Application of ring finger protein coding gene OsRF in regulation and control of rice grain shape and grain weight

Abstract

The invention provides application of a ring finger protein coding gene OsRF in regulation and control of rice grain shape and grain weight, the gene OsRF for coding the ring finger protein is separated and cloned from rice Nipponbare, and the gene is targeted by rice small RNA OsmiR530. In order to prevent the OsmiR530 from being cut and improve the expression level of the OsmiR530, artificial synonymous mutation is carried out on the OsRF recognized sequence by the OsmiR530, a mutant OsRF fragment (mOsRF) of which the recognition sequence is subjected to synonymous mutation is created by utilizing overlapping PCR (Polymerase Chain Reaction), an overexpression vector of the mOsRF is constructed and is overexpressed in rice, and the grain width and the grain weight of transgenic rice are obviously increased. Therefore, the overexpression of the mOsRF in the rice is of great significance for obtaining round-grain rice and increasing the rice yield, and a new resource and a new research method are provided for rice grain shape improvement breeding.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to the application of a RING finger protein encoding gene OsRF in regulating rice grain shape and grain weight. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Rice is one of the most important food crops in the world, and its yield and quality have an important impact on global food security. Rice yield is mainly regulated by three factors: the number of effective panicles, the number of grains per panicle, and the thousand-grain weight. Among them, the thousand-grain weight is mainly determined by the shape, size, and filling fullness of the grains. The grain shape of rice can affect the volume of the endosperm and determine its weight, and its weight is directly related to the grain weight, so the grain shape of rice is a key factor in determining rice yield. The grain shape of rice is determined by the length, width, and length-to-width ratio. Affected by light and temperature conditions, the rice in southern my country is mainly long-grained indica rice, while the round-grained japonica rice is mainly planted in the north. The nutritional components of the two are not much different in terms of protein, vitamins, carbohydrates, fats, magnesium, iron, zinc and other elements, but the content of vitamin B2 and calcium in round-grained japonica rice is slightly higher, and the selenium content and niacin in long-grain rice are better.

[0004] Existing studies have shown that rice grain shape is controlled by multiple genes, and there is a phenomenon of one cause and multiple effects. There are currently six known rice grain shape regulation pathways, namely the E3 ubiquitin ligase degradation pathway, the MAPK cascade signaling pathway, the G protein signaling pathway, the transcription factor pathway, the plant hormone pathway, and the epigenetic pathway. The study of the ubiquitination pathway regulating rice grain shape was relatively early. Through the map-based cloning technology, multiple ubiquitination pathway-related genes that regulate grain shape have been found. For example, the functional loss of the E3 ubiquitin ligase GW2 significantly increases the width and weight of rice grains; the deubiquitinating enzyme WTG1 can cause cells to become narrower, thereby forming slender grains; ubiquitin-specific protease 15 (OsUBP15) has deubiquitination activity. Reducing its expression will produce smaller grains, while increasing its expression will obtain the opposite phenotype. OsMAPK6 in the MAPK cascade pathway affects grain size by regulating processes such as cell proliferation and BR signal transduction. OsMKKK70 is upstream of OsMKK4-OsMAPK6, and overexpression of OsMKKK70 can significantly increase grain length. The D1 gene encodes the Gα subunit of rice G protein, and its nonsense mutation leads to dwarf plants, smaller grains, and upright leaves. RGG2 encodes the B-type γ subunit, and its overexpression will lead to dwarf plants and smaller grains. In addition, multiple transcription factors, including AP2, bHLH, WRKY, and GRF family genes, are involved in the regulation of grain shape.

[0005] So far, the regulatory mechanisms of cloned grain shape regulatory genes have not been deeply explored. Due to their associated effects on other traits, the application of these genes in breeding is limited. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention aims to provide an application of the RING finger protein encoding gene OsRF in regulating rice grain shape and grain weight.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides an application of the RING finger protein encoding gene OsRF or its mutant mOsRF in regulating rice grain shape and grain weight. The nucleotide sequence of the RING finger protein encoding gene OsRF is shown in SEQ ID NO: 1; the nucleotide sequence of the mutant mOsRF is shown in SEQ ID NO: 3.

