Application of OsMYBR1 gene in regulating rice quality

Abstract

The application discloses application of an OsMYBR1 gene in regulating rice quality and belongs to the technical field of genetic engineering. In Nipponbare, the OsMYBR1 gene is knocked out by using CRISPR / Cas9 technology, and compared with a wild type, the chalkiness and chalky grain rate of the knockout mutant rice are significantly increased, and the appearance quality is deteriorated. However, the amylose content of the mutant rice is reduced, the pasting temperature of endosperm starch is reduced, the hardness and chewiness of rice are reduced, and the viscosity is significantly increased, which indicates that the cooking and eating quality of the knockout mutant rice is improved. Compared with the wild type, the chalkiness and chalky grain rate of the overexpression strain of the OsMYBR1 gene are not significantly different. The amylopectin content of the rice is significantly increased, the alkali-dissolving value of endosperm starch is increased, and the pasting entropy value is reduced, which indicates that the overexpression of the OsMYBR1 gene can improve the cooking and eating quality of the rice without affecting the appearance quality, and can be used for breeding and improving the quality of rice.

Description

TECHNICAL FIELD

[0001] The application relates to the field of genetic engineering, in particular to application of an OsMYBR1 gene in regulating rice quality. BACKGROUND

[0002] Rice (Oryza sativa L.) is one of the important food crops in China, and with the improvement of people's living standards, the demand for rice quality is also higher. Starch is the main component of rice, and its composition and structure determine the quality of rice. Rice quality traits mainly include processing, appearance, cooking taste quality and nutritional quality and other aspects. Chalky (chalkiness and chalky grain rate) is an important indicator for evaluating the appearance quality of rice, and directly affects the market price of rice. Chalky refers to the white opaque part in the endosperm of rice. Rice with chalky is easy to break during fine grinding, which directly affects the processing quality such as head rice rate. The chalky trait is a quantitative trait regulated by multiple genes, and is also easily affected by environmental factors. Rice cooking taste quality includes amylose content, gelatinization temperature and gel consistency of rice. Rice cooking taste quality is an important physicochemical index for evaluating rice quality, which determines the taste of rice. Mutations of endosperm starch synthesis related enzyme genes, such as OsGBSS I, OsSS IIIa and OsBE IIb, will cause obstacles in rice endosperm starch synthesis, changes in starch structure, increase in endosperm chalkiness and changes in cooking taste quality. Therefore, finding transcription factor genes involved in regulating rice endosperm starch synthesis, and using gene editing or overexpression to regulate rice endosperm starch synthesis, has important theoretical value and production application potential for improving rice quality. SUMMARY

[0003] The application aims to provide application of the OsMYBR1 gene in regulating rice quality, so as to solve the problems in the prior art. By overexpressing the OsMYBR1 gene in rice, the cooking taste quality of rice can be significantly improved under the premise of unchanged appearance quality. The application provides technical support for improving rice quality by using the OsMYBR1 gene.

[0004] To achieve the above-mentioned purpose, the application provides the following scheme.

[0005] The application provides application of the OsMYBR1 gene in any one of the following aspects:

[0006] (1) application in regulating rice quality;

[0007] (2) application in rice breeding;

[0008] The nucleotide sequence of the OsMYBR1 gene is shown in SEQ ID No. 1.

[0009] The application also provides application of the protein coded by the OsMYBR1 gene in any of the following aspects:

[0010] (1) application in regulating rice quality;

[0011] (2) application in rice breeding;

[0012] The amino acid sequence of the protein is shown in SEQ ID No. 2.

[0013] The application also provides application of the recombinant vector containing the OsMYBR1 gene in any of the following aspects:

[0014] (1) application in regulating rice quality;

[0015] (2) application in rice breeding;

[0016] The nucleotide sequence of the OsMYBR1 gene is shown in SEQ ID No. 1.

