CN106244596A - A kind of rice paddy seed dormant gene OsDOG1L3 and application thereof - Google Patents

Abstract

The invention discloses a rice seed dormancy gene OsDOG1L3 and its application. The present invention isolates and clones the homologous DOG1 gene from N22 strongly dormant rice, and names it OsDOG1L3. The inventor connects the full-length cDNA of the OsDOG1L3 genome to the expression vector initiated by the overexpression vector, and uses Agrobacterium to infiltrate and transform Rice non-dormant variety Nanjing 35, it was found that the above-mentioned genes can enhance the dormancy of the non-dormant variety Nanjing 35.

Description

A rice seed dormancy gene OsDOG1L3 and its application

technical field

The invention belongs to the field of genetic engineering, and in particular relates to a gene OsDOG1L3 for regulating rice seed dormancy and its application.

Background technique

Seed dormancy refers to the phenomenon that seeds with normal vitality cannot germinate under suitable environmental conditions (light, temperature, moisture, oxygen, etc.). In crops, especially rice and wheat, if there is high temperature and rainy weather before harvest, ear germination often occurs, which seriously affects the yield and quality of crops, and brings huge losses to agricultural production. The strength of seed dormancy directly determines whether the seed will germinate.

There are many factors that affect seed dormancy, and environmental factors mainly include temperature, humidity, light and air. Temperature is the main external factor affecting rice seed dormancy. Temperature mainly affects the formation of dormancy inducers during seed maturation. During seed development, seeds formed at high temperatures have low or no dormancy, while those formed at low temperatures will have low dormancy. enhanced. Moisture is also an important factor affecting the strength of seed dormancy. With the increase of seed maturity, the moisture at the seed dormancy point will decrease, and the seed dormancy can be broken under a certain humidity environment; at the same time, the study found that moisture only affects the dormancy of freshly harvested seeds. Seed dormancy is also affected by light, for example, red light can promote germination, while far-red light and blue light can inhibit germination. In the case of too much carbon dioxide and insufficient oxygen, the germ of rice seeds can grow normally, but the growth of radicle is not normal. It is generally believed that hybrid rice seeds will enter a dormant state when carbon dioxide exceeds 30%. Intrinsic factors mainly include post-ripening, seed coat disorder, regulation of plant hormones and effects of dormancy-related genes. Dormancy is broken after dry mature cereal seeds have been stored at a certain temperature (the temperature at which the seeds were harvested) for a period of time (typically several months) due to the effect of afterripening. Abscisic acid (ABA) and gibberellin (GA) among the endogenous hormones are the main factors regulating seed germination and dormancy. Among them, GA ₃ can relieve seed dormancy and promote seed germination, while ABA inhibits seed germination and induces dormancy. GA and ABA have antagonistic effects during seed development. Seed dormancy is controlled by multiple genes. In recent years, some new seed dormancy genes have been isolated through QTLs mapping and cloning. DELAY OF GERMINATION1 (DOG1) is the first dormant QTL cloned in Arabidopsis, which encodes a protein of unknown function. In addition to this gene cloning method of forward genetics, more and more researchers use the method of reverse genetics to obtain the required genes through the analysis of transcriptome and proteome data. So far, some progress has been made in the mapping and cloning of rice dormancy genes, but the regulatory mechanism of rice seed dormancy is still unclear, which requires us to locate and clone more dormancy genes to further reveal the mechanism of seed dormancy.

Contents of the invention

The purpose of the present invention is to disclose a rice seed dormancy-related gene OsDOG1L3, the nucleotide sequence of which is shown in SEQ ID NO: 1, containing 738bp. Its genome sequence is identical to the CDS sequence.

The second object of the present invention is also to provide the protein sequence encoded by the rice seed dormancy-related gene OsDOG1L3, the amino acid sequence of which is shown in SEQ ID NO.2, containing 245 amino acids.

The gene encoding said protein is preferably the DNA molecule described in 1) or 2) or 3) as follows:

1) The DNA molecule shown in SEQ ID NO.1;

2) a DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes the protein;

3) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) or 2) and encoding a plant seed dormancy-related protein.

Recombinant expression vectors containing any of the above genes also belong to the protection scope of the present invention.

An existing plant expression vector can be used to construct a recombinant expression vector containing the gene.

The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. The plant expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage The untranslated region transcribed at the 3' end of protein gene) has similar functions.

When using the gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, maize Ubiquitin promoters (Ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors, enhancers, including translation enhancers or transcription enhancers, can also be used, These enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) Genes, etc.), antibiotic resistance markers (gentamycin markers, kanamycin markers, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

The recombinant expression vector can be a recombinant plasmid obtained by inserting the gene (OsDOG1L3) at the recombination site of the vector pCAMBIA1390 double-digested with restriction endonucleases KpnI and SpeI. The pCAMBIA1390 containing OsDOG1L3 was named pCAMBIA1390-OsDOG1L3, and this recombinant expression vector can overexpress the gene OsDOG1L3.

