Rice enolase gene OsENO and application thereof

Abstract

The invention provides a rice enolase gene OsENO and application thereof. The nucleotide sequence of the gene OsENO is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the gene OsENO is shown as SEQ ID NO.2 in the sequence table. The gene OsENO can be applied to rice so as to improve the water logging resistance of the rice.

Description

Technical Field

[0001] The invention relates to the field of seed biotechnology, in particular to a rice enolase gene OsENO and an application thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world and is widely planted in southern China. Compared with traditional manual transplanting, direct-seeding rice cultivation has the advantages of reducing labor intensity, reducing labor time, and promoting mechanization of rice production. With the large-scale transfer of rural labor in my country, direct-seeding rice has developed rapidly. However, direct-seeding rice faces problems such as low seedling rate, serious weeds, and easy lodging in the middle and late stages of production, which may lead to yield losses. Enolase (ENO) participates in the glycolysis pathway and is an important factor in regulating carbohydrate metabolism and energy. There are no reports on the regulation of rice flooding tolerance by enolase genes, nor on the application of enolase genes in screening and breeding flood-tolerant rice varieties. Summary of the invention

[0003] The main purpose of the present invention is to provide a rice enolase gene OsENO, which is applied in rice to provide flooding resistance of rice.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is: a rice enolase gene OsENO, characterized in that its nucleotide sequence is shown in the sequence list SEQ ID NO.1, and its amino acid sequence is shown in the sequence list SEQ ID NO.2.

[0005] The present invention also provides an application of a rice enolase gene OsENO in improving the waterlogging resistance of rice varieties, characterized in that its nucleotide sequence is shown in the sequence table SEQ ID NO.1, and its amino acid sequence is shown in the sequence table SEQ ID NO.2.

[0006] The present invention also provides a method for constructing a rice enolase gene OsENO mutant, which specifically comprises the following steps:

[0007] Step 1, obtaining a rice enolase gene OsENO, whose nucleotide sequence is shown in the sequence list SEQ ID NO.1, and whose amino acid sequence is shown in the sequence list SEQ ID NO.2;

[0008] Step 2: Select the target and design the primer according to the target;

[0009] Step 3: Perform PCR amplification using pCBC-MT1T2 as a template, purify and recover the PCR product to obtain the MT1T2-PCR vector;

[0010] Step 4: Digest the pHUE411 vector with BsaI, and construct the MT1T2-PCR gel recovery product onto the pHUE411 vector using homologous recombination to obtain the pHUE411+MT1T2-PCR vector;

[0011] Step 5: using the obtained plasmid containing pHUE411+MT1T2-PCR vector to transform into Agrobacterium; and transforming the Agrobacterium carrying the transformation plasmid into rice;

[0012] Step 6: Sequencing the PCR amplified product and comparing it with the wild-type gene sequence to screen homozygous mutants. The upstream primer sequence used is shown in the sequence listing SEQ ID NO.11, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.12.

[0013] The present invention also provides a method for constructing an overexpression material of the rice enolase gene OsENO, which specifically comprises the following steps:

[0014] Step 1, obtaining a rice enolase gene OsENO, whose nucleotide sequence is shown in the sequence list SEQ ID NO.1, and whose amino acid sequence is shown in the sequence list SEQ ID NO.2;

[0015] Step 2: Perform PCR amplification using rice cDNA as a template, purify and recover the PCR product to obtain the OsENO CDS fragment of the gene, wherein the upstream primer sequence of the PCR is shown in the sequence listing SEQ ID NO.3, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.4;

[0016] Step 3: amplify the CDS fragment of the OsENO gene obtained in step 2 by PCR using primers with homologous recombination adapters, purify and recover the PCR product, and obtain a CDS fragment with homologous recombination adapters;

[0017] Step 4: construct the CDS fragment with homologous recombination linker obtained in step 3 into the pCAMBIA1300 vector to obtain a recombinant plasmid;

[0018] Step 5, transferring the recombinant plasmid obtained in step 4 into Agrobacterium, and then transferring the Agrobacterium into rice;

[0019] Step 6: Screen positive overexpression materials by amplifying the hygromycin resistance tag using PCR. The upstream primer sequence used is shown in the sequence listing as SEQ ID NO.15, and the downstream primer sequence is shown in the sequence listing as SEQ ID NO.16.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention isolated and cloned the OsENO gene from rice, and demonstrated for the first time that the gene was involved in regulating the flooding tolerance of rice seeds by constructing a CRISPR / Cas9 mutant;