[0017] The application also provides application of the host bacterium containing the recombinant vector in any of the following aspects:

[0018] (1) application in regulating rice quality;

[0019] (2) application in rice breeding;

[0020] The recombinant vector contains the OsMYBR1 gene, and the OsMYBR1 gene is integrated into the genome of the host bacterium through the recombinant vector, and the nucleotide sequence of the OsMYBR1 gene is shown in SEQ ID No. 1.

[0021] Optionally, the rice quality includes chalkiness, chalky grain rate, amylose content, amylopectin content, endosperm starch alkali digestion value, gelatinization temperature, gelatinization entropy value and gel consistency of rice.

[0022] Optionally, the regulation mode includes (1) increasing the amylopectin content and endosperm starch alkali digestion value in rice by overexpressing the OsMYBR1 gene in rice, so as to improve the rice quality; or (2) reducing the amylose content and gelatinization temperature of endosperm starch in rice by knocking out the OsMYBR1 gene in rice, so as to improve the rice quality; and the nucleotide sequence of the OsMYBR1 gene is shown in SEQ ID No. 1.

[0023] The application also provides a method for improving the cooking and eating quality of rice, comprising (1) overexpressing the OsMYBR1 gene in rice to increase the amylopectin content and the alkali swelling value of endosperm starch in rice; or (2) knocking out the OsMYBR1 gene in rice to reduce the amylose content and the gelatinization temperature of endosperm starch in rice; the nucleotide sequence of the OsMYBR1 gene is shown as SEQ ID No. 1.

[0024] The application also provides a method for cultivating transgenic rice with improved cooking and eating quality, comprising (1) overexpressing the OsMYBR1 gene in rice to increase the amylopectin content and the alkali swelling value of endosperm starch in rice, so that the transgenic rice constructed has improved cooking and eating quality; or (2) knocking out the OsMYBR1 gene in rice to reduce the amylose content and the gelatinization temperature of endosperm starch in rice, so that the transgenic rice constructed has improved cooking and eating quality; the nucleotide sequence of the OsMYBR1 gene is shown as SEQ ID No. 1.

[0025] The application discloses the following technical effects:

[0026] (1) The application finds that the OsMYBR1 gene is a gene for regulating rice quality, and can be applied to genetic improvement of rice quality.

[0027] (2) The application provides the function of the OsMYBR1 gene in regulating rice quality. In the Nipponbare background, the OsMYBR1 gene is knocked out by using the CRISPR / Cas9 technology, and compared with the wild type, the chalkiness and chalky grain rate of the OsMYBR1 gene knockout mutant strain are extremely significantly increased, and the appearance quality is poor; but the amylose of the mutant rice is reduced, and the gelatinization temperature of endosperm starch is reduced, which can reduce the hardness and chewiness of rice and significantly increase the viscosity, indicating that the cooking and eating quality of the knockout mutant rice is increased.

[0028] (3) In the Nipponbare background, the OsMYBR1 gene is overexpressed, and compared with the wild type, the chalkiness and chalky grain rate of the overexpression transgenic strain are not significantly different, the amylopectin content of rice is significantly increased, and the alkali swelling value is increased, indicating that overexpression of the OsMYBR1 gene can make starch more gelatinized without affecting the appearance quality, improve the cooking and eating quality of rice, and can be used for breeding and improvement of rice quality. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and other accompanying drawings can also be obtained by those skilled in the art without any creative effort on the basis of these accompanying drawings.

[0030] Figure 1 Figure 2 is the relative expression level of OsMYBR1 gene in different tissues of rice; in the figure, Root is young root, Shoot is young stem, Leaf is young leaf, Spikelet is young spike, and 3DAF-24DAF is rice seed from 3d to 24d after flowering;

[0031] Figure 2 Figure 3 is the construction of OsMYBR1 gene knockout and overexpression lines; A: plant phenotype of OsMYBR1 gene knockout and overexpression lines; B: target site of OsMYBR1 gene knockout by CRISPR / Cas9 technology and base variation information of homozygous mutant obtained; C: relative expression level of OsMYBR1 gene in wild type, OsMYBR1 gene knockout mutant (osmybr1-1 and osmybr1-2) and overexpression lines (OE-MYBR1-1 and OE-MYBR1-2);

