, also known as Wx protein. It is mainly present in storage tissues such as endosperm and embryo sac. The Wx gene is not expressed in glutinous rice, so the amylose content in glutinous rice is extremely low, almost non-existent. The non-weak gene Wx is dominant to the glutinous gene wx, and there is a relatively obvious dosage effect. Peng Jisong and others believe that soluble starch synthase plays an important role in the ratio of amylose content to amylopectin content. Zhong Lianjin and others also showed that the activity of starch granule-bound starch synthase GBSS is closely related to the difference in amylose content. Most people prefer rice with medium amylose content, but many varieties cannot meet this condition. Therefore, it is necessary to fine-tune the amylose content. In order to improve the competitiveness of rice and meet the needs of the market and consumers, improving the taste quality of rice has become the focus of the development of the rice industry. 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 the taste quality of rice. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the Osmads91 gene in regulating the taste quality of rice to solve the problems existing in the above-mentioned prior art. After the Osmads91 gene is knocked out by the CRISPR / Cas9 technology, the content of amylose in rice is significantly reduced, the hardness of rice after cooking is reduced, the viscosity of rice after cooking is enhanced, and the taste quality of rice is significantly improved, but the plant height, tiller number and thousand-grain weight of rice are not significantly affected. The present invention provides new gene resources for high-quality breeding of cereal crops such as rice, and also provides 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 an application of Osmads91 gene, protein encoded by Osmads91 gene or biological material with knocked-out Osmads91 gene in regulating rice taste quality, wherein 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;

[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 having the function of knocking out the Osmads91 gene or a recombinant microorganism having the function 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 the 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 having the function of knocking out the Osmads91 gene or a recombinant microorganism having the function 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, and the amino acid sequence of the protein 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 above-mentioned Osmads91 gene mutant or the above-mentioned 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] The present invention identifies a gene Osmads91 (also known as MADS-box91) that controls the taste quality of rice. After knocking out the gene using CRISPR / Cas9 technology, the content of amylose in rice can be significantly reduced, the hardness of rice after cooking can be reduced, the viscosity of rice after cooking can be increased, and the taste quality of rice can be significantly improved, but there is no significant effect on the plant height, tiller number and thousand-grain weight of rice. The present invention provides new gene resources for high-quality breeding of rice and other cereal crops, and also provides 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 drawings required for use in the embodiments will be briefly introduced below. 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 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 A is a schematic diagram of the structure of the SK-gRNA vector and the pC1300-Cas9 vector; A is a schematic diagram of the structure of the SK-gRNA vector; B is a schematic diagram of the structure of the pC1300-Cas9 vector;

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

[0031] Figure 4 Figure 1 is the growth status and agronomic traits test results of the homozygous mutant strains of Osmads91 gene and wild-type NIP; A is the growth status of the homozygous mutant strains of Osmads91 gene and wild-type NIP; B is the plant height of the homozygous mutant strains of Osmads91 gene and wild-type NIP; C is the tillering number of the homozygous mutant strains of Osmads91 gene and wild-type NIP; D is the thousand-grain weight of the homozygous mutant strains of Osmads91 gene and wild-type NIP;

[0032] Figure 5 A is the detection result of the relative expression of Wx gene and the content of GBSSI protein in the homozygous mutant strain of Osmads91 gene and wild-type NIP; A is the detection result of the relative expression of Wx gene in the homozygous mutant strain of Osmads91 gene and wild-type NIP; B is the detection result of the content of GBSSI protein in the homozygous mutant strain of Osmads91 gene 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 7A is the test results of rice taste quality of Osmads91 gene homozygous mutant strains and wild-type NIP; A is the rice chalkiness of Osmads91 gene homozygous mutant strains and wild-type NIP; B is the rice amylose content of Osmads91 gene homozygous mutant strains and wild-type NIP; C is the rice hardness of Osmads91 gene homozygous mutant strains and wild-type NIP; D is the rice viscosity of Osmads91 gene homozygous mutant strains 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 is the japonica rice variety Nipponbare (NIP).

[0042] 2. Osmads91 gene: Use the RAP-DB website (https: / / rapdb.dna.affrc.go.jp) and 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 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 relative expression of Osmads91 gene in different tissues (root, stem, leaf, sheath) and grains at different growth stages (3d, 5d, 10d, 15d) of NIP was detected to analyze the spatiotemporal expression profile of Osmads91 gene. 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) on the Osmads91 gene of NIP. The first three bases of the PAM sequence are the mutation sites of the CAS9 protein. The selected target sequence is: CACCGGCGCTAGCGTCGCCGTGG (SEQ ID NO. 3), and the PAM sequence is 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 DNAs is as follows:

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

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

[0055] (2) Intermediate vector construction:

[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. Mix and connect the vector with sticky ends and the fragment with sticky ends at a molar ratio of 1:(3-10), and transform them into E. coli DH5α. Use primer T3 and g-- to perform colony PCR positive detection.

