Rice cytochrome b5 gene OsCyb5 and application thereof

Abstract

The invention provides a rice cytochrome b5 gene OsCyb5 and application thereof, a nucleotide sequence of the rice cytochrome b5 gene OsCyb5 is shown as a sequence table SEQ ID NO.1, an amino acid sequence of the rice cytochrome b5 gene OsCyb5 is shown as a sequence table SEQ ID NO.2, and the cytochrome b5 gene OsCyb5 is applied to rice to improve the salt tolerance of rice seeds.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a rice cytochrome b5 gene OsCyb5 and its application. Background Art

[0002] Rice (Oryza sativa L.) is one of the food crops with the longest cultivation history in China. In recent years, with the development of the economy, the rural labor force has become increasingly scarce, and the production of direct-seeded rice has become more and more common. Rice seeds are relatively sensitive to salt damage during the germination stage. Under salt stress, problems such as low germination rate, poor seedling establishment rate, and weak growth of seedlings will occur, seriously affecting the yield of direct-seeded rice. Cytochrome is an important factor regulating plant growth and development. There is no report on the regulation of rice salt tolerance by cytochrome b5 gene, and there is also no report on the application of using cytochrome b5 gene to screen and cultivate salt-tolerant rice varieties. Summary of the Invention

[0003] The main purpose of the present invention is to provide a rice cytochrome b5 gene OsCyb5 and its application. Applying OsCyb5 to rice can improve the salt tolerance of rice seeds.

[0004] To achieve the above object, the technical solution adopted by the present invention is: a rice cytochrome b5 gene OsCyb5, characterized in that its nucleotide sequence is as shown in SEQ ID NO.1 in the sequence listing, and its amino acid sequence is as shown in SEQ ID NO.2 in the sequence listing.

[0005] The present invention also provides an application of a rice cytochrome b5 gene OsCyb5 in improving the salt tolerance of rice seeds during the germination stage, characterized in that the nucleotide sequence of the rice cytochrome b5 gene OsCyb5 is as shown in SEQ ID NO.1 in the sequence listing, and the amino acid sequence is as shown in SEQ ID NO.2 in the sequence listing.

[0006] The present invention also provides a preparation method of a mutant based on the rice cytochrome b5 gene OsCyb5, characterized in that it specifically includes the following steps:

[0007] Step 1: Obtain the rice cytochrome b5 gene OsCyb5, the nucleotide sequence of the rice cytochrome b5 gene OsCyb5 is as shown in SEQ ID NO.1 in the sequence listing, and the amino acid sequence is as shown in SEQ ID NO.2 in the sequence listing;

[0008] Step 2: Select a target site and design primers according to the target site sequence;

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

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

[0011] Step 5: Transfer the plasmid containing the pHUE411 + MT1T2-PCR vector obtained into Agrobacterium, and then transfer the Agrobacterium carrying the transformed plasmid into wild-type rice;

[0012] Step 6: Use PCR amplification product sequencing to screen for homozygous mutants. The upstream primer sequence used for sequencing is as shown in SEQ ID NO.11 in the sequence listing, and the downstream primer sequence is as shown in SEQ ID NO.12 in the sequence listing.

[0013] The present invention also provides a method for constructing an overexpression material of rice cytochrome b5 gene OsCyb5, specifically including the following steps:

[0014] Step 1: Obtain the rice cytochrome b5 gene OsCyb5. The nucleotide sequence of the rice cytochrome b5 gene OsCyb5 is as shown in SEQ ID NO.1 in the sequence listing, and the amino acid sequence is as shown in SEQ ID NO.2 in the sequence listing;

[0015] Step 2: Use the rice cDNA as a template for PCR amplification, purify and recover the PCR product to obtain the OsCyb5 gene CDS fragment. The upstream primer sequence of the PCR is as shown in SEQ ID NO.3 in the sequence listing, and the downstream primer sequence is as shown in SEQ ID NO.4 in the sequence listing;

[0016] Step 3: Use the obtained OsCyb5 gene CDS fragment as a template, and perform PCR amplification with primers with homologous recombination adapters, purify and recover the PCR product to obtain a CDS fragment with homologous recombination adapters. The upstream primer sequence of the PCR is as shown in SEQ ID NO.13 in the sequence listing, and the downstream primer sequence is as shown in SEQ ID NO.14 in the sequence listing;

[0017] Step 4: Use homologous recombination to construct the target fragment with the OsCyb5 gene obtained in Step 3 onto the pCAMBIA1300-HA vector to obtain a recombinant plasmid;

[0018] Step 4: Transfer the recombinant plasmid into Agrobacterium, and transfer the Agrobacterium carrying the recombinant plasmid into wild-type rice;

[0019] Step 5. Use PCR amplification of the hygromycin resistance tag to screen positive overexpression materials. The upstream primer sequence used is as shown in SEQ ID NO.15 of the sequence listing, and the downstream primer sequence is as shown in SEQ ID NO.16 of the sequence listing.