[0032] Figure 3 Figure 4 is the seed phenotype of OsMYBR1 gene knockout and overexpression lines; A: appearance phenotype of rice of OsMYBR1 gene knockout mutant and overexpression lines; B-D: respectively represent grain length, grain width and grain thickness of seeds of wild type material Nipponbare NIP, OsMYBR1 gene knockout mutant lines (osmybr1-1 and osmybr1-2) and OsMYBR1 gene overexpression transgenic lines (OE-MYBR1-1 and OE-MYBR1-2); E and F: respectively represent statistical results of chalky grain rate and chalkiness degree; G: represents 1000-grain weight of seeds of wild type rice Nipponbare NIP, OsMYBR1 gene knockout mutant lines (osmybr1-1 and osmybr1-2) and OsMYBR1 gene overexpression transgenic lines (OE-MYBR1-1 and OE-MYBR1-2); different letters represent significant difference of p<0.05, and the difference is statistically analyzed by one-way ANOVA test;

[0033] Figure 4 Figure 5 is the effect of OsMYBR1 gene knockout and overexpression on endosperm starch granules of rice;

[0034] Figure 5Comparison results of rice eating quality of wild type rice Nipponbare NIP, OsMYBR1 gene knockout mutant and overexpression transgenic plants; A: amylose content, B: amylopectin starch content, C: total starch content, D: total protein content, E: alkali digestion value determination results, F: peak gelatinization temperature, G: gelatinization entropy value of rice flour, H: determination results of gel consistency of rice flour; different letters represent significant difference of p < 0.05, and the difference is tested by one-way ANOVA. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not a limitation of the present application.

[0036] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a stated value of the parameter in range form. These are only examples of the various values of the parameter, which are encompassed by or are included in this application. Other examples of the parameter excluded from the range are also included in the scope of this application.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0038] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0039] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0040] Example 1 Analysis of tissue expression pattern of OsMYBR1 gene

[0041] Total RNA was extracted from rice roots, stems, leaves, young panicles and seeds of 3d, 10d, 18d and 24d after flowering using TRIzol reagent (Invitrogen, Carlsbad, CA). The concentration of the extracted total RNA was determined using the instrument Nanodrop 2000, and then the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (ReverTra Ace qPCR RT Kit, Toyobo, Osaka, Japan). Real-time fluorescent quantitative PCR was performed using a SYBR Green Master Mix (Toyobo Co. Ltd, Japan) kit and an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA) fluorescent quantitative PCR instrument. UBIQUITIN (LOC_Os03g13170) gene was used as an internal reference. The fluorescent quantitative RT-qPCR program was as follows: 95.0°C for 3 min; 95.0°C for 10 s, 60.0°C for 30 s, 40 cycles; melting curve 65.0°C-95.0°C, 0.5°C measurement once, and the temperature increasing rate was 0.5°C / s. The primer sequences are shown in Table 1.

[0042] The results are shown in Figure 1 Real-time fluorescent quantitative PCR showed that the OsMYBR1 gene was specifically expressed in rice seeds.

[0043] Table 1 Real-time fluorescent quantitative PCR primer sequences

[0044]

[0045] Example 2 Construction of OsMYBR1 gene transgenic lines

[0046] 1. Construction of OsMYBR1 gene CRISPR / Cas9 knockout transgenic lines

[0047] The sequence of the OsMYBR1 gene (Locus number LOC_Os10g20990) was obtained using the RGAP (http: / / rice.plantbiology.msu.edu / ) database, the nucleotide sequence is shown as SEQ ID No. 1, and the amino acid sequence encoded by the OsMYBR1 gene is shown as SEQ ID No. 2.

[0048] The OsMYBR1 gene knockout target sequence (SEQ ID No. 7) is: 5'-CCGCTGGAAGGAGATTGGCCGGG-3'.

