Application of rice osarf10 gene in improving seed vigor and grain shape

Abstract

The application belongs to the field of plant genetic engineering, and discloses a rice auxin response factor coding gene OsARF10 The application discloses the application of the auxin response factor coding gene in seed vigor and grain shape. OsARF10 The nucleotide sequence of the auxin response factor coding gene is shown as SEQ ID NO. 1, and the encoded amino acid sequence is shown as SEQ ID NO. 2. OsARF10 After overexpression of the auxin response factor coding gene, the seed germination rate and seedling rate of rice under normal, salt and PEG conditions are significantly improved, and the grain length and 1000-grain weight of the rice are significantly improved. OsARF10 The overexpression of the auxin response factor coding gene can improve the seed vigor and yield of rice, and the overexpression of the gene can help the genetic improvement of the seed vigor and grain shape of rice, and the gene can be applied to the improvement of the seed vigor and yield of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and relates to the gene encoding the rice auxin response factor. OsARF10 The application of cloning and overexpression of [a specific compound] in improving rice seed vigor and grain shape, specifically involving overexpression... OsARF10 It can improve rice seed vigor, increase rice grain length and thousand-grain weight, and be used for genetic improvement of rice seed vigor and grain shape to promote crop yield. Background Technology

[0002] Seed vigor is an important standard for measuring seed quality. High-vigor seeds germinate quickly, resulting in robust seedlings with better resistance to adverse conditions. Rice is one of my country's important food crops. Rice base-ascorbic acid transporter protein... OsNAT9 Hedgehog interaction similar proteins OsHIPL1 Isopropyl malate synthase OsIPMS1 pyruvate kinase OsPK5 transcription factors OsNAC3 , OsbZIP23 and OsbZIP42 Genes such as [these genes] play an important role in regulating seed vigor and will be helpful for the genetic improvement of rice seed vigor and the breeding of new high-vigor seed varieties.

[0003] Grain shape is a key factor determining crop yield, a quantitative trait controlled by multiple genes, including grain length, grain width, grain thickness, and thousand-grain weight. Regarding the genetic regulation of rice grain shape, numerous genes have been cloned and reported, such as... GW2 , GL2 / OsGRF4 , GS3 , GL3.1 / qGL3 , LGY3 , qsw5 / GW5 G S5 , GS6 , TGW6 , GW6a , GL7 / GW7 , GLW7 , GW8 and GS9 However, the molecular regulatory mechanisms of grain shape are complex, and further in-depth research is needed to identify key genes controlling rice grain shape in order to improve rice yield in actual production. Summary of the Invention

[0004] To address the aforementioned technical problems in existing technologies, this invention, based on the crucial regulatory role of plant hormones (auxins) in plant growth and development, utilizes a rice auxin response factor encoding gene. OsARF10 Regulate seed vigor, OsARF10Overexpression significantly improved seed vigor, promoted seed germination, seedling growth, and resistance to abiotic stress. Meanwhile, agronomic trait surveys showed that… OsARF10 The overexpression of the material significantly increased grain length and thousand-grain weight, demonstrating potential for increased yield and providing important genetic resources for the genetic improvement of rice seed vigor and grain shape.

[0005] The technical solution of this invention is as follows: The first objective of this invention is to provide a gene encoding an overexpression rice auxin response factor. OsARF10 Or overexpression of rice auxin response factor encoding genes OsARF10 Application of substances in improving rice seed vigor and / or regulating rice seed grain shape.

[0006] Furthermore, the improvement of rice seed vigor refers to improving rice seed vigor under normal conditions and / or abiotic stress; preferably, the abiotic stress is salt stress or drought stress.

[0007] The improvement in seed vigor includes enhancing or improving the genetic characteristics of germination rate, seedling rate, and germination index of rice under normal and abiotic stresses during germination.

[0008] In a particular embodiment, enhancing rice seed viability also includes promoting rice germination and seedling formation.

[0009] Furthermore, the regulation of rice seed shape is to increase the length and / or weight of rice seeds.

