Application of rice OsUBC48 gene or its encoded protein in regulating rice seed germination

Abstract

The present invention discloses the application of rice OsUBC48 gene or the protein encoded by it in regulating rice seed germination, and relates to the field of bioengineering technology. The base sequence of the OsUBC48 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the OsUBC48 gene is shown in SEQ ID NO.2. By overexpressing the OsUBC48 gene in rice, the germination rate of the transgenic plant is higher than that of the wild-type strain, and the shoot length and root length of the transgenic plant are significantly higher than those of the wild-type strain.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to application of rice OsUBC48 gene or protein encoded by the gene in regulating rice seed germination. Background Art

[0002] Rice (Oryza sativa L.) is one of the important grains in my country. As a country with a large population, it is of great significance to ensure our food security. Therefore, increasing rice yield and improving rice quality have been the research focus in recent years. Germination is an important factor that determines the later growth, development and yield of rice. Exploring rice germination-related genes is crucial to improving rice yield. Rice germination is an important and complex process, which is regulated by many plant hormones. The currently accepted conclusion is that abscisic acid (ABA) inhibits seed germination, while gibberellin (GA) promotes seed germination. ABA and GA play a key role in the seed germination process.

[0003] Ubiquitination is an important post-translational modification of proteins and the main pathway for intracellular protein degradation. It plays an important role in the growth and development, morphological construction, stress resistance and disease resistance of eukaryotic organisms (Hershko, 2005, The ubiquitin system for protein degradation and some of its roles in the control of the cell-division cycle (nobel lecture). Angewandte Chemie International Edition, 44 (37): 5932-5943.). The system consists of ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, ubiquitin ligase E3, proteasome and deubiquitinating enzymes (DUBs). Ubiquitin conjugating enzymes E2s (UBCs) catalyze the transfer of ubiquitin to substrates or E3 ligases and are key enzymes for target protein ubiquitination modification. However, rice (Oryza sativa L.) is one of the world's largest staple food and a model plant for botanical research, but there is little research on the genes of the rice UBC gene family. Currently, 48 UBC family genes (OsUBC1-48) have been identified in rice, of which 37 have true ubiquitin-conjugating enzyme catalytic activity.Protein ubiquitination modification plays a regulatory role in seed development, dormancy and germination, and root development (Koiwai H, Tagiri A, Katoh S, et al (2007). RING-H2 type ubiquitin ligase EL5 is involved in root development through the maintenance of cell viability in rice. The Plant Journal, 51 (1): 92-104.); ectopic expression of the wild rice gene OgUBC in Arabidopsis can enhance the plant's resistance to Botrytis cinerea and UV-B radiation (Jeon, Pak et al. 2012, Ectopic expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsis thaliana. Biochem Biophys Res Commun, 427(2): 309-314.); Patent application with publication number CN114703226A discloses the application of rice OsUBC27 gene or its encoded protein in improving rice yield. The constructed plant overexpression vector pCUbi1390-OsUBC27 was expressed in wild-type Nipponbare. The number of tillers of transgenic plants was significantly increased compared with wild-type plants. Compared with wild-type plants, the grain length and thousand-grain weight of rice were increased.

[0004] Although the roles of several plant UBCs have been reported, our understanding of the biological functions of most of these enzymes is still very limited, especially in food crops such as rice. There are also no reports on the role of ubiquitination in rice seed germination. Summary of the invention

[0005] The present application provides the application of rice OsUBC48 gene or the protein encoded by it in regulating the growth and development of rice. The rice OsUBC48 protein encoding gene OsUBC48, its nucleotide sequence is: SEQ ID NO.1. The constructed plant overexpression vector pCUbi1390-OsUBC48 was expressed in the wild-type Nipponbare, and it was found that the seed germination rate of the transgenic plants was higher than that of the wild-type plant seeds, and the bud length and root length of the transgenic plant seeds were longer than those of the wild-type plant seeds, with significant differences. It can be seen that the rice OsUBC48 protein encoding gene OsUBC48 described in the present invention is a potential target for genetic engineering, which can improve rice seed germination and promote rice yield, and has important application value.