[0049] OsMYBR1 gene mutant plants in Nipponbare background were obtained by Agrobacterium-mediated genetic transformation using the pCAMBIA1305 vector containing the OsMYBR1 gene. The sequence of the OsMYBR1 gene mutant was confirmed by PCR and sanger sequencing.

[0050] 2. Construction of OsMYBR1 gene overexpression transgenic lines

[0051] According to the characteristics of the pCAMBIA1305 vector, primers containing (Kpn I and BamH I) enzyme digestion sites were designed (F: 5'-TTCTGCACTAGGTACCATGCGTGTGGGGAAGGCAA-3', SEQ ID No. 8; R: 5'-TGCTCACCATGGATCCCTATCTTCGCAGGCGGAAGT-3', SEQ ID No. 9), and the OsMYBR1 gene was PCR amplified using the cDNA of young seeds of Nipponbare as the template. The PCR product was gel recovered and ligated to the pCAMBIA1305 vector using recombinase. The plasmid was sent to Wuhan EIDIAN Crystal Biotechnology Co., Ltd. for genetic transformation, and transgenic overexpression T0 generation lines in Nipponbare background were obtained. The RNA of T0 generation plants was extracted, and the above extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (ReverTra Ace qPCR RT Kit, Toyobo, Osaka, Japan). At the same time, the wild type Nipponbare leaf material was used as a control, the plant leaf RNA was extracted, and the cDNA was reverse transcribed. Real-time fluorescent quantitative PCR was performed using the SYBR Green Master Mix (Toyobo Co. Ltd, Japan) kit, and the real-time fluorescent quantitative PCR analysis was performed on the ABI PRISM 7900HT (Applied Biosystems, Foster City, CA) fluorescent quantitative PCR instrument, with UBIQUITIN (LOC_Os03g13170) gene as the internal reference. The T0 generation plants overexpressing the OsMYBR1 gene were selected, and the T2 generation plants were used for phenotype identification. The fluorescent quantitative RT-qPCR program was: 95.0℃ 3min; 95.0℃ 10s, 60.0℃ 30s, 40 cycles; melting curve 65.0℃-95.0℃: 0.5℃ measurement once, with a temperature increase rate of 0.5℃ / s; the primer sequences are shown in Table 1.

[0052] As shown in Figure 2 , the gene expression in the OsMYBR1 gene knockout transgenic lines was significantly reduced, and the gene expression in the overexpression transgenic lines was significantly increased.

[0053] Phenotype detection of OsMYBR1 gene knockout and overexpression transgenic lines

[0054] The wild type NIP material, OsMYBR1 gene knockout and overexpression transgenic material were planted in the test field, and the conventional water and fertilizer management was carried out. After the mature seeds were harvested, they were naturally dried. The grain length, grain width and grain thickness of the seeds were measured by using a vernier caliper; 1000 full seeds were randomly selected for thousand seed weight determination; the rice was hulled by using a rice huller to obtain brown rice, and the brown rice was milled into polished rice, and the whole polished rice was selected, and the rice chalkiness, chalky grain rate and thousand seed weight were measured by using a Wanshen SC-G grain tester.

[0055] As shown in Figure 3 Compared with the wild type control, there was no significant difference in grain length and grain width, the grain thickness of the OsMYBR1 gene knockout mutant and the overexpression transgenic line was reduced, resulting in the decrease of the thousand seed weight; the chalkiness and chalky grain rate of the OsMYBR1 gene knockout mutant rice were significantly increased, and the chalkiness and chalky grain rate of the overexpression transgenic line seed had no significant difference with the wild type control.

[0056] Example 4: Effect of OsMYBR1 gene knockout and OsMYBR1 gene overexpression on rice endosperm starch granules

[0057] The formation of endosperm chalkiness is often closely related to the structure of endosperm starch granules and the arrangement of starch granules. In the present application, the cross-sectional starch granule morphology of the wild type NIP, the OsMYBR1 gene knockout line and the OsMYBR1 gene overexpression transgenic line was observed by using a scanning electron microscope.