[0010] Furthermore, the gene encoding the rice auxin response factor... OsARF10 The nucleotide sequence is shown in SEQ ID NO.1: Furthermore, the rice auxin response factor encoding gene OsARF10 The amino acid sequence is shown in SEQ ID NO.2: MELAGPTEGDGGGSVDSQLWAACAGSMSSVPPVGAAVYYFPQGHAEQASAAVDLSSARVP PLVPCRVVAVRFMADAESDEVFAKIRLVPLRPGDAVVDVGEAAAAEARREEENSRPRPTS FAKTLTQSDANNGGGFSVPRFCAETIFPELDYSSEPPVQSVCAKDVHGVEWTFRHIYRGT PRRHLLTTGWSPFVNKKQLTAGDSIVFMRDEGGNIHVGLRRAKRGFCSIGGDDESLSSIP GWDQYRGLMRRNATATATGGRTPPKGKVPPENVLTAATRATTGQPFEVLYYPRASTPEFC VRAAAVRTAMAVQWCPGMRFKMAFETEDSSRISWFMGTVAGVQASDPVRWPQSPWRLLQVTWDEPELLQNVKRVCPWLVELVSSMPNLHLPSFSPPRKKPRNPPYAELPLEGQIFTGPVF PPNPMAHDHHHHHGFPFLPFPDSSAQPAGIQGARHAQFASPFPEFHIGNLQPNLMLYAGI RLPPADRAAPAPRPPRIIIISTDLTIGSPGKPDDAACSPSSGGKKIDDTKPRGFLLFGQAI LTEEQIKNGNSDGRPASPNWDAEKAPNTSEGSDSGVTQGSPTKNTTPSWSLPYFGGNNIS RASEYELNPGQCKVFVESETVGRSLDLSALSSFEELYACLSDMFSIGSDELRSHLVYRSP AGEVKHAGDEPFCAFVKSARKLRILTDAGSDNLGD* Furthermore, the overexpression of the rice auxin response factor encoding gene OsARF10 The substance is the gene encoding the rice auxin response factor. OsARF10 Overexpression vectors.

[0011] Furthermore, the rice auxin response factor encoding gene OsARF10 The overexpression vector was based on the pCAMBIA1300s plasmid. Primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 were used to amplify the rice auxin response factor encoding gene shown in SEQ ID NO. 1. OsARF10 The coding region was ligated into the pCAMBIA1300s plasmid. CPN I / Room The I restriction site was constructed.

[0012] 1300s-OsARF10-F is shown in SEQ ID NO. 5: AGCTTTCGCGAGCTCGGTACCATGGAGCTCGCGGG; 1300s-OsARF10-R is shown in SEQ ID NO. 6: CTTGCATGCCTGCAGGTCGACCTAATCTCCCAGGTTGTCACTGC.

[0013] A second objective of this invention is to provide a method for improving rice seed vigor and yield, wherein the method involves constructing a gene encoding a rice auxin response factor. OsARF10 Overexpression of transgenic lines or corresponding overexpression materials can be used to obtain plants with improved seed vigor and grain shape.

[0014] Furthermore, the method includes the following steps: (1) Rice genes OsARF10 Overexpression transgenic construction: Construction of a rice auxin response factor encoding gene OsARF10 Overexpression vectors are used to transform rice, resulting in rice. OsARF10 Overexpression lines; (2) Screening and identification of overexpression lines in rice: Design specific detection primers to amplify rice OsARF10 cDNA was overexpressed in the young leaves of the strains, and conventional PCR products and / or real-time quantitative RT-qPCR expression levels were performed to screen for positive and stable overexpression plants, which are the plants that improve rice seed vigor and grain shape.

[0015] Furthermore, the gene encoding the rice auxin response factor... OsARF10 The primers for identifying the vector in overexpressing conventional PCR-positive plants are shown in SEQ ID NO.7 and SEQ ID NO.8. The gene encoding the rice auxin response factor... OsARF10Primers for real-time quantitative RT-qPCR detection of overexpression are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0016] Furthermore, the gene encoding the rice auxin response factor was amplified. OsARF10 The primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0017] The forward primer is shown in SEQ ID NO.3: CCACTAGTCCACAAACCCCTCC; The reverse primer is shown in SEQ ID NO.4: TTTCGTGAAACAAATTAAGGCTCAA.

[0018] The forward primer is shown in SEQ ID NO.7: ATGCCATCATTGCGATAAAG; The reverse primer is shown in SEQ ID NO.8: CCGGTAGATGTGCCGGAA.

[0019] As shown in SEQ ID NO.9: CAGCATTGGCAGCGATGAACTG; SEQ ID NO.10 shows: ATTTCACGAACGCGCAGAATGG.

[0020] The technical solution of this invention has the following beneficial effects: This invention discloses a method for improving rice seed vigor and grain shape by overexpressing the gene encoding the rice auxin response factor. OsARF10 This increases the expression level of the gene in rice, thereby promoting the genetic improvement of rice seed vigor and grain shape.