[0009] The present invention isolates and clones a gene OsRF encoding a ring finger protein from rice Nipponbare, and the gene is targeted by rice small RNA OsmiR530. In order to avoid being cut by OsmiR530 and improve its expression level, the OsRF recognition sequence of OsmiR530 is artificially mutated synonymously, and a mutant OsRF fragment (mOsRF) with a synonymous mutation in the recognition sequence is created by overlapping PCR. An overexpression vector of mOsRF is constructed and overexpressed in rice, and the grain width of the transgenic rice is significantly increased and the grain weight is increased. Therefore, overexpression of mOsRF in rice is of great significance for obtaining round-grain rice and improving rice yield, which also provides new resources and research methods for rice grain type improvement breeding.

[0010] In some embodiments, the regulation of rice grain shape and grain weight is specifically to reduce the length-to-width ratio of rice and increase the rice grain size.

[0011] In a second aspect, the present invention provides a rice overexpression vector containing the mutant mOsRF.

[0012] In a third aspect, the present invention provides the use of the RING finger protein encoding gene OsRF or mutant mOsRF in rice breeding.

[0013] In some embodiments, the application includes the following steps:

[0014] Constructing a rice overexpression vector of the RING finger protein encoding gene OsRF or mutant mOsRF;

[0015] Transforming the constructed rice overexpression vector of the RING finger protein encoding gene OsRF or mutant mOsRF into rice or rice cells;

[0016] Cultivate and screen to obtain transgenic rice with target grain shape and grain weight.

[0017] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0018] (1) The present invention demonstrates for the first time the role of the RING finger protein OsRF in regulating rice grain shape and grain weight, and provides a method for reducing the length-to-width ratio of rice grains and increasing grain weight by overexpressing a synonymous mutant OsRF (mOsRF).

[0019] (2) The OsRF of the present invention can successfully regulate rice grain shape and grain weight, which helps to clarify the mechanism of action of RING finger proteins in regulating plant grain shape.

[0020] (3) Due to the high conservation of plant RING finger protein sequences and functions, the results of the present invention are of great value for studying the role of RING finger proteins RF in grain shape regulation in cereal crops such as rice and other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 This is the sequence of the OsRF of the present invention before and after the miR530 targeted segment mutation.

[0023] Figure 2 The schematic diagram of the construction of the mOsRF overexpression vector of the present invention is shown in Figure 1. The mutant OsRF amplified by overlapping PCR is cloned into the pRicefox vector by homologous recombination, so that mOsRF is positively inserted into the maize ubiquitin gene promoter (Pubi), i.e., the pRicefox-ubi-mOsRF plant expression vector.

[0024] Figure 3 The results of detecting the relative expression level of mOsRF transcripts in T3 transgenic rice positive plants. OsRF-ox represents plants transfected with osa-miR530, #8, #14 and #23 represent independent positive transgenic lines; WT represents wild-type rice plants. EF-1a was used as the internal reference gene for fluorescence quantitative PCR analysis.

[0025] Figure 4The figure shows the grain shape comparison between rice plants overexpressing mOsRF and wild-type plants at maturity. Figure A shows the grain shape comparison between wild-type plants (NIP) and three mOsRF overexpressing lines at maturity; Figure B shows the grain length comparison statistics. NIP represents the wild-type control, and #8, #14, and #23 represent three independent positive transgenic lines overexpressing mOsRF. ** indicates that the difference is extremely significant (P<0.01) according to TTEST statistical analysis. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] The present invention takes a rice ring finger protein OsRF as the research object, and studies the role and application of OsRF in changing rice grain shape and grain weight. The coding region sequence of the OsRF gene is a 1104bp nucleic acid sequence (SEQ ID NO: 1).