Expression cassettes, transgenic cell lines and recombinant bacteria containing any of the above genes (OsDOG1L3) all belong to the protection scope of the present invention.

The primer pair for amplifying the full length or any fragment of the gene (OsDOG1L3) also belongs to the protection scope of the present invention, and the primer pair is preferably Primer1/Primer2, Primer3/Primer4.

The application of at least one of the gene (OsDOG1L3), the protein, the recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant bacterium in plant breeding of the present invention.

A method for cultivating moderate dormancy of rice seeds, comprising introducing the gene into a non-dormant rice variety to obtain a transgenic rice with enhanced dormancy; the germination rate of the non-dormant rice variety is close to 100% rice; the dormancy The germination rate of the enhanced transgenic rice was 80% lower than that of the transgenic plants.

The method of the present invention preferably introduces the gene into the non-dormant rice through the recombinant expression vector.

A method for cultivating a transgenic plant with enhanced seed dormancy, comprising overexpressing the gene of the present invention in a target plant to obtain a transgenic plant with enhanced dormancy; the target plant is a plant carrying the gene.

Beneficial effect:

The present invention isolates and clones the homologous DOG1 gene from N22 strongly dormant rice, and names it OsDOG1L3. The inventor connects the full-length cDNA of the OsDOG1L3 genome to the expression vector initiated by the overexpression vector, and uses Agrobacterium to infiltrate and transform Rice non-dormant variety Nanjing 35, it was found that the above-mentioned genes can enhance the dormancy of the non-dormant variety Nanjing 35. The gene is used to obtain varieties with moderate dormancy through plant genetic engineering technology, so that the quality of rice and rice seeds under harsh conditions can be guaranteed.

Description of drawings

Figure 1 is a comparison of the amino acid sequences of rice and Arabidopsis DOG1.

Fig. 2 is a plasmid map of the overexpression vector pCAMBIA1390.

Figure 3 is the germination of transgene overexpression plants.

detailed description

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1. Sequence analysis, cloning and vector construction of rice OsDOG1L3 gene

1. Sequence analysis and cloning of rice dormancy genes

Through bioinformatics analysis, the inventor cloned the rice OsDOG1L3 gene in rice N22 as shown in SEQ ID NO.1. The rice OsDOG1L3 gene is located on the first chromosome. The open reading frame of OsDOG1L3 is 738bp, encoding 245aa as shown in SEQ ID NO.2.

According to the found rice OsDOG1L3 gene sequence, primers were designed and cloned. The cloning method is as follows:

(1) Extraction of DNA:

① Take about 0.2 grams of young rice leaves, put them in a 2.0ml Eppendorf tube, put a steel ball in the tube, freeze the Eppendorf tube with the sample in liquid nitrogen for 5 minutes, and put it on a 2000-type GENO/GRINDER instrument to crush it Sample 1min.

②Add 660μl extract solution (contains 100mM Tris-Hcl (PH 8.0), 20mM EDTA (PH 8.0), 1.4M NaCl, 0.2g/ml CTAB solution), vortex vigorously on a vortex mixer, and ice-bath for 30min.

③ Add 40 μl of 20% SDS, incubate at 65°C for 10 minutes, and gently invert up and down every two minutes to mix well.

④Add 100μl 5M NaCl and mix gently.

⑤Add 100μl 10×CTAB, incubate at 65°C for 10min, and mix by gently inverting up and down intermittently.

⑥Add 900μl chloroform, mix thoroughly, and centrifuge at 12000rpm for 3min.

⑦ Transfer the supernatant to a 1.5mL Eppendorf tube, add 600μl isopropanol, mix well, and centrifuge at 12000rpm for 5min.

⑧ Discard the supernatant, rinse the precipitate once with 70% (volume percent) ethanol, and dry it at room temperature.

⑨ Add 100 μl 1×TE (121 g Tris dissolved in 1 liter water, adjust the pH value to 8.0 with hydrochloric acid) to dissolve the DNA.

⑩ Take 2 μl of electrophoresis to detect the quality of DNA, and use a DU800 spectrophotometer to determine the concentration (Beckman Instrument Inc. U.S.A).

(2) Cloning of OsDOG1L3 gene:

OsDOG1L3 primer sequence:

Upstream primer Primer1: ATGCCGGCGTCGTACCTGCAG (SEQ ID NO.3)

Downstream primer Primer2: CTAGAGGCCCCGCCGGCCGCCGT (SEQ ID NO.4)

Perform PCR amplification using the above-mentioned extracted DNA product as a template;

PCR reaction system (50 μl) was amplified with KOD enzyme (purchased from TOYOBO): DNA ~ 200ng, upstream primer (10pmol/ul) 1.5ul, downstream primer (10pmol/ul) 1.5ul, 2x PCR Buffer for KOD FX 25ul, dNTPs (2mM) 10ul, KOD FX (1.0U/ul) 1ul, supplemented with ddH ₂ O, a total of 50ul.