[0022] (2) The present invention provides a basis for screening flood-tolerant rice varieties and also provides important gene resources for improving the flood tolerance of rice seeds, which is of great significance to production;

[0023] In conclusion, the use of enolase OsENO, which is involved in the regulation of rice seed flooding tolerance, can help screen and breed flooding-tolerant rice varieties, and is of great significance to direct-seeded rice production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 , a bar graph showing the expression of rice OsENO gene at different flooding times;

[0025] Figure 2 , Actual photos of the performance of rice OsENO mutants and overexpressing materials under flooding conditions. DETAILED DESCRIPTION

[0026] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0027] Embodiment 1

[0028] The cDNA of indica rice variety Zhonghua 11 was used as a template for PCR to clone the sequence of gene OsENO, the upstream primer sequence of PCR is shown in the sequence list SEQ ID NO.3, and the downstream primer sequence is shown in the sequence list SEQ ID NO.4. The nucleotide sequence and amino acid sequence of rice OsENO gene were obtained, the nucleotide sequence is shown in the sequence list SEQ ID NO.1, and the amino acid sequence is shown in the sequence list SEQ ID NO.2.

[0029] Analysis of OsENO gene expression in rice seeds flooded at different times:

[0030] Using the indica rice variety Zhonghua 11, 15 healthy and full seeds were selected each time, and the surface was disinfected with 0.1% mercuric chloride solution for 5 minutes, rinsed with distilled water 3 times, and the surface of the seeds was wiped dry, placed in a test tube, and 8 cm deep distilled water was added. It was placed under 25°C conditions and cultured for 12 hours in light / darkness. After flooding for 0, 12, 24, 48, 60, 72, and 96 hours, samples were taken. The samples were frozen with liquid nitrogen and quickly ground into powder, and the samples were stored at -80°C. The experiment was repeated 3 times.

[0031] RNA from each sample was extracted using the TransZol Plant kit (Transgen, www.transgen.com); II Reverse Transcriptase system (Vazyme Biotech Co., Ltd) kit was used to reverse transcribe the cDNA, which was used as a template; fluorescence quantitative PCR was used for analysis, and the primer sequence of OsENO was detected by fluorescence quantitative PCR. The upstream primer sequence is shown in the sequence list SEQ ID NO.17, and the downstream primer sequence is shown in the sequence list SEQ ID NO.18. The primers for the rice internal reference gene OsActin were used. The sequence of the upstream primer is shown in the sequence list SEQ ID NO.19, and the sequence of the downstream primer is shown in the sequence list SEQ ID NO.20. The results showed that the expression level of the OsENO gene showed an upward trend during the seed flooding process. The expression of the OsENO gene was the highest when the seeds were flooded for 60-72 hours. Figure 1 It can be seen that the gene is induced to express during the seed flooding process, and gene expression plays an important role in improving the flooding tolerance of seeds.

[0032] Embodiment 2

[0033] This example is to construct a mutant of OsENO CRISPR / Cas9, which specifically includes the following steps:

[0034] Step 1, log in to the website http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html, screen the target, the target sequence is such as SEQ ID NO.5 and SEQ ID NO.6 in the sequence list, design primers based on the target sequence, the primer structure corresponding to SEQ ID NO.5 is such as SEQ ID NO.7 and SEQ ID NO.8 in the sequence list; the primer structure corresponding to SEQ ID NO.6 is such as SEQ ID NO.9 and SEQ ID NO.10 in the sequence list, use pCBC-MT1T2 as a template for PCR amplification, purify and recover the PCR product, and obtain the MT1T2-PCR vector;

[0035] Step 2: Digest the pHUE411 vector with BsaI, and construct the MT1T2-PCR gel recovery product onto the pHUE411 vector using homologous recombination to obtain the pHUE411+MT1T2-PCR vector;

[0036] Step 3: using the obtained plasmid containing pHUE411+MT1T2-PCR vector to transform into Agrobacterium; using the Agrobacterium carrying the transformation plasmid to transform the wild-type indica rice variety Zhonghua 11;

[0037] Step 4: Sequencing the PCR amplified product and comparing it with the wild-type gene sequence to screen homozygous mutants. The upstream primer sequence used is shown in the sequence listing SEQ ID NO.11, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.12.