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

[0021] The present invention applies the OsCyb5 gene to rice, which can improve the salt tolerance of rice seeds. Description of the Drawings

[0022] Figure 1 Expression of the rice OsCyb5 gene at different times under salt stress;

[0023] Figure 2 Performance of rice OsCyb5 mutants and overexpression materials after salt stress treatment at the bud stage. Detailed Embodiments

[0024] 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 in the following description are only examples, and those skilled in the art can think of other obvious variations.

[0025] Example 1: Cloning and Expression Analysis of the OsCyb5 Gene

[0026] Cloning of the OsCyb5 gene:

[0027] Using the cDNA of the japonica rice variety Nipponbare as a template, the sequence of the OsCyb5 gene was cloned by PCR. The upstream primer sequence of the PCR was as shown in SEQ ID NO.3 of the sequence listing, and the downstream primer sequence was as shown in SEQ ID NO.4 of the sequence listing. The nucleotide sequence and amino acid sequence of the rice OsCyb5 gene were obtained. Its nucleotide sequence was as shown in SEQ ID NO.1 of the sequence listing, and its amino acid sequence was as shown in SEQ ID NO.2 of the sequence listing.

[0028] Expression analysis of the OsCyb5 gene:

[0029] Using the japonica rice variety Nipponbare, 60 healthy and plump seeds were selected for each repetition, surface sterilized with 3% hydrogen peroxide solution for 5 min, rinsed 3 times with distilled water, dried the seed surface, placed in a 9 cm petri dish, added 10 ml of distilled water, and cultured at 25 °C with 12 h of light / dark for 48 h. Take 24 seeds with a bud length of 2 mm and no roots, place them on a 0.3% agar medium containing 120 mM NaCl, and sample at 0, 12, 18, 24, 48, and 72 h respectively. After the samples were frozen in liquid nitrogen, they were quickly ground into powder, and the samples were stored at -80 °C. The experiment was repeated 3 times.

[0030] Extract the RNA of the powder of each sample using the TransZol Plant kit (Transgen, www.transgen.com); use the II Reverse Transcriptase system (Vazyme Biotech Co., Ltd) kit to reverse transcribe into cDNA and use it as a template; perform analysis by fluorescence quantitative PCR. The primer sequences for detecting OsCyb5 by fluorescence quantitative PCR are as shown in SEQ ID NO.17 in the sequence listing for the upstream primer sequence and as shown in SEQ ID NO.18 in the sequence listing for the downstream primer sequence. Use the rice internal reference gene OsActin primers, with the sequence of the upstream primer as shown in SEQ ID NO.19 in the sequence listing and the sequence of the downstream primer as shown in SEQ ID NO.20 in the sequence listing. The results show that during the salt treatment at the bud stage, the expression level of the OsCyb5 gene shows an upward trend of change ( Figure 2 ), and the expression of the OsCyb5 gene is the highest at 18 h of seed salt treatment. It can be seen that this gene is induced to express during the seed bud stage salt treatment, and gene expression plays an important role in improving the salt tolerance of seeds.

[0031] Example 2: Construct an OsCyb5 CRISPR / Cas9 mutant, specifically using the following steps:

[0032] Step 1: Log in to the website http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html to screen for target sites. The target site sequences are as shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing. Design primers based on the target site sequences. The gRNA target site sequences (19bp target fragments in the OsCyb5 gene) of the CRISPR / Cas9 mutant are as shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing. The primer structures corresponding to SEQ ID NO.5 are as shown in SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing, and the primer structures corresponding to SEQ ID NO.6 are as shown in SEQ ID NO.9 and SEQ ID NO.10 in the sequence listing.

[0033] Step 2: Perform four-primer PCR amplification using pCBC-MT1T2 as a template, purify and recover the PCR product to obtain the MT1T2-PCR vector;

[0034] Step 3: Digest the pHUE411 vector with BsaI, and use homologous recombination to construct the MT1T2-PCR gel recovery product onto the pHUE411 vector to obtain the pHUE411 + MT1T2-PCR vector;

[0035] Step 4: Transfer the plasmid containing pHUE411 + MT1T2 - PCR vector obtained into Agrobacterium, and then transfer the Agrobacterium with the transformed plasmid into the wild - type japonica rice variety Nipponbare;

[0036] Step 5: Use PCR amplification product sequencing, compare with the wild - type, and screen for homozygous mutants. The upstream primer sequence used is as shown in SEQ ID NO.11 in the sequence listing, and the downstream primer sequence is as shown in SEQ ID NO.12 in the sequence listing.