[0021] Cloned by this invention OsARF10 Genes also provide new genetic resources for breeding high-viability seeds and high-yield rice varieties, which has important guiding significance for the breeding of high-viability seeds and high-yield rice. Attached Figure Description

[0022] Figure 1 OsARF10 Identification of transgenic positive plants with overexpression OsARF10 Expression level analysis. The control wild-type material was the Nipponbare variety. a) is a gel image of positive identification of transgenic plants of the overexpression material; b) is the expression level of transgenic plants of the overexpression material. OsARF10 Expression level. ** indicates a significant difference at the 0.01 level.

[0023] Figure 2 Under normal (water) conditions OsARF10Germination phenology of overexpressing transgenic seeds. a shows germination photos on days 3 and 5; be and b represent seed germination rate (GP), seedling survival rate (SP), germination index (GI), and time required for 50% seed germination, respectively. T 50 Bar = 1 cm. * and ** indicate significant differences at the 0.05 and 0.01 levels, respectively, and ns indicates no significant difference.

[0024] Figure 3 Under 200mM NaCl conditions OsARF10 Germination phenology of overexpressing transgenic seeds. a shows germination photos on days 7 and 10; be and be represent seed germination rate (GP), seedling survival rate (SP), germination index (GI), and time required for 50% seed germination, respectively. T 50 Bar = 1 cm. * and ** indicate significant differences at the 0.05 and 0.01 levels, respectively, and ns indicates no significant difference.

[0025] Figure 4 Under 15% PEG conditions OsARF10 Germination phenology of overexpressing transgenic seeds. a shows germination photos on days 7 and 10; be and be represent seed germination rate (GP), seedling survival rate (SP), germination index (GI), and time required for 50% seed germination, respectively. T 50 Bar = 1 cm. * and ** indicate significant differences at the 0.05 and 0.01 levels, respectively, and ns indicates no significant difference.

[0026] Figure 5 OsARF10 Seed shape of overexpressing transgenic plants. 'a' shows photos of seed length and width; 'be' shows phenotypic data for seed length, width, thickness, and thousand-seed weight, respectively. Bar = 1 cm. * and ** indicate significant differences at the 0.05 and 0.01 levels, respectively; ns indicates no significant difference. Detailed Implementation

[0027] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0028] Unless otherwise specified, all methods used in the examples are conventional methods. Primers and sequencing were performed by Nanjing Qingke Biotechnology Co., Ltd. Various restriction endonucleases, ligases, DNA ladders, high-fidelity enzymes, vectors, etc. were purchased from Baoriyi Biotechnology (Beijing) Co., Ltd. RNA extraction kits and reverse transcription kits were purchased from Novizan Biotechnology Co., Ltd. Plasmid extraction kits, gel extraction kits, and genome extraction kits were purchased from Tiangen Biotech Co., Ltd. All methods were performed in accordance with the instructions.

[0029] Example 1 Rice OsARF10 Cloning of genes Total RNA was extracted from seedlings of the japonica rice variety Nipponbare and reverse transcribed into cDNA as a template. Using the upstream primer (sequence shown in SEQ ID NO.3) and the downstream primer (sequence shown in SEQ ID NO.4), cDNA was cloned via PCR. OsARF10 Gene coding sequence. After sequencing, the following was obtained. OsARF10 The nucleotide and amino acid sequences of the gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0030] Example 2 Construction of rice OsARF10 overexpression vector (1) With amplified OsARF10 Using the gene coding region sequence (SEQ ID NO.1) as a template, and utilizing the enzyme restriction sites... CPN I and Room The homologous recombination primers 1300s-OsAAH-F (SEQ ID NO. 5) and 1300s-OsARF10-R (SEQ ID NO. 6) amplified enzymes containing the restriction enzyme sites. OsARF10 The coding region product was subjected to PCR product gel recovery.

[0031] (2) Using restriction endonucleases CPN I and Room The plant overexpression vector pCAMBIA1300s was digested with enzyme I, and the PCR product was recovered by gel extraction.

[0032] (3) Using the ClonExpress II One Step Cloning Kit (C112-01) from Novizan, the gel-recovered products described in (1) and (2) were recombinated at 37°C for 30 minutes. The recombinant products were transformed into *E. coli*. Plasmids from correctly sequenced *E. coli* were extracted and then transformed into *Agrobacterium*. *Agrobacterium* culture was used to infect callus tissue from Nipponbare materials, and tissue culture was employed to obtain… OsARF10 Transgenic plants that are overexpressed.

[0033] Example 3 Identification of transgenic positive plants of rice with OsARF10 overexpression Forward primers, as shown in SEQ ID NO. 7, were designed on the pCAMBIA1300s vector using Primer Premier 5 software. OsARF10 The reverse primers shown in SEQ ID NO.8 were designed for gene sequencing. PCR was performed using genomic DNA from rice seedlings as a template, and the PCR products were then subjected to agarose gel electrophoresis to screen rice varieties. OsARF10 Overexpression of positive transgenic lines.