[0006] The technical solution of the present invention is as follows:

[0007] The invention provides application of rice OsUBC48 gene or protein encoded by the gene in regulating rice seed germination.

[0008] The regulation of rice seed germination is reflected in increasing the rice seed sprout length, root length and germination rate.

[0009] Preferably, the base sequence of the OsUBC48 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the OsUBC48 gene is shown as SEQ ID NO.2.

[0010] The invention provides a method for cultivating transgenic rice, which overexpresses the OsUBC48 gene in rice crops.

[0011] Preferably, the base sequence of the OsUBC48 gene is shown in SEQ ID NO.1.

[0012] A method for cultivating transgenic rice comprises the following steps:

[0013] (1) Construction of a vector overexpressing the OsUBC48 gene;

[0014] (2) Construction: The OsUBC48 gene overexpression vector of step (1) is introduced into rice cells to overexpress the OsUBC48 gene having a base sequence as shown in SEQ ID NO.1, and transgenic rice plants are obtained after cultivation.

[0015] Preferably, the plant expression vector is pCUbi1390.

[0016] When transforming the recipient plant, the Agrobacterium-mediated transformation method can be used. The Agrobacterium can specifically be the Agrobacterium EHA105 strain. In step (2), after the gene overexpression vector is transferred into the Agrobacterium, the rice cells are infected.

[0017] The recipient plant is rice. The variety of the rice can be japonica rice Nipponbare, but is not limited to Nipponbare.

[0018] The gene overexpression was used to genetically transform rice, and tests showed that the overexpression of the OsUBC48 gene could increase the sprout length and root length of rice seeds during germination.

[0019] Preferably, the cell source infected by Agrobacterium is callus induced from rice seeds. In addition to callus induced from rice seeds, callus induced from tissue samples obtained from rice plants, or other methods that can cultivate plants after genetic modification are also acceptable.

[0020] Beneficial effects of the present invention:

[0021] The invention overexpresses the OsUBC48 gene in rice, so that the germination rate of the transgenic plants is higher than that of the wild-type strains, and the shoot length and root length of the transgenic plants are significantly higher than those of the wild-type strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a hygromycin PCR identification diagram of transgenic plants; wherein, M: marker; 1 is the pCUbi1390 plasmid, which is used as a positive control; 2-7 are T0 generation transgenic lines that positively overexpress the OsUBC48 gene; and 8 is a non-transgenic wild-type Nipponbare line.

[0023] Figure 2 This is the qRT-PCR identification diagram of the OsUBC48 gene expression level in the T1 generation transgenic line.

[0024] Figure 3 This is the qRT-PCR identification diagram of the OsUBC48 gene expression level in the T2 generation transgenic line.

[0025] Figure 4 Figure 2 is a graph showing the germination of seeds of the OsUBC48 overexpression strain and the wild-type strain; A is a graph showing the appearance of seed buds and roots of the OsUBC48 overexpression strain and the wild-type strain; B is a graph showing the germination rate of seeds of the OsUBC48 overexpression strain and the wild-type strain; C is a graph showing the comparison of seed bud lengths of the OsUBC48 overexpression strain and the wild-type strain. DETAILED DESCRIPTION

[0026] Example 1

[0027] Cloning and transformation of rice OsUBC48 gene.

[0028] RNA was extracted from young rice leaves and reverse transcribed into cDNA using MLV reverse transcription reagent (Takara) for later use.

[0029] Total RNA was extracted using the NucleoZOL kit (MACHEREY-NAGEL, Germany) as follows:

[0030] (1) Take 100 mg of leaves and put them into 2 mL sterile centrifuge tubes. Add a small steel ball to each tube, quickly put the sample into liquid nitrogen, and break the leaf tissue with a sampler;

[0031] (2) Add 1 mL of NucleoZOL extract, mix well, add 400 μL RNase-free water per 1 mL of NucleoZOL extract to lyse the leaves, shake vigorously for 15 seconds, and incubate at room temperature for 5-15 minutes;

[0032] (3) Centrifuge the sample at 12000 rpm for 15 min at room temperature;