[0028]

[0029] The sequence of the segment targeted by OsmiR530 is (5'-CAGATGAAGGTGCAAATGCA-3', SEQ ID NO: 2), and the cleavage site is located between the 10th and 11th bases of the targeting sequence. The present invention uses the cDNA of rice Nipponbare leaves as a template, designs a synonymous mutation type intermediate primer, uses an overlapping PCR method to amplify the mOsRF gene (SEQ ID NO: 3) in which the rice OsRF undergoes a synonymous mutation, connects it forward to the plant expression vector pRicefox, and transforms rice by an Agrobacterium-mediated genetic transformation method, thereby obtaining a transgenic rice plant overexpressing mOsRF. In the T3 generation of transgenic plants, the grains of the positive plants become round, the grain width increases significantly, the grain length does not change significantly, and the grain weight also increases accordingly.

[0030]

[0031] The present invention will be further described below in conjunction with the embodiments.

[0032] Example 1: Cloning of a synonymous mutant OsRF gene DNA fragment

[0033] Total RNA was extracted from leaves of rice cultivar Nipponbare seedlings using TRIZOL reagent (Invitrogen).

[0034] The specific steps are as follows: put 20 mg of leaves into a mortar pre-cooled with liquid nitrogen, add liquid nitrogen and quickly grind into powder, put the powder into a 1.5 ml centrifuge tube, quickly add 1 ml Trizol (Invitrogen), invert and mix, and let stand at room temperature for 5 minutes.

[0035] Centrifuge at 12000 rpm for 10 minutes at 4°C, transfer the supernatant to a new 1.5 ml centrifuge tube, add 200 μl of chloroform, shake vigorously by hand for 15 seconds, and let stand at room temperature for 2-3 minutes.

[0036] Centrifuge at 4°C, 12000rpm for 15 minutes. Take the colorless aqueous phase to a new 1.5ml centrifuge tube, add 250μl isopropanol and 250μl high salt solution, mix by inversion, and let stand at room temperature for 10 minutes. Centrifuge at 4°C, 12000rpm for 10 minutes, and remove the supernatant.

[0037] Add 1 ml of ice-cold 75% ethanol, invert several times, centrifuge at 4°C, 7500 rpm for 5 minutes, discard the supernatant, and dry at room temperature until the precipitate becomes transparent. Add an appropriate amount of DEPC water (usually 60 μl) to dissolve the precipitate, and measure the RNA concentration using a UV spectrophotometer.

[0038] It was reverse transcribed into cDNA using reverse transcriptase SuperScript II (Invitrogen), and the specific steps were as follows: 1 μl of 500 μg / ml oligo(dT)12-18, 2 μg of total RNA, 1 μl of 10 mM dNTP mixture and DEPC water were added to 12 μl, placed in a 65°C water bath for 5 minutes, quickly placed in an ice bath for 5 minutes, and slightly centrifuged to collect the sample at the bottom of the tube.

[0039] Then, 4 μl of 5× first-strand buffer, 2 μl of 0.1 M DTT and 1 μl of RNaseOUT (40 U / μl) were added in sequence at 42° C. for 2 minutes.

[0040] Then add 1 μl SuperScript II, mix gently, react at 42°C for 50 minutes, and then incubate in a 70°C water bath for 15 minutes to inactivate the enzyme. This will synthesize the first-strand cDNA, and use the first-strand cDNA as a template to amplify the target gene.

[0041] The OsRF CDS sequence was found through the website https: / / rice.uga.edu / , and the specific sequence information is shown in SEQ ID NO.1.

[0042] Mutant forward primers RF-OE-MS (5'-GGGCGCCAACGCTGGAGCT-3', SEQ ID NO: 4) and reverse primers RF-OE-MAS (5'-GCTCCAGCGTTGGCGCCCTCA-3', SEQ ID NO: 5) were designed in the OsRF targeted region by OsmiR530. At the same time, forward primers RF-OE-FSfi with homology arms were designed at the 5' and 3' ends of the OsRF CDS.

[0043] (5'-CTCCTGCAGGGGCCAAATCGGCCATGGATGATCACATGGG AAG-3', SEQ ID NO: 6) and reverse primer RF-OE-Rsfi (5'-ATCCTCTAGAGGCCCTTATGGCCCTGGGTAGAACATTCATG GAG-3', SEQ ID NO: 7).