[0038] Embodiment 3

[0039] This embodiment is to construct an overexpression material of the gene OsENO, which specifically includes the following steps:

[0040] Step 1, using rice cDNA as a template for PCR amplification, purifying and recovering the PCR product to obtain the OsENO CDS gene fragment, wherein the upstream primer sequence of the PCR is shown in the sequence listing SEQ ID NO.3, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.4;

[0041] Step 2: amplify the CDS fragment of the OsENO gene obtained in step 1 by PCR using primers with homologous recombination adapters, purify and recover the PCR products, and obtain a CDS fragment with homologous recombination adapters;

[0042] Step 3: construct the CDS fragment with homologous recombination linker obtained in step 2 into the pCAMBIA1300 vector to obtain a recombinant plasmid;

[0043] Step 4: transferring the recombinant plasmid obtained in step 3 into Agrobacterium, and then transferring the Agrobacterium into rice;

[0044] Step 5: Screening positive overexpression materials by PCR amplification of hygromycin resistance tag, the upstream primer sequence used is shown in the sequence listing SEQ ID NO.15, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.16.

[0045] Embodiment 4

[0046] The constructed OsENO CRISPR / Cas9 mutants oseno-1 and oseno-2 were used to overexpress OE-1, OE-2 and the wild-type Zhonghua 11 (WT) rice variety for flooding tolerance test. The specific method is as follows: 15 healthy and plump seeds were selected for each repetition, the surface was disinfected with 0.1% mercuric chloride solution for 5 minutes, and the seeds were rinsed with distilled water 3 times. The surface of the seeds was wiped dry and placed in a test tube. 8 cm deep distilled water was added and cultured at 25°C for 12 hours in light / darkness for 5 days. Finally, the length of the coleoptile was counted. The experiment was repeated 3 times. The results showed that compared with the wild type, the mutant OsENO gene significantly reduced the length of the coleoptile under seed flooding conditions, and overexpression of the OsENO gene could significantly increase the length of the coleoptile under seed flooding conditions, such as Figure 2It can be seen that this gene plays an important role in improving seed flooding tolerance.

[0047] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A rice enolase gene OsENO, characterized in that: The nucleotide sequence is shown in the sequence list as SEQ ID NO.1, and the amino acid sequence is shown in the sequence list as SEQ ID NO.

2.

2. An application of a rice enolase gene OsENO in improving the flooding resistance of rice seeds, characterized in that: The nucleotide sequence is shown in the sequence list as SEQ ID NO.1, and the amino acid sequence is shown in the sequence list as SEQ ID NO.

2.

3. A method for constructing a rice enolase gene OsENO mutant, comprising the following steps: Step 1, obtaining a rice enolase gene OsENO, whose nucleotide sequence is shown in the sequence list SEQ ID NO.1, and whose amino acid sequence is shown in the sequence list SEQ ID NO.2; Step 2: selecting a target site based on the obtained rice enolase gene OsENO, and designing primers based on the target site; Step 3: Perform PCR amplification using pCBC-MT1T2 as a template, purify and recover the PCR product to obtain the MT1T2-PCR vector; Step 4: Digest the pHUE411 vector with BsaI, and construct the MT1T2-PCR gel recovery product onto the pHUE411 vector using homologous recombination to obtain the pHUE411+MT1T2-PCR vector; Step 5: using the obtained plasmid containing pHUE411+MT1T2-PCR vector to transform into Agrobacterium; and transforming the Agrobacterium carrying the transformation plasmid into rice; Step 6: Sequencing the PCR amplified product and comparing it with the wild-type gene sequence to screen homozygous mutants. The upstream primer sequence used is shown in the sequence listing SEQ ID NO.11, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.

12.

4. A method for constructing an overexpression material of rice enolase gene OsENO, comprising the following steps: Step 1, using rice cDNA as a template for PCR amplification, purifying and recovering the PCR product to obtain the OsENO CDS gene fragment, wherein the upstream primer sequence of the PCR is shown in the sequence listing SEQ ID NO.3, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.4; Step 2: amplify the CDS fragment of the OsENO gene obtained in step 2 by PCR using primers with homologous recombination adapters, purify and recover the PCR products, and obtain a CDS fragment with homologous recombination adapters; Step 3: construct the CDS fragment with homologous recombination linker obtained in step 3 into the pCAMBIA1300 vector to obtain a recombinant plasmid; Step 4: transferring the recombinant plasmid obtained in step 4 into Agrobacterium, and then transferring the Agrobacterium into rice; Step 5: Screening positive overexpression materials by PCR amplification of hygromycin resistance tag, the upstream primer sequence used is shown in the sequence listing SEQ ID NO.15, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.16.

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