[0033] (4) Transfer 1 mL of supernatant to a new 2 mL centrifuge tube;

[0034] (5) Add 1 mL of isopropanol to each 1 mL of supernatant to precipitate RNA, incubate at room temperature for 10 min, then centrifuge the sample at 12000 rpm for 10 min and discard the supernatant;

[0035] (6) Add 500 μL of 75% ethanol (v / v) to wash the RNA, centrifuge at 6000 rpm for 2-3 minutes, remove the ethanol with a pipette, repeat the ethanol wash once, and place in a fume hood to blow air to remove residual ethanol;

[0036] (7) Add 30 μL of RNase-free water to dissolve the RNA and vortex at room temperature for 3 minutes to improve the dissolution efficiency;

[0037] Primers were designed based on the sequence information of gene OsUBC48 (search number LOC_Os01g42040) provided by the Rice Gene Database (https: / / www.ricedata.cn / gene / ):

[0038] UBC48-CDS-F: 5′-TTACTTCTGCACTAGGTACCATGGCTCAGGCTGCAAGACT-3′,

[0039] UBC48-CDS-R: 5′-GAATTCCCGGGGATCCTTAGCTTCTTGGTTTTCCAGCCAATC-3′;

[0040] PCR amplification was performed using leaf cDNA of rice "Nipponbare" as a template. The complete ORF (open reading frame) was constructed into the linearized vector pCUbi1390 after KpnI digestion by homologous recombination using the infusion method, transformed into Escherichia coli DH5α competent cells, and spread on LB solid medium containing 100 μg / mL kanamycin resistance. After culturing at 37°C for 12-16 hours, a single clone was picked, and the clones positive for colony PCR detection were sent for sequencing verification. The verified correct vector clone was named pCUbi1390-OsUBC48, which is the expression vector of gene OsUBC48. pCUbi1390-OsUBC48 was transformed into Agrobacterium tumefaciens EHA105 competent cells to obtain pCUbi1390-OsUBC48 Agrobacterium bacterial solution.

[0041] The OsUBC48 gene overexpression strain was constructed by Agrobacterium transformation.

[0042] In order to obtain the overexpression material of gene OsUBC48, the genetic transformation method mediated by Agrobacterium was used. The specific operation steps are as follows:

[0043] (1) Callus induction: Select about 20g of mature and plump seeds, shell and select them (free of pathogens and black spots). The selected seeds are then disinfected and cleaned (in a clean bench, treated with 75% alcohol (v / v) for 1min, washed with sterile water 5 times, soaked in 50% pasteurized solution (v / v) for 30min, and then washed with sterile water until there is no foam). The sterilized seeds are placed on sterile filter paper and the surface moisture is blown dry in a clean bench (about 4h). The seeds are then spread on an induction medium (N6D medium) and cultured in the dark at 28°C for one month for callus culture;

[0044] (2) Agrobacterium infection: About 300 μL of revived Agrobacterium culture (containing pCUbi1390-OsUBC48) was added to 150 mL of AAM medium containing 50 mg / L acetosyringone and cultured at 28°C until OD 600 The concentration of the culture medium was about 0.1, and then fresh callus was infected. The callus was immersed in the Agrobacterium solution for 30-60 seconds, and the callus was taken out and washed with sterile deionized water for 8-10 times. After infection, the callus was cultured for 3 days (N6D-As medium, sealed, and cultured in the dark at 28°C).

[0045] (3) Screening of resistant callus: After washing the above callus tissue with sterile water containing 400 mg / L carbenicillin for about 8 times, the callus tissue was spread on sterile filter paper and the surface water was drained; finally, the callus tissue was spread on N6D solid medium containing 400 mg / L carbenicillin and 50 mg / L hygromycin B;

[0046] (4) Differentiation and rooting: The resistant callus (resistant tissue is yellow in color) is transferred to a differentiation medium containing 50 mg / L hygromycin B and 250 mg / L carbenicillin; the seedlings that have grown about 5 cm on the differentiation medium are transferred to a rooting medium containing 50 mg / L hygromycin B and 200 mg / L carbenicillin; the above operations are all performed in a sterile operating table;

[0047] (5) Transgenic detection: The obtained transgenic seedlings were tested; the OsUBC48 gene overexpression strain was tested by PCR using hygromycin primers. The primer information is as follows:

[0048] hygF: 5′-GTGCTTGACATTGGGGAGTT-3′,

[0049] hygR: 5′-GATGTTGGCGACCTCGTATT-3′.