[0044] The cDNA of young leaves of rice Nipponbare obtained by reverse transcription was used as a template, and two pairs of primers RF-OE-FSfi and RF-OE-MAS, RF-OE-MS and RF-OE-Rsfi were used to amplify the target fragment under the action of Takara Ex Taq (TaKaRa). The PCR reaction conditions were: 94℃ pre-denaturation for 3 minutes; 94℃ for 1 minute, 54℃ for 30 seconds, 72℃ for 20 seconds, 30 cycles, and 72℃ for 10 minutes. After the amplified product was recovered and purified, the two tubes of recovered products were mixed 1:1, and then the recovered mixture was used as a template, and the primers RF-OE-FSfi and RF-OE-Rsfi were used for the second round of amplification. After gel recovery, the mutant OsRF (mOsRF) gene fragment (sequence shown in SEQ ID NO: 3) was obtained, and the fragment was named mOsRF cDNA.

[0045] Example 2: Construction and genetic transformation of plant expression vector of synonymous mutant mOsRF gene

[0046] In order to better analyze the function of OsRF and prevent it from being cut and inactivated by OsmiR530, the applicant increased the expression level of mOsRF in rice through overexpression technology. Since the mutation is a synonymous mutation, it will not cause changes in the coding protein sequence. The function of the gene is studied based on the agronomic characteristics of transgenic plants.

[0047] The method for constructing a synonymous mutant OsRF gene plant overexpression vector is as follows: first, the plant expression vector pRicefox is digested with SfiI to recover the vector fragment, and then the target fragment mOsRF cDNA obtained in Example 1 and the recovered vector fragment are subjected to homologous recombination reaction and transformed into Escherichia coli DH5ɑ. Positive clones are screened by PCR detection and sequencing analysis to obtain a plant expression vector named pRicefox-ubi-mOsRF (see Figure 1 pRicefox is a commonly used plant genetic transformation vector in the world (see Figure 1 ). pRicefox-ubi-mOsRF was transformed into Agrobacterium strain EHA105.

[0048] The rice genetic transformation system mediated by Agrobacterium (see the following examples of the present invention) was introduced into the rice variety Nipponbare, and transgenic plants were obtained through pre-culture, infection, co-cultivation, screening of callus with hygromycin resistance, differentiation, rooting, and transplanting. The rice genetic transformation system mediated by Agrobacterium was improved on the basis of the method reported by Hiei et al. (Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Yukoh Hiei, shozo Ohta, Toshihiko Komari, Takashi Kumashiro, 1994, Plant J., 6: 271-282). A total of 16 independent transgenic rice plants were obtained by transformation.

[0049] The specific steps are as follows:

[0050] (1) Callus induction: The shelled wild-type Nipponbare rice seeds were surface-sterilized with 70% ethanol for 1 minute; surface-sterilized with 5% (active chlorine content) NaClO solution for 20 minutes; rinsed with sterile water 4-5 times; sown on callus induction medium to induce callus tissue (the composition is shown below), and cultured in the dark at 25-26°C for 4-7 days. Primary callus tissue was induced from the scutellum of the mature embryo, and the embryonic buds growing on the embryo were removed with tweezers. The seeds were subcultured on the callus induction medium and cultured for another 2 weeks until embryonic callus tissue with a light yellow color, hard texture and granular texture was grown.

[0051] (2) Pre-culture of callus tissue: transfer the callus tissue to fresh callus induction medium and culture it in the dark at 25-26°C for 4 days.

[0052] (3) Agrobacterium culture: Pick a single clone of Agrobacterium and inoculate it into 5 mL YEP liquid medium (containing 50 mg / L kanamycin) at 28°C, 220 rpm, and culture it until the late logarithmic growth period (about 18-24 hours). Transfer the obtained bacterial liquid to 50 mL fresh AB liquid medium (composition see below) containing 50 mg / L kanamycin at a 1% inoculum amount; culture it at 28°C, 220 rpm until the OD600 value is about 0.5 (culture for 5-6 hours).