PCR reaction program: denaturation at 94.0°C for 2 min; denaturation at 98.0°C for 10 s, annealing at 55°C for 30 s, extension at 68°C for 1 min, and a total of 35 cycles; extension at 68°C for 7 min; storage at 10°C. The PCR reaction was carried out in a PTC-200 (MJ Research Inc.) PCR instrument.

After the reaction, perform agarose gel electrophoresis. After detecting the target band, cut the gel and perform gel recovery. Take the above product and connect it to the pEASY-T1 cloning vector. conduct. Then the ligated product was transformed into Escherichia coli DH5α strain (purchased from Tiangen Company) by heat shock method, and grown overnight on LB plates containing ampicillin. Pick a single white colony and culture it in LB liquid medium. Plasmid DNA was extracted and sequenced.

2. Construction of overexpression vector

Using the correct plasmid with the OsDOG1L3 gene as a template, perform PCR amplification to obtain the OsDOG1L3(N22) gene. The PCR primer sequences are as follows:

Primer3 (the underlined sequence is the Kpn I recombination site):

5'— TGCACTAGGTACC ATGCCGGCGTCGTACC—3' (SEQ ID NO.5)

Primer4 (the underlined sequence is the Spe I recombination site):

5'— CGTTAACACTAGTCTAGAGGCCCCGCCGG —3' (SEQ ID NO. 6)

The above primers are located at the start of the coding region and the terminator of the coding region of the gene shown in SEQ ID NO.2, the amplified product contains the complete coding region of the gene, and the PCR product is recovered and purified. use The HD Cloning Kit recombination kit (Takara Company) cloned the PCR product into the vector pCAMBIA1390 (Fig. 2).

In-Fusion recombination reaction system (10 μL): PCR product 10-200 ng, pCAMBIA1390 vector 50-200 ng recovered by Kpn I and Spe I double digestion, 5×In-Fusion HD Enzyme Premix 2 μL, Deionized water to 10 μL. Pipette the tip of the pipette to mix well, react the mixed system at 50°C for 15 min, and place it on ice. Take 2 μL of the reaction system and transform Escherichia coli DH5α competent cells (Tiangen Company) by heat shock method. Spread all transformed cells evenly on LB solid medium containing 100 mg/L kanamycin. Incubate at 37°C for 12-16 hours, pick positive clones and sequence them.

The sequence determination results showed that the fragment obtained by the PCR reaction contained the nucleotide sequence shown in SEQ ID NO.1, encoding a protein (SEQ ID NO.2) consisting of 245 amino acid residues. The protein represented by SEQ ID NO.3 was named OsDOG1L3(N22).

Embodiment 2, the acquisition and identification of overexpression transgenic plants

1. Obtaining recombinant Agrobacterium

The pCAMBIA1390-OsDOG1L3(N22) was transformed into Agrobacterium EHA105 strain (purchased from Handsome Company, USA) by electric shock method to obtain a recombinant strain, and the plasmid was extracted for identification by PCR and enzyme digestion. The recombinant strains identified correctly by PCR and enzyme digestion were named EH-pCAMBIA1390-OsDOG1L3(N22), respectively.

The acquisition of transgenic plants

Transform EH-pCAMBIA1390-OsDOG1L3(N22) into rice Nanjing 35, the specific method is:

(1) Cultivate EH-pCAMBIA1390-OsDOG1L3(N22) at 28°C for 16 hours, collect the bacteria, and dilute them into N6 liquid medium (Sigma, C1416) containing 100 μmol/L to a concentration of OD ₆₀₀ ≈0.5, and obtain bacteria liquid;

(2) The mature embryogenic callus of Nanjing 35 rice that has been cultivated for one month is mixed with the bacterial solution of step (1) to infect for 30 minutes, and the bacterial solution is blotted by filter paper and then transferred to a co-cultivation medium (N6 solid co-cultivation Medium, Sigma Company), co-cultivated at 24°C for 3 days;

(3) the callus of step (2) is inoculated on the N6 solid selection medium containing 100mg/L paromomycin (Phyto Technology Laboratories company) for the first time selection (16 days);

(4) pick healthy callus and transfer to the second selection on the N6 solid selection medium containing 100mg/L paromomycin, subculture once every 15 days;

(5) pick healthy callus and transfer to the third screening on the N6 solid selection medium containing 50mg/L paromomycin, subculture once every 15 days;

(6) pick the resistant callus and transfer it to the differentiation medium for differentiation;

The T ₀ generation positive plants differentiated into seedlings were obtained. Nanjing 35 was used as negative control.