[0058] As shown in Figure 4 Compared with the control NIP, the endosperm starch granules of the OsMYBR1 gene knockout mutant rice were irregular round and arranged loosely, while the endosperm starch granules of the wild type and the OsMYBR1 overexpression transgenic plant rice were polyhedral and arranged closely.

[0059] Example 5: Method for detecting and analyzing the eating quality of rice of OsMYBR1 gene knockout mutant and overexpression transgenic lines

[0060] (1) Amylose content determination: The milled rice of wild type, OsMYBR1 knockout mutant and overexpression transgenic lines were ground into rice flour, sieved through 100 mesh, and 0.050 g of each was placed in a 50 mL volumetric flask. Meanwhile, standard samples (concentrations of 0.4%, 10.6%, 16.2% and 26.5% amylose) were prepared and treated in the same manner. 0.5 mL of 95% ethanol was slowly added, and the volumetric flask was shaken gently. Then, 4.5 mL of 1 mol / L NaOH solution was added, and the mixture was allowed to stand overnight at room temperature. The next day, deionized water was added to make up the volume to 50 mL. After mixing and standing for 20 min, 0.5 mL of the sample and standard sample was accurately pipetted into a 10 mL test tube, and 5 mL of ddH2O, 100 μL of 1 M acetic acid solution, 200 μL of KI-I2 (2 g I2+20 g KI+1000 mL ddH2O) solution and 4.2 mL of ddH2O were added in turn. The mixture was mixed well using a shaker, and allowed to stand at room temperature for 20 min. Then, 0.2 mL of the mixture was pipetted into a transparent ELISA plate, and the absorbance of the sample at 620 nm was measured using an enzyme marker. The amylose content of each sample was calculated according to the linear equation of the absorbance and the amylose content. Figure 5 in the middle A).

[0061] (2) Total starch content determination: The total starch content in the milled rice of wild type, OsMYBR1 knockout mutant and overexpression transgenic lines was determined using the Megazyme total starch assay kit K-TSTA (Megazyme, Ireland, UK) (see Figure 5 in the middle C). The content of amylopectin in the milled rice was calculated as total starch content-amylose content, and the results are shown in Figure 5 in the middle B.

[0062] (3) Total protein content determination: 0.1 g of milled rice was weighed into a 100 mL digestion tube, and 5 mL of concentrated sulfuric acid was added. The digestion tube was placed in a 290°C digestion furnace, and the timing was started. The tube was taken out and shaken every 15 min, and this was repeated about 4 times. The digestion tube was removed, and 0.75 mL of hydrogen peroxide solution was added. The tube was placed in the 290°C digestion furnace again, and the solution was observed for clarity. When the solution became clear, the tube was removed and cooled to room temperature. The volume was made up to 100 mL. The absorbance of the sample and standard sample at 280 nm was measured using an enzyme marker. The nitrogen content of the sample was calculated according to the linear equation of the nitrogen content and the absorbance OD value of the standard sample, and the protein content was converted. The results are shown in Figure 5 in the middle D.

[0063] (4) Alkali value determination: 6 full and intact milled rice grains were selected and placed in a square box, 10 mL of 1.70% potassium hydroxide solution was added, the lid was covered, and it was placed in a 30°C constant temperature box for about 23 h. The endosperm decomposition of the rice grains was observed. The results were recorded according to the edible rice variety quality NT / T593-2021 standard, and the results are shown in Table 2. Figure 5 Table 2

[0064] (5) Determination of gelatinization temperature: 0.005 g of milled rice powder was weighed into an aluminum sample cup, 10 μL of distilled water was added, sealed, and the gelatinization temperature of wild type NIP, OsMYBR1 gene knockout mutant and OsMYBR1 overexpression transgenic lines of rice was determined by Mettler-Toledo differential scanning calorimeter (DSC). The results are shown in Tables 3 and 4. Figure 5 Table 3