[0050] The results showed that the hygromycin test of the 6 randomly selected transgenic plants was positive, which preliminarily indicated that the pCUbi1390-OsUBC48 overexpression vector had been successfully transferred into the rice plants ( Figure 1 ).

[0051] Example 2

[0052] Identification of OsUBC48 gene expression levels in overexpression lines.

[0053] Seeds of hygromycin-positive transgenic T0 rice plants were collected and planted in transgenic fields for two consecutive generations (2021-2022). Young leaves were selected during the tillering stage of rice to extract total RNA, which was reverse transcribed into cDNA for qRT-PCR (the steps were the same as in Example 1). The rice constitutively expressed gene Actin was used as an internal reference, and the primer sequences were as follows:

[0054] ActinF: 5′-GAAATGGAGACTGCCAAGACC-3′,

[0055] ActinR: 5′-TTGGCAATCCACATCTGCTG-3′;

[0056] The qRT-48F and qRT-48R primers were used to detect the expression of the OsUBC48 gene in different T1 and T2 rice lines. The sequences of the qRT-48F and qRT-48R primers are as follows:

[0057] qRT-48F: 5′-TGACCCAAACCCTGACGATG-3′,

[0058] qRT-48R: 5′-TAGCGCCAATTGCTGAAGGA-3′.

[0059] The results showed that the expression level of OsUBC48 gene in two lines of T1 transgenic lines numbered OE154, OE154-1 and OE154-2, was significantly higher than that in the wild-type line ( Figure 2 ). OE154-2 was used to continue planting the T2 generation, and two T2 overexpression lines 141-1 and 141-2 were randomly selected. The results of qRT-PCR showed that the expression levels of the OsUBC48 gene were significantly higher than those of the wild-type lines, indicating that the overexpression of the OsUBC48 gene can be stably inherited in multiple generations of transgenic lines ( Figure 3 ).

[0060] Example 3

[0061] Seed germination test.

[0062] The T1 generation seeds OE154-2 strain and wild-type seeds in Example 2 were collected, and dormancy was broken by 7 days in a 45°C oven. 30 fresh seeds that were full and free of pests and diseases were selected, disinfected with 0.5% NaClO (v / v) for 25 min, rinsed repeatedly with deionized water for 8-10 times, and then placed in a culture dish with 3 layers of double-circle qualitative filter paper (diameter 9em) added, and 15mL of distilled water was added. Put it in a light incubator (temperature 28°C, light 16h, dark 8h) for germination experiment. The seeds that germinated were counted with whitening as the standard, and the number of germinations was counted every 24h. The statistics were observed continuously for 7 days, and the length of the buds and roots was measured on the seventh day. Three biological replicates were set for each material.

[0063] The results showed that the germination rate of the OsUBC48 overexpression strain OE154-2 was higher than that of the wild-type WT strain from 24h to 72h; at 96h, the germination rates of the wild-type WT and the OsUBC48 overexpression strain OE154-2 were close ( Figure 4 B), at this time, the shoot length and root length of the wild-type WT and the OsUBC48 gene expression strain OE154-2 showed obvious differences, and the shoot length and root length of the OE154-2 material were significantly higher than those of the wild-type WT strain ( Figure 4 A, C). On the seventh day, the shoot lengths of the wild-type strain and the overexpression strain were measured and recorded, and it was found that the shoot length of the overexpression strain OE154-2 was significantly longer than that of the wild-type strain, indicating that the OsUBC48 gene can promote seed germination.

Claims

1. Application of overexpressed rice OsUBC48 gene or protein encoded by the gene in regulating rice seed germination, wherein the base sequence of the OsUBC48 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the OsUBC48 gene is shown in SEQ ID NO.2; The regulation of rice seed germination is reflected in increasing the length of rice seed sprouts and roots.

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