Application of rice DNA-binding single zinc finger protein OsDOF22 gene in improving primary root length of direct seeding rice

Abstract

The application discloses a DNA binding single-zinc finger protein OsDOF22 The application discloses a DNA binding single-zinc finger protein OsDOF22 The nucleotide sequence is shown as SEQ ID NO. 1, and the corresponding protein amino acid sequence is shown as the sequence table SEQ ID NO. 2. The application reports for the first time that OsDOF22 The gene can regulate the growth of primary roots in the germination stage, and experiments show that the mutation of the gene reduces the length of the primary roots of rice, and the overexpression of the gene improves the root growth ability of rice in the germination stage. It is proved that the application OsDOF22 The gene regulates the length of the primary roots of rice in the germination stage, and the use of the gene is helpful to screening and cultivating high-activity rice varieties and is beneficial to the direct seeding rice production.

Description

Technical Field

[0001] This invention relates to the field of seed biotechnology, and in particular to the application of the rice DNA-binding single zinc finger protein OsDOF22 gene in improving the root length of primary roots in direct-seeded rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of the oldest cultivated grain crops in my country. In recent years, with economic development and a growing shortage of rural labor, direct-seeded rice production has become increasingly common. The taproot, as the primary root system formed after seed germination, plays a crucial role in the early development of rice seedlings. Good taproot development anchors the seedling, provides mechanical support, promotes water and nutrient absorption, and ensures the initial energy needs of the seedling. DNA-binding single zinc finger proteins are important transcription factors regulating plant growth and development. However, there are no reports on the regulation of primary root elongation in rice using the OsDOF22 gene, nor on its application in screening and breeding suitable direct-seeded rice varieties. Summary of the Invention

[0003] The main objective of this invention is to provide an application of the rice DNA-binding single zinc finger protein OsDOF22 gene in improving the root length of primary roots in direct-seeded rice. The OsDOF22 gene is applied to direct-seeded rice to increase the root length of primary roots.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The application of the rice DNA-binding single zinc finger protein OsDOF22 gene in improving the root length of primary roots in direct-seeded rice, wherein the nucleotide sequence of the OsDOF22 gene is shown in SEQ ID NO.1 and its amino acid sequence is shown in SEQ ID NO.2.

[0005] Compared with the prior art, the present invention has the following beneficial effects: This invention has discovered that the OsHSL1 gene, when applied to direct-seeded rice, can increase the root length of primary roots in direct-seeded rice. Attached Figure Description

[0006] picture Figure 1 Expression of the rice OsDOF22 gene at different time points; Figure 2 The root length of primary roots in rice Osdof22 mutant materials; Figure 3 The expression of primary root growth in rice OsDOF22 overexpression material. Detailed Implementation

[0007] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0008] Example 1 The application of the rice DNA-binding single zinc finger protein OsDOF22 gene in improving the root length of primary roots in direct-seeded rice, wherein the nucleotide sequence of the OsDOF22 gene is shown in SEQ ID NO.1 and its amino acid sequence is shown in SEQ ID NO.2.

[0009] Example 2 In this embodiment, the OsDOF22 gene sequence was cloned by PCR using cDNA from the japonica rice variety Nipponbare as a template. The upstream primer sequence for PCR is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4. The nucleotide and amino acid sequences of the rice OsDOF22 gene were obtained, and the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0010] Example 3 This embodiment analyzes the expression of the OsDOF22 gene at different time points during seed germination, and specifically includes the following steps: Step 1: Using the Japanese rice variety Nipponbare, select 30 healthy and plump seeds each time. Step 2: Disinfect the selected seeds with a 3% hydrogen peroxide solution for 5 minutes, rinse them 3 times with distilled water, and wipe the seed surface dry. Step 3: Place in a 9 cm petri dish, add 10 ml of distilled water, and incubate at 25 ℃ under light / dark conditions for 12 h each. Take samples after 0, 6, 12, 24, 36, 48, 60 and 72 h. Step 4: After each sample of seeds is frozen in liquid nitrogen, it is quickly ground into powder and stored at -80℃. Step 5: Repeat the above steps 3 times; Step Six: Analyze samples from different time points: Extract RNA from each sample using the TransZol Plant kit (Transgen, www.transgen.com); use HiScript... II. The Reverse Transcriptase System (Vazyme Biotech Co., Ltd) kit was used to reverse transcribe cDNA, which was then used as a template. Analysis was performed using quantitative real-time PCR. The primer sequences for OsDOF22 were detected by quantitative real-time PCR. The upstream primer sequence is shown in SEQ ID NO. 17, and the downstream primer sequence is shown in SEQ ID NO. 18. Primers for the rice internal reference gene OsActin were also used. The upstream primer sequence is shown in SEQ ID NO. 19, and the downstream primer sequence is shown in SEQ ID NO. 20.