[0065] (6) The determination of gel consistency (GC) was carried out according to the edible rice variety quality NT / T593-2021 standard. All data were based on 3 biological replicates, and the values were the average values, and the error was the standard error (SEM) of the average value. Different lowercase letters represent significant differences (P < 0.05), and the difference was tested by one-way ANOVA, and the results are shown in Table 5. Figure 5 Table 5

[0066] The results showed that compared with wild type rice, the amylose content in OsMYBR1 gene knockout mutant rice was significantly reduced, the alkali value was increased, and the gelatinization temperature was significantly reduced; the amylopectin content, total starch content, total protein content and gel consistency had no significant change. Compared with wild type rice, the amylopectin content of OsMYBR1 gene overexpression transgenic lines of rice was significantly increased (total starch content had no significant change, amylose content was significantly reduced), alkali value was improved; total protein content, gelatinization temperature and gel consistency had no significant change. The above results showed that the starch was more easily gelatinized, and the cooking and taste quality was improved.

[0067] The above experimental results of the examples showed that the OsMYBR1 gene could regulate multiple indicators affecting rice quality, and it was a gene regulating rice quality, which could be applied to genetic improvement of rice quality.

[0068] The sequence of OsMYBR1 gene is as follows (SEQ ID No. 1):

[0069]

[0070] The protein sequence encoded by OsMYBR1 gene is as follows (SEQ ID No. 2):

[0071] MRVGKAKRRLDLSTRRGTDEGGRTNCWKTATSAPSLARATAVRVLATDPDPSGVACVRCDVNGERVRAWSCGAKTCGMGAGRKLKTHRRNQRWADKAYKKSHFGNEWKKPFAGSSHAKGIVLEKIGIEAKQPNSAICKCARVQLVKNGKKIAAFVPNDGCLNFIKENEVAYVDGIYGIKVLVDALSSSRLRGRDGSGGDHGDATAAAAASREVAPPPRPRDVYHRPFTARDDDELLRLHYRLGDRWKEIGRAVYGRTSRVMKHRWRELRRGGFLAAAARKELAALDMADDMVETSEVEEPADQSLPSLELQRSTLADTLASSFGSCSLATDHVMDPLAGSLALGKYQFFTIIKIEDVFTGTLVRHPWLPRLVKENGFRHWSRVARIMPRRSARLCRDRWCHHLARDVYHRPFTARDDDELLRLHYRLGDCWKKIGHAVYGRTSRVMNHRWRELRRSGFLAAAARTEQKLDMADDMVESEVEESDQSLPTTRKSIIATGRKGSLQAGQPSACKPTTVKIADLRRRGGRPPAKIIFAGGRWLVRSACEKKNRPPTKKNFRLRR.

[0072] The above-described embodiments are merely preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the present application, which are apparent to those skilled in the art, are intended to fall within the scope of the present application defined by the appended claims.

Claims

1. The application of OsMYBR1 gene knockout in reducing amylose content and endosperm starch gelatinization temperature in rice, characterized in that... The nucleotide sequence of the OsMYBR1 gene is shown in SEQ ID No.

1.

2. The application of OsMYBR1 gene knockout in rice breeding, characterized in that, The application involves knocking out the OsMYBR1 gene in rice to reduce the amylose content and endosperm starch gelatinization temperature, thereby improving rice quality; the nucleotide sequence of the OsMYBR1 gene is shown in SEQ ID No.

1.

3. A method for improving the flavor and quality of cooked rice, characterized in that, By knocking out the OsMYBR1 gene in rice, the amylose content in rice and the gelatinization temperature of endosperm starch are reduced; the nucleotide sequence of the OsMYBR1 gene is shown in SEQ ID No.

1.

4. A method for cultivating transgenic rice with improved cooking and eating quality, characterized in that, By knocking out the OsMYBR1 gene in rice, the content of amylose and the gelatinization temperature of endosperm starch in rice are reduced, thereby improving the cooking quality of the constructed transgenic rice; the nucleotide sequence of the OsMYBR1 gene is shown in SEQ ID No. 1.

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