[0053] (4) Agrobacterium infection: Transfer 50 mL of bacterial solution into a centrifuge tube, centrifuge at 4°C, 4000g for 10 minutes, discard the supernatant, and add an equal volume of AAM medium to resuspend the bacteria. Immerse the embryonic callus tissue of Nipponbare from (2) in the above AAM bacterial solution, infect for 2 minutes, and shake slowly. Blot the callus tissue dry with sterile absorbent paper, place it on the co-cultivation medium (lay a layer of sterile filter paper on the medium), and co-cultivate at 26°C in the dark for 2-3 days.

[0054] (5) Callus washing and selection culture: After co-cultivation, the callus tissue was washed 4 times with sterile water, and then washed twice with sterile water containing 500 mg / L carbenicillin Cb, and then dried with sterile absorbent paper and placed on a workbench for 30 minutes. The callus tissue was placed on a solid screening medium (containing 25 mg / L hygromycin and 400 mg / L carbenicillin) and cultured in the dark at 26°C for 2 weeks. Then it was transferred to a solid screening medium (containing 30 mg / L hygromycin and 300 mg / L carbenicillin), cultured in the dark at 26°C, subcultured every 2 weeks, and screened for 4 weeks.

[0055] (6) Differentiation culture: The resistant callus was transferred to differentiation medium and cultured at 28°C under light for 7 days. After one transfer, the culture was continued until regenerated seedlings were produced.

[0056] (7) Seedling hardening and transplanting: The regenerated plantlets are transferred to fresh 1 / 2MS culture medium and rooted in the culture bottle. When the seedlings grow to about 10 cm, open the sealing film, harden the seedlings for 2-3 days, and then transfer the regenerated seedlings to the soil for cultivation.

[0057] Reagent formula:

[0058] (1) Abbreviations of reagents and solutions: The abbreviations of the plant hormones used in the present invention are as follows: Cb (Cabenicillin); KT (Kinetin); NAA (Napthalene acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS (Acetosyringone); DMSO (Dimethyl sulfoxide).

[0059] (2) Culture medium formulation for rice genetic transformation:

[0060] 1) YEP liquid medium: 2 g Bacto-peptone, 2 g yeast powder, 1 g NaCl, add water to 200 mL, and adjust the pH to 7.0 with 5 N NaOH.

[0061] 2) Callus induction medium: N6 macro, N6 micro, iron salt, N6 vitamin, 0.5 g / L acid hydrolyzed casein, 30 g / L sucrose, 2 mg / L 2,4-D, Gelrite (Sigma) 4 g / L, pH 5.8.

[0062] 3) AB liquid culture medium: 3g / L K2HPO4, 1g / L NaH2PO4, 1g / L NH4Cl, 300mg / L MgSO4·7H2O, 150mg / L KCl, 10mg / L CaCl2·2H2O, 2.5mg / LFeSO4·7H2O, 5g / L glucose, pH 7.0.

[0063] 4) AAM medium: AA macro, AA trace, 0.9 g / L L-glutamine, 0.3 g aspartic acid, MS vitamins, 0.5 g / L acid hydrolyzed casein, 36 g / L glucose, 68.5 g / L sucrose, 20 mg / L AS, pH 5.2.

[0064] 5) Co-culture medium: N6 macro, N6 micro, iron salt, N6 vitamins, 30 g / L sucrose, 10 g / L glucose, 0.5 g / L acid hydrolyzed casein, 2 mg / L 2,4-D, 20 mg / L AS, Gelrite (Sigma) 4 g / L, pH 5.8.

[0065] 6) Solid screening medium: N6 macro, N6 micro and N6 vitamins, 0.5 g / L acid hydrolyzed casein, 30 g / L sucrose, 2 mg / L 2,4-D, Gelrite (Sigma) 4 g / L, pH 5.8, appropriate concentrations of hygromycin and carbenicillin.

[0066] 7) Differentiation medium: MS macro, MS micro, iron salts and MS vitamins, 2 g / L acid hydrolyzed casein, 30 g / L sucrose, 30 g / L sorbitol, 2 mg / L KT, 0.2 mg / L NAA, pH 5.8, 30 mg / L hygromycin B, 200 mg / L carbenicillin.