[0037] Example 3: Construction of OsCyb5 gene over - expression material

[0038] Step 1: Perform PCR amplification using rice cDNA as a template, purify and recover the PCR product to obtain the CDS fragment of the OsCyb5 gene. The upstream primer sequence of the PCR is as shown in SEQ ID NO.3 in the sequence listing, and the downstream primer sequence is as shown in SEQ ID NO.4 in the sequence listing;

[0039] Step 2: Use the obtained CDS fragment of the OsCyb5 gene as a template, perform PCR amplification using primers with homologous recombination adapters, purify and recover the PCR product to obtain a CDS fragment with homologous recombination adapters. The upstream primer sequence of the PCR is as shown in SEQ ID NO.13 in the sequence listing, and the downstream primer sequence is as shown in SEQ ID NO.14 in the sequence listing;

[0040] Step 3: Use homologous recombination to construct the target fragment with the OsCyb5 gene obtained in Step 2 onto the pCAMBIA1300 - HA vector to obtain a recombinant plasmid;

[0041] Step 4: Transfer the recombinant plasmid into Agrobacterium, and transfer the Agrobacterium with the recombinant plasmid into the wild - type japonica rice variety Nipponbare;

[0042] Step 5: Use PCR amplification of the hygromycin resistance tag to screen for positive over - expression materials. The upstream primer sequence used is as shown in SEQ ID NO.15 in the sequence listing, and the downstream primer sequence is as shown in SEQ ID NO.16 in the sequence listing.

[0043] Example 4: Phenotypic analysis of gene mutants and over - expression transgenic materials

[0044] Using the constructed OsCyb5 CRISPR / Cas9 mutants OsCyb5-1, OsCyb5-2, OsCyb5-3, overexpression lines OE-1, OE-2 and the wild-type Nipponbare (WT) rice variety, a salt tolerance test at the germination stage was conducted. The specific method is as follows: Each repetition selected 60 healthy and plump seeds, surface-sterilized them with 3% hydrogen peroxide solution for 5 min, rinsed them 3 times with distilled water, dried the seed surface, placed them in a 9 cm Petri dish, added 10 ml of distilled water, and cultured them for 48 h under 12 h light / 12 h dark at 25°C. Take 24 seeds with a bud length of 2 mm and no root growth, place them on a 0.3% agar medium containing 120 mM NaCl, and treat them under 12 h light / 12 h dark at 25°C for 3 d. After 3 d, take out the seeds from the medium, rinse them 3 times with distilled water, transfer the seeds to a 96-well plate with cut edges, place them in rice nutrient solution for 7 d to recover, and count the survival rate, root length, and shoot length. The experiment was repeated 3 times. The results showed that compared with the wild type, the mutated OsCyb5 gene significantly reduced the root length after recovery from salt treatment of the seeds, and overexpression of the OsCyb5 gene could significantly increase the root length after recovery from salt treatment ( Figure 1 ). It can be seen that this gene plays an important role in improving the salt tolerance of seeds.

[0045] 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 by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A rice cytochrome b5 gene OsCyb5, 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 cytochrome b5 gene OsCyb5 in improving the salt tolerance of rice seeds at the germination stage, characterized in that: The nucleotide sequence of the rice cytochrome b5 gene OsCyb5 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.

3. A method for preparing a mutant based on rice cytochrome b5 gene OsCyb5, characterized in that: The specific steps include: Step 1, obtaining a rice cytochrome b5 gene OsCyb5, wherein the nucleotide sequence of the rice cytochrome b5 gene OsCyb5 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; Step 2: Select the target and design the primer according to the target sequence; Step 3: Perform four-primer 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: The obtained plasmid containing pHUE411+MT1T2-PCR vector is transferred into Agrobacterium, and then the Agrobacterium carrying the transformation plasmid is transferred into wild-type rice; Step 6: Sequencing the PCR amplified products to screen homozygous mutants. The upstream primer sequence used for sequencing is shown in the sequence listing as SEQ ID NO.11, and the downstream primer sequence is shown in the sequence listing as SEQ ID NO.

12.

4. A method for constructing a rice cytochrome b5 gene OsCyb5 overexpression material, comprising the following steps: Step 1, obtaining a rice cytochrome b5 gene OsCyb5, wherein the nucleotide sequence of the rice cytochrome b5 gene OsCyb5 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; Step 2, using the rice cDNA as a template to perform PCR amplification, purifying and recovering the PCR product to obtain the CDS fragment of the OsCyb5 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; Step 3: Using the obtained OsCyb5 gene CDS fragment as a template, performing PCR amplification using primers with homologous recombination adapters, purifying and recovering the PCR products to obtain a CDS fragment with homologous recombination adapters, wherein the upstream primer sequence of the PCR is shown in the sequence listing SEQ ID NO.13, and the downstream primer sequence is shown in the sequence listing SEQ ID NO.14; Step 4: Use homologous recombination to construct the target fragment with OsCyb5 gene obtained in step 3 into pCAMBIA1300-HA vector to obtain a recombinant plasmid; Step 4: Transform the recombinant plasmid into Agrobacterium, and then transform the Agrobacterium carrying the recombinant plasmid into wild-type rice; Step 5: 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.

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