[0059] D. Use common 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. The 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 connected to the pC1300-Cas9 vector after restriction digestion, and then transformed into Escherichia coli DH5α.

[0062] The transformed E. coli was cultured, and the colony PCR positive detection was performed using primers T3 and g--, and 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] The DNA of leaves of transgenic T0 plants was extracted, and PCR amplification was performed using primers Osmads91-F (SEQ ID NO.6) and Osmads91-R (SEQ ID NO.7), and the PCR products were sequenced. Two homozygous mutant lines were obtained by sequencing, both of which terminated prematurely at 82aa and 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 in the two homozygous mutant strains and the amino acid sequences of the proteins encoded by them are as follows:

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

[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 the 82nd amino acid):

[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 homozygous mutant strains of Osmads91 gene

[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 It can be seen that there is no significant difference in plant height, tiller number and 1000-grain weight between the homozygous mutant strain and the wild-type NIP, indicating that the Osmads91 gene does not affect the agronomic traits of rice.

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

[0081] DNA from leaves of homozygous mutant strains osmads91-1, osmads91-2 and wild-type NIP was extracted, and PCR amplification was performed using primer pairs Wx-F and Wx-R. Ubiquitin was used as the internal reference gene to calculate the relative expression of the Wx gene. Western Blot technology was used to detect the content of GBSSI protein in the three strains. The sequences of the primer pairs used are as follows:

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

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

[0084] Results Figure 5 It can be seen that compared with wild-type NIP, the expression of Wx genes in rice was significantly reduced after knocking out the Osmads91 gene, and the content of GBSSI protein was also reduced. This shows 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 the Taste Quality of Rice with the Homozygous Mutant Line of Osmads91 Gene

[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 out 0.0500g of rice flour and place it in a 50mL volumetric flask. Slightly shake the volumetric flask to shake the rice flour attached to the wall of the volumetric flask to the bottom of the bottle. First add 500μL of anhydrous ethanol and mix slightly, then add 4.5mL of 1mol / L NaOH solution along the bottleneck of the volumetric flask and let it stand at room temperature for 23h. Then make up to 50mL with deionized water and shake evenly. Pipette 0.5mL of sample solution and 5mL of water into a test tube, add 100μL of 1mol / L acetic acid solution, 200μL of KI-I2 and 4.2mL of H2O to the test tube, shake vigorously on the oscillator to mix, and let it stand for 20min. 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 at a wavelength of 620nm on the spectrophotometer and measure the absorbance value 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 and simmer 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 TA.XTplus physical property tester (Stable MicroSystems, UK) was used for the measurement. The measurement conditions are as follows: probe: P / 36R; induction force: 20.0g; pre-measurement speed: 1.0mm / s, test speed: 1.0mm / s, post-measurement speed: 1.0mm / s; a secondary compression test was used, with the first compression ratio of 40%, the second compression ratio of 75%, and the interval between the two compressions of 5s. During the test, 3 whole rice grains were randomly selected from the middle part of the rice sample each time, and placed in a ring shape directly under the probe of the texture analyzer stage for testing. Each sample was measured 6 times, the maximum and minimum values ​​were removed, and the average value was 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 the 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 the 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 knocking out 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Osmads91 gene, protein encoded by Osmads91 gene or biological material with knockout of Osmads91 gene in regulating the taste quality of rice, 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.

2. The use according to claim 1, characterized in that: 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.

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

4. Use of the Osmads91 gene, the protein encoded by the Osmads91 gene or the biological material with the Osmads91 gene knocked out 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.

5. The use according to claim 4, characterized in that: The application is to knock out the Osmads91 gene in rice to construct transgenic rice with low amylose content, low hardness or high viscosity.

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

7. An Osmads91 gene mutant, characterized in that: The nucleotide sequence of the Osmads91 gene mutant is shown in SEQ ID NO.8 or SEQ ID NO.

10.

8. The protein encoded by the Osmads91 gene mutant according to claim 7, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.9 or SEQ ID NO.

11.

9. Use of the Osmads91 gene mutant according to claim 7 or the protein according to claim 8 in regulating the taste quality of rice, characterized in that: The eating 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.

10. Use of the Osmads91 gene mutant according to claim 7 or the protein according to claim 8 in cultivating any of the following transgenic rice: (1) Transgenic rice with low amylose content; (2) Transgenic rice with low hardness; (3) High viscosity genetically modified rice.

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