[0067] 8) 1 / 2MS culture medium: 1 / 2MS macro, 1 / 2MS micro, MS vitamins, 30g / L sucrose, 4g / L Gelrite, 30mg / L hygromycin B, 200mg / L carbenicillin, pH 5.8. (3) Main solution formula:

[0068] 1) N6 macroelements (10×)

[0069]

[0070] 2)N6 trace (1000×):

[0071] 3) N6 vitamin (1000×)

[0072]

[0073] 4) MS macroelements (10×)

[0074]

[0075] 5) MS trace (1000×):

[0076] 6) MS vitamins (1000×)

[0077]

[0078]

[0079] 7) Iron salt (200×)

[0080] FeSO4.7H2O 5.56g

[0081] Na2EDTA.2H2O 7.46 g;

[0082] 8) AA large quantity (200×)

[0083]

[0084] 9) AA trace (1000×)

[0085]

[0086] 10) 2,4-D stock solution (2 mg / ml)

[0087] Weigh 100 mg of 2,4-D, dissolve it in 1 ml of DMSO, add distilled water to make it up to 49 ml, then add 0.5 N NaOH until it is completely dissolved, and store it at -20°C.

[0088] 11) Kinetin stock solution (0.2 mg / ml)

[0089] Weigh 10 mg of Kinetin, dissolve it in 1 ml of 1N KOH, add distilled water to make up to 50 ml, and store at 4°C.

[0090] 12) NAA storage solution (0.2 mg / ml)

[0091] Weigh 10 mg of NAA, dissolve it in 0.5 ml of 1N KOH, add distilled water to make up to 50 ml, and store it at 4°C.

[0092] 13) Acetosyringone (100 mg / ml)

[0093] Weigh 100 mg of acetosyringone, dissolve in 1 ml of DMSO, and store at -20°C.

[0094] 14) Kanamycin (50 mg / ml)

[0095] Weigh 500 mg of kanamycin, dissolve it in 8 ml of distilled water, add distilled water to make it up to 10 ml, then filter and sterilize it with a 0.22 μm filter and store it at -20°C.

[0096] Example 3: Detection of OsRF expression levels in transgenic rice plants and wild-type rice

[0097] Using wild-type rice Nipponbare and three independent T3 transgenic rice plants as materials, RNA was extracted from rice leaves at the four-leaf stage, and the transcript level of OsRF in rice leaves was detected by RT-PCR. The specific method is as follows: Total RNA was extracted from 100 mg of rice leaves at the four-leaf and one-heart stage using the Takara MiniBEST Universal RNA Extraction Kit (Takara), and genomic DNA contamination was removed by a genomic DNA removal centrifuge column provided in the kit. The specific steps are as described in Example 1.

[0098] According to PrimeScript TM Synthesize the first strand of cDNA using the RT Enzyme Mix I (TaKaRa) instruction manual. The specific steps are as follows: Add 1 μg of Total RNA to an ice-cold RNase-free reaction tube, up to 5 μg, 5× PrimeScript TM Buffer 4ul, RT Primer Mix4ul, PrimeScript TMRT Enzyme Mix I 1ul, add RNase Free dH2O to make the total volume 20μl. Gently mix the above reaction solution with a pipette, react at 42℃ for 30 minutes after brief centrifugation, and react at 85℃ for 5 seconds. The obtained reaction solution cDNA can be directly used in the next Real Time PCR experiment, or it can be frozen at -20℃ or lower.