[0034] The results showed that 2 were obtained. OsARF10 Overexpression of transgenic positive lines (OE1, OE2, and OE3), electrophoresis results are as follows: Figure 1 As shown in a. OsARF10 Overexpression of transgenic lines OE-1, OE-2, and OE-3 showed distinct bands at 1000 bp, but no bands were observed in the control wild-type (Nipponbare, WT). This indicates that OE-1, OE-2, and OE-3 are all transgenic lines. OsARF10 Overexpression of transgenic positive lines.

[0035] Example 4: Rice OsARF10 overexpression transgenic positive plants OsARF10 Expression level identification Using an RNA kit, extract OsARF10 Total RNA from overexpressed plants (OE-1, OE-2, and OE-3) and their wild-type (Nipponbare, WT) three-leaf stage seedlings was reverse transcribed to obtain the corresponding cDNA. Based on... OsARF10 Based on the coding region sequence of the gene, RT-qPCR primers were designed as shown in SEQ ID NO. 9 and SEQ ID NO. 10, and internal control primers as shown in SEQ ID NO. 11 (CAGCATTGGCAGCGATGAACT) and SEQ ID NO. 12 (ATTTCACGAACGCGCAGAATGG) were used for detection. OsARF10 Gene expression analysis in rice OsARF10 Overexpression of transgenic positive plants OsARF10 Expression level.

[0036] The results showed that rice OsARF10 Overexpression of transgenic positive lines OE-1, OE-2, and OE-3 OsARF10 Expression levels were significantly higher than those of the control wild-type (Nipponbare, WT), such as Figure 1 As shown in b, this indicates that strains OE-1, OE-2, and OE-3 are... OsARF10 Overexpression of transgenic positive material.

[0037] Example 5: Seed vigor analysis of rice plants with positive OsARF10 overexpression (a) Seed germination phenotype under normal conditions Select OsARF10 Seed germination experiments were conducted on rice varieties with overexpression lines and the control wild type (Nipponbare, WT) as follows: For each replicate, 30 healthy, plump seeds were selected, surface-sterilized with 2% sodium hypochlorite solution for 10 min, rinsed three times with distilled water, dried, and evenly spread in a petri dish (9 cm in diameter). 10 mL of distilled water was added, and the dishes were incubated at 25℃ under light / dark conditions for 12 h each time for 7 days. Germination and seedling emergence were then recorded. The experiment was repeated three times.

[0038] The results showed that rice OsARF10 The overexpression lines (OE-1, OE-2, and OE-3) showed significantly higher seed germination rate (GP), seedling survival rate (SP), and germination index (GI) than the Nipponbare wild-type control (WT) material. The time required for 50% seed germination (T) was also significantly higher. 50 The values ​​were significantly lower than those of the wild-type control material of Nipponbare, such as... Figure 2 As shown. It can be seen that under normal conditions... OsARF10 Overexpression can significantly improve the germination and seedling formation of rice seeds.

[0039] (II) Seed germination phenotype under salt stress Select OsARF10 Seed germination experiments were conducted on rice varieties with overexpression lines and the control wild type (Nipponbare, WT) as follows: For each replicate, 30 healthy, plump seeds were selected, surface-sterilized with 2% sodium hypochlorite solution for 10 min, rinsed three times with distilled water, dried, and placed in a 9 cm diameter petri dish containing 40 mL of quartz sand. 20 mL of 200 mM NaCl was added, and the dishes were incubated at 25℃ under light / dark conditions for 10 days (12 h light / 12 h darkness). The seedling survival rate was then calculated. The experiment was repeated three times.

[0040] The results showed that rice OsARF10 The overexpression lines (OE-1, OE-2, and OE-3) showed significantly higher seed germination rate (GP), seedling survival rate (SP), and germination index (GI) than the Nipponbare wild-type control (WT) material. The time required for 50% seed germination (T) was also significantly higher. 50 The values ​​were significantly lower than those of the wild-type control material of Nipponbare, such as... Figure 3 As shown. It can be seen that under salt stress... OsARF10 Overexpression can significantly improve the germination and seedling formation of rice seeds.

[0041] (III) Seed germination phenotypes under PEG stress Select OsARF10Seed germination experiments were conducted on rice varieties with overexpression lines and the control wild type (Nipponbare, WT) as follows: For each replicate, 30 healthy, plump seeds were selected, surface-sterilized with 2% sodium hypochlorite solution for 10 min, rinsed three times with distilled water, dried, and evenly spread in a petri dish (9 cm in diameter). 10 mL of 15% PEG solution was added, and the dishes were incubated at 25℃ under light / dark conditions for 10 days (12 h light / 12 h darkness). The seedling survival rate was then calculated. The experiment was repeated three times.