4. Identification of transgenic plants

1. PCR molecular identification

Genomic DNA is extracted from the T ₀ generation positive plants obtained in step 3, using genomic DNA as a template, using the primer Primer3 near the left border of the insertion site of SEQ ID NO.2 on pCAMBIA1390 and the primer Primer4 on SEQ ID NO.2 as a primer pair Amplification was performed (Primer3: 5'- TGCACTAGGTACC ATGCCGGCGTCGTACC-3' (SEQ ID NO.5) and Primer4: 5'- CGTTAACACTAGT CTAGAGGCCCCGCCGG-3' (SEQ ID NO.6)), and the amplified length was 764bp. PCR reaction system: DNA (20ng/ul) 2ul, Primer5 (10pmol/ul) 2ul, Primer6 (10pmol/ul) 2ul, 10xBuffer (MgCl ₂ free) 2ul, dNTP (10mM) 0.4ul, MgCl ₂ (25mM) 1.2ul , rTaq (5u/ul) 0.4ul, ddH ₂ O 10ul, total volume 20ul. The amplification reaction was carried out on a PTC-200 (MJ Research Inc.) PCR instrument: 94°C for 3min; 94°C for 30sec, 55°C for 45sec, 72°C for 1min, 35 cycles; 72°C for 5min.

The PCR product was purified and recovered with a kit (Beijing Tiangen Company). PCR products were detected by 1% agarose electrophoresis. (N22) plants.

2. Phenotype identification

Transgenic EH-pCAMBIA1390-OsDOG1L3(N22) plants of the T ₀ generation, Nanjing 35 and N22 were planted at the Rice Experimental Station of Nanjing Agricultural University, and the transgenic plants 35 days after heading were harvested to identify the dormant phenotype. The germination rate of Nanjing 35 plants was about 97%, and the germination rate of transgenic plants transferred to EH-pCAMBIA1390-OsDOG1L3(N22) was separated, among which the germination rate of Y2906-1-5 was about 13%, and that of Y2907-1-9 The germination rate was about 39% (Figure 3).

Claims (10)

1. an adjusting and controlling rice seed dormant gene OsDOG1L3, it is characterised in that: deriving from Oryza sativa L., its nucleotide sequence is such as Shown in SEQ ID NO.1.

2. the albumen of claim 1 coding, it is characterised in that: aminoacid sequence is as shown in SEQ ID NO.3.

3. the gene of albumen described in coding claim 2, it is characterised in that: it is selected from following 1) or 2) or 3) or 4) DNA divide Son:

1) DNA molecular shown in SEQ ID NO.1；

2) DNA molecular shown in SEQ ID NO.2；

3) under strict conditions with 1) or 2) the DNA sequence hybridization that limits and the DNA molecular of encoding said proteins；

4) with 1) or 2) or 3) DNA sequence that limits has more than 90% homology, and coding rice paddy seed dormant trait albumen DNA molecular.

4. contain the recombinant expression carrier of gene described in claim 1 or 3, expression cassette, transgenic cell line or recombinant bacterium.

Recombinant expression carrier the most according to claim 4, it is characterised in that: described recombinant expression carrier be The restructuring that gene described in claim 1 or 3 obtains is inserted between Kpn I and the Spe I double enzyme site of pCAMBIA1390 carrier Plasmid.

6. the total length of gene described in amplification claim 1 and the primer pair of any fragment thereof, preferably Primer1/Primer2 and Primer3/Primer4；Described Primer1 sequence as shown in SEQ ID NO.3, described Primer2 sequence such as SEQ ID Shown in NO.4, described Primer3 sequence as shown in SEQ ID NO.5, described Primer4 sequence such as SEQ ID NO.6 institute Show.

7. gene described in claim 1 or 3, albumen described in claim 2, recombinant expression carrier, expression described in claim 4 At least one in box, transgenic cell line or recombinant bacterium application in plant breeding.

8. the method cultivating rice paddy seed appropriateness dormant trait, is without dormancy Oryza sativa L. by channel genes described in claim 1 or 3 In kind, obtain the transgenic paddy rice that dormant trait strengthens；The germination percentage of described rice varieties without the dormancy Oryza sativa L. close to 100%； The germination percentage of the transgenic paddy rice that described dormant trait the strengthens transgenic plant less than 80%.

Method the most according to claim 7, it is characterised in that: gene described in claim 1 or 2 passes through claim 4 or 5 Described recombinant expression carrier imports without in dormancy Oryza sativa L..

10. cultivate the method for transgenic plant that Grain Dormancy strengthens, be in process LAN purpose plant claim 1 or Gene described in 3, obtains the transgenic plant that dormant trait strengthens；Described purpose plant is to carry gene described in claim 1 or 3 Plant.