[0099] Using the above cDNA as template, according to The Top Green qPCR SuperMix (+Dye I / +DyeII) fluorescence quantitative detection kit (Quanshijin) was used to identify the expression level of OsRF in wild-type rice and transgenic rice transformed with pRicefox-ubi-mOsRF at the four-leaf and one-heart stage. The forward primer OsRF-RS (5'-GAATAAGGAAGGCAGATG-3', SEQ ID NO: 8) and the reverse primer OsRF-RAS (5'-AATTCCTCCTGGTATCTC-3', SEQ ID NO: 9) were designed and synthesized. The specific steps of OsRF quantitative PCR are as follows: Thaw at room temperature in the dark. TopGreen qPCR SuperMix (+Dye I / +Dye II) Top Green qPCRSuperMix (+Dye I / +Dye II) was gently inverted up and down to mix, avoiding bubbles, and was gently centrifuged before use. A new sterilized centrifuge tube was placed on ice and the following components were prepared for fluorescence quantitative PCR analysis: Top Green qPCR SuperMix (+Dye I / +Dye II) 10ul, OsRF-RS (10μM) 0.4μl, OsRF-RAS (10μM) 0.4μl, RNA first-strand cDNA no more than 2μl, supplemented with Nuclease-free Water to 20μl. At the same time, rice EF-1a RNA was used as the internal reference gene for fluorescence quantitative PCR analysis, and its specific upstream primer was OsEF-1a qF1 (5'-TTTCACTCTTGGTGTGAAGCAGAT-3', SEQID NO: 10), and the downstream primer was OsEF-1a qR1 (5'-GACTTCCTTCACGATTTCATCGTAA-3', SEQ ID NO: 11). The PCR reaction conditions were 94℃ pre-denaturation for 30 seconds, 94℃ denaturation for 5 seconds, 60℃ annealing and extension for 34 seconds, and 40 cycles. After the PCR reaction was completed, the amplification curve and melting curve of each sample were analyzed to determine the credibility of the experimental results. Then, the Ct values ​​obtained by fluorescence quantitative PCR were processed using Excel by the 2-△△Ct method and the standard error was calculated. The final processing results were plotted. The results showed that the transcript level of osa-miR530 in the overexpression strain was 3-7 times that of the non-transgenic rice plants, proving that the osa-miR530 precursor gene had been integrated into the rice genome and was expressed in large quantities in rice ( Figure 3 ).

[0100] Example 4: The grains of transgenic rice with overexpression of mOsRF became wider and heavier

[0101] This example analyzes the grain shape of transgenic rice plants and wild-type rice plants at maturity. The specific steps are as follows: the seeds of transgenic plants (#8, #14 and #23) and non-transgenic wild-type plants were surface-sterilized with 70% ethanol for 1 minute; 5% (active chlorine content) NaClO solution for 20 minutes, then rinsed with sterile water 4-5 times, and germinated in dark conditions for 3 days. Then they were transferred to soil and grown to the three-leaf stage, transplanted to the field and cultured to maturity, and the grain shape of transgenic plants and wild-type plants was measured at maturity, and photos were taken ( Figure 4 A), and the significance of the difference between wild-type and transgenic plants was analyzed by TTEST. Compared with the grains of non-transgenic wild-type plants, the grain width and weight of transgenic rice plants overexpressing mOsRF at maturity increased, but the grain length did not change much ( Figure 4 B-4D).

[0102] The above results showed that overexpression of mOsRF caused rice grains to become rounder and their weight to increase.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of the RING finger protein encoding gene OsRF or its mutant mOsRF in regulating rice grain shape and grain weight. The nucleotide sequence of the RING finger protein encoding gene OsRF is shown in SEQ ID NO: 1; the nucleotide sequence of the mutant mOsRF is shown in SEQ ID NO:

3.

2. The use according to claim 1, characterized in that: The regulation of rice grain shape and grain weight specifically involves reducing the length-to-width ratio of rice and increasing the rice grain size.

3. A rice overexpression vector containing the RING finger protein encoding gene OsRF or its mutant mOsRF according to claim 1.

4. Use of the RING finger protein encoding gene OsRF or its mutant mOsRF according to claim 1 in rice breeding.

5. The use according to claim 4, characterized in that: The following steps are involved: Constructing a rice overexpression vector of the RING finger protein encoding gene OsRF or its mutant mOsRF; Transforming the constructed rice overexpression vector of the RING finger protein encoding gene OsRF or its mutant mOsRF into rice or rice cells; Cultivate and screen to obtain transgenic rice with target grain shape and grain weight.

Images