[0042] The results showed that rice OsARF10 The overexpression lines (OE-1, OE-2, and OE-3) showed significantly higher seed germination rate (GP), seedling survival rate (SP), and germination index (GI) than the Nipponbare wild-type control (WT) material. The time required for 50% seed germination (T) was also significantly higher. 50 The values ​​were significantly lower than those of the wild-type control material of Nipponbare, such as... Figure 4 As shown. It can be seen that under drought stress... OsARF10 Overexpression can significantly improve the germination and seedling formation of rice seeds.

[0043] In summary, OsARF10 Overexpression can enhance rice seed vigor and significantly promote seed germination rate and seedling growth under both normal and abiotic stress conditions. Example 6 Rice OsARF10 Overexpression of positive plant grain shape and thousand-grain weight Select OsARF10 Seeds from the overexpression line and the control wild type (Nipponbare, WT) were used to determine seed length, width, thickness, and thousand-seed weight: uniformly sized seeds were collected. OsARF10 Seeds from the overexpression lines and the control wild type (Nipponbare, WT) were measured for length, width, and thickness using calipers, and for thousand-seed weight using an electronic balance. Simultaneously, photographs were taken of the seeds arranged according to their length. Three biological replicates were used for each experimental line.

[0044] The results showed that rice OsARF10 The overexpression lines (OE-1, OE-2, and OE-3) had significantly longer grains and a thousand-grain weight than the Nipponbare wild-type control (WT), while grain width and thickness showed no significant difference from the Nipponbare wild-type control. Figure 5 As shown. It can be seen that, OsARF10 Overexpression can significantly increase grain length and thousand-grain weight, thus promoting crop yield.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression of rice auxin response factor encoding gene OsARF10 Or overexpression of rice auxin response factor encoding genes OsARF10 Application of substances in improving rice seed vigor and / or regulating rice seed grain shape.

2. The application according to claim 1, characterized in that, The improvement of rice seed vigor refers to improving rice seed vigor under normal conditions and / or abiotic stress; preferably, the abiotic stress is salt stress and / or drought stress.

3. The application according to claim 1, characterized in that, The regulation of rice seed shape is to increase the length and / or weight of rice seeds.

4. The application according to claim 1, characterized in that, The gene encoding the rice auxin response factor OsARF10 The nucleotide sequence is shown in SEQ ID NO.

1.

5. The application according to claim 1, characterized in that, The rice auxin response factor encoding gene OsARF10 The amino acid sequence is shown in SEQ ID NO.

2.

6. The application according to claim 1, characterized in that, The overexpression of the rice auxin response factor encoding gene OsARF10 The substance is the gene encoding the rice auxin response factor. OsARF10 Overexpression vectors.

7. The application according to claim 6, characterized in that, The rice auxin response factor encoding gene OsARF10 The overexpression vector was based on the pCAMBIA1300s plasmid. Primers shown in SEQ ID NO. 5 and SEQ ID NO. 6 were used to amplify the rice auxin response factor encoding gene shown in SEQ ID NO.

1. OsARF10 The coding region was ligated into the pCAMBIA1300s plasmid. Kpn I / Sal The I restriction site was constructed.

8. A method for improving rice seed vigor and yield, characterized in that, The method described is for constructing a gene encoding a rice auxin response factor. OsARF10 Overexpression of transgenic lines or corresponding overexpression materials can be used to obtain plants with improved seed vigor and grain shape.

9. The method according to claim 8, characterized in that, The method includes the following steps: (1) Rice genes OsARF10 Overexpression transgenic construction: Construction of a rice auxin response factor encoding gene OsARF10 Overexpression vectors are used to transform rice, resulting in rice. OsARF10 Overexpression lines; (2) Screening and identification of overexpression lines in rice: Design specific detection primers to amplify rice OsARF10 cDNA was overexpressed in the young leaves of the strains, and the expression levels were detected by conventional PCR products and / or real-time quantitative RT-qPCR. Positive and stable overexpressing plants were screened, which are the plants that improve rice seed vigor and grain shape.

10. The method according to claim 9, characterized in that, Rice auxin response factor encoding gene OsARF10 The primers for identifying the vector in overexpressing conventional PCR-positive plants are shown in SEQ ID NO.7 and SEQ ID NO.

8. The gene encoding the rice auxin response factor... OsARF10 Primers for real-time quantitative RT-qPCR detection of overexpression are shown in SEQ ID NO.9 and SEQ ID NO.10.

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