[0011] The results showed that during germination, the expression level of the OsDOF22 gene gradually increased from 0 to 24 hours, reaching its peak at 24 hours, and then gradually decreased. Figure 1 As shown in the figure, this gene is induced to express during the early germination process of seeds, and gene expression plays an important role in the growth of primary roots.

[0012] Example 4 This embodiment is used to construct mutants, and specifically includes the following steps: Step 1: Log in to the website http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html, filter for targets, and the target sequences are shown in SEQ ID NO.5 and SEQ ID NO.6 of the sequence listing; Step 2: Design primers based on the target sequence. The primer structures are shown in the sequence list SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10. Step 3: Perform four-primer PCR amplification using pCBC-MT1T2 as a template, and purify and recover the PCR product after amplification to obtain the MT1T2-PCR vector. Step 4: Digest the pHUE411 vector with BsaI enzyme, and construct the MT1T2-PCR gel-recovered product into the pHUE411 vector using homologous recombination method to obtain the pHUE411+MT1T2-PCR vector. Step 5: Transform Agrobacterium into the obtained plasmid containing the pHUE411+MT1T2-PCR vector; transform the Agrobacterium with the transformation plasmid into the wild-type japonica rice variety Nippon Harunaka; sequence the PCR amplification products and compare them with the wild type to screen for homozygous mutants. The upstream primer sequence is shown in SEQ ID NO.11 of the sequence listing, and the downstream primer sequence is shown in SEQ ID NO.12 of the sequence listing.

[0013] Example 5 This embodiment is used to construct an overexpression seed for the OsDOF22 gene, and specifically includes the following steps: Step 1: PCR amplification was performed using rice cDNA as a template. The PCR product was purified and recovered to obtain the CDS fragment of the OsDOF22 gene. The upstream primer sequence of the PCR is shown in SEQ ID NO.3 of the sequence listing, and the downstream primer sequence is shown in SEQ ID NO.4 of the sequence listing. Step 2: Using the obtained OsDOF22 gene CDS fragment as a template, PCR amplification is performed using primers with homologous recombination adapters. The PCR product is purified and recovered to obtain a CDS fragment with homologous recombination adapters. The upstream primer sequence of the PCR is shown in SEQ ID NO.13 of the sequence listing, and the downstream primer sequence is shown in SEQ ID NO.14 of the sequence listing. Step 3: Using homologous recombination, the target fragment carrying the OsDOF22 gene is constructed into the pCAMBIA35S-4×Myc-CMV-3×FLAG vector to obtain the recombinant plasmid. Step 4: Transform the recombinant plasmid into Agrobacterium, and then transform the Agrobacterium carrying the transformation plasmid into the wild-type japonica rice variety Nipponbare. Use PCR to amplify the hygromycin resistance tag to screen for positive overexpression seeds. The upstream primer sequence is shown in SEQ ID NO.15 of the sequence listing, and the downstream primer sequence is shown in SEQ ID NO.16 of the sequence listing.

[0014] Example 6 This example is used to analyze the gene mutants obtained in Example 4 and the positive overexpression gene materials obtained in Example 5.

[0015] Using the constructed OsDOF22 CRISPR / Cas9 mutants OsDOF22-1, OsDOF22-2, and OsDOF22-3, and overexpressing them in seeds of OE-1, OE-2, and wild-type Nipponbare (WT) rice varieties, a primary root length identification experiment was conducted. The specific methods are as follows: Sixty healthy, plump seeds were selected for each trial. The seeds were surface-sterilized with 3% hydrogen peroxide solution for 5 min, rinsed three times with distilled water, dried, and placed in 9 cm petri dishes. 10 L of distilled water was added, and the dishes were incubated at 25 ℃ for 12 h light / dark for 24 h. Twenty-four seeds were collected, and the mutant and overexpression seeds were transferred to 96-well plates with the bottom cut off. These were treated at 25 ℃ for 12 h light / dark for 5 days. After 5 days, the seeds were removed from the culture medium, rinsed three times with distilled water, and root and seedling lengths were recorded. The experiment was repeated three times. The results showed that, compared with the wild type, the mutant OsDOF22 gene significantly reduced the length of the primary roots. Figure 2Overexpression of the OsDOF22 gene can significantly increase the length of primary roots. Figure 3 It is evident that this gene plays a crucial role in increasing the length of primary roots in seeds.

[0016] The foregoing has shown and described 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 embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. Application of the rice DNA-binding single zinc finger protein OsDOF22 gene in improving the root length of primary roots in direct-seeded rice, wherein the nucleotide sequence of the OsDOF22 gene is shown in SEQ ID NO.1 and its amino acid sequence is shown in SEQ ID NO.2.

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