Application of rice JAZ protein gene OsJAZ12 in pre-harvest sprouting resistance

Abstract

The invention provides application of a rice JAZ protein gene OsJAZ12 in pre-harvest sprouting resistance, a nucleotide sequence of the rice JAZ protein gene OsJAZ12 is shown in a sequence table SEQ ID NO.1, an amino acid sequence of the rice JAZ protein gene OsJAZ12 is shown in a sequence table SEQ ID NO.2, the rice JAZ protein gene OsJAZ12 mutant can be applied to rice pre-harvest sprouting resistance, and the possibility of sprouting in the rice seed dormancy period is reduced.

Description

Technical Field

[0001] The present invention relates to the field of seed biotechnology, and in particular to application of rice JAZ protein gene OsJAZ12 in anti-ear sprouting. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops. Seed dormancy refers to the inability of viable seeds to germinate under suitable conditions. Weak dormancy can lead to pre-harvest germination, known as panicle sprouting. Panicle sprouting severely impacts seed quality and yield. The rice JAZ family comprises 15 members, all of which possess highly conserved NT, TIFY, and Jas domains. While reports primarily focus on their involvement in regulating rice spikelet development, little research has yet to investigate the molecular mechanisms by which JAZ proteins regulate seed dormancy. Summary of the Invention

[0003] The main purpose of the present invention is to provide an application of rice JAZ protein gene OsJAZ12 in anti-ear sprouting.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is: a method for preparing a rice JAZ protein gene OsJAZ12 mutant, which specifically comprises the following steps:

[0005] Step 1: Obtain the rice JAZ protein gene OsJAZ12, the nucleotide sequence of the rice JAZ protein gene OsJAZ12 is shown in the sequence listing SEQ ID NO.1, and the amino acid sequence is shown in the sequence listing SEQ ID NO.2;

[0006] Step 2: Design the target site and primers based on the nucleotide sequence and amino acid sequence obtained in step 1, perform PCR amplification using pCBC-MT1T2 as a template, purify and recover the PCR product to obtain the MT1T2-PCR vector. The target site sequences are shown in the sequence listing as SEQ ID NO.3 and SEQ ID NO.4, and the primer structures are shown in the sequence listing as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8;

[0007] Step 3: Digest the pHUE411 vector with BsaI, and construct the MT1T2-PCR vector gel recovery product obtained in step 2 onto the pHUE411 vector using homologous recombination to obtain the pHUE411+MT1T2-PCR vector;

[0008] Step 4: transforming the plasmid of pHUE411+MT1T2-PCR vector obtained in step 3 into Agrobacterium; and transforming the Agrobacterium carrying the transformation plasmid into rice;

[0009] Step 5: Screening and identifying the rice mutants obtained in step 4.

[0010] The present invention also provides a rice JAZ protein gene OsJAZ12 mutant, which is prepared by the preparation method.

[0011] The present invention also provides an application of a rice JAZ protein gene OsJAZ12 mutant in ear sprout resistance.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention isolated and cloned the OsJAZ12 gene from rice, and demonstrated for the first time that the gene is involved in regulating rice seed dormancy by constructing a CRISPR / Cas9 mutant.

[0014] The present invention provides a basis for screening rice varieties resistant to ear sprouting and also provides important gene resources for improving rice ear sprouting, which is of great significance to production.

[0015] Therefore, the use of the JAZ protein gene OsJAZ12, which is involved in the regulation of rice seed dormancy, can help screen and cultivate rice varieties resistant to panicle germination, which is of great significance to rice production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the expression of rice OsJAZ12 gene at different times after spikelet filling;

[0017] Figure 2 This is a photo of the panicle germination of the rice OsJAZ12 mutant material;

[0018] Figure 3 This is the germination status of rice OsJAZ12 mutant and wild type seeds. DETAILED DESCRIPTION

[0019] The following description is intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are merely examples, and those skilled in the art may conceive of other obvious variations.

[0020] Example 1

[0021] Using the japonica rice variety Zhonghua 11, seeds were collected 5, 10, 15, 20, 25, 30, and 35 days after grain filling. The seeds were frozen in liquid nitrogen and quickly ground into powder. The samples were stored at -80°C. The experiment was repeated three times.

[0022] RNA of each sample was extracted using 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 OsJAZ12 was detected by fluorescence quantitative PCR. The upstream primer sequence 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. The rice internal reference gene OsActin primer was used. The sequence of the upstream primer is shown in the sequence listing SEQ ID NO.13, and the sequence of the downstream primer is shown in the sequence listing SEQ ID NO.14. The results showed that the expression level of the OsJAZ12 gene showed a trend of first increasing and then decreasing during seed development ( Figure 2 The OsJAZ12 gene reached its highest expression level 25 days after grain filling. This suggests that OsJAZ12 regulates spikelet development and thus influences seed dormancy. The nucleotide sequence of the rice OsJAZ12 gene is shown in SEQ ID NO. 1, and its amino acid sequence is shown in SEQ ID NO. 2.

[0023] Example 2

[0024] This example is used to construct a mutant of the rice JAZ protein gene OsJAZ12, and specifically includes the following steps:

[0025] Step 1: obtaining the nucleotide sequence and amino acid sequence of the rice JAZ protein gene OsJAZ12;

[0026] Step 2: Design a target and primers based on the nucleotide sequence and amino acid sequence obtained in step 1, perform PCR amplification using pCBC-MT1T2 as a template, purify and recover the PCR product, and obtain an MT1T2-PCR vector. The target sequence is as shown in the sequence listing as SEQ ID NO.3 and SEQ ID NO.4, and the primer structure is as shown in the sequence listing as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, wherein the upstream primer and downstream primer corresponding to the target of SEQ ID NO.3 in the sequence listing correspond to SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing, respectively, and the upstream primer and downstream primer corresponding to the target of SEQ ID NO.4 in the sequence listing correspond to SEQ ID NO.7 and SEQ ID NO.8 in the sequence listing, respectively;

[0027] Step 3: Digest the pHUE411 vector with BsaI, and construct the MT1T2-PCR vector gel recovery product obtained in step 2 onto the pHUE411 vector using homologous recombination to obtain the pHUE411+MT1T2-PCR vector;

[0028] Step 4: transforming the plasmid of pHUE411+MT1T2-PCR vector obtained in step 3 into Agrobacterium; and transforming the Agrobacterium carrying the transformation plasmid into rice;

[0029] Step 5: Screen and identify the rice mutants obtained in step 4. The upstream primer sequence used to screen homozygous mutants is shown in the sequence listing as SEQ ID NO. 9, and the downstream primer sequence is shown in the sequence listing as SEQ ID NO. 10. The rice panicle germination phenotype is also identified.

[0030] Example 3

[0031] The mutants obtained in Example 2 were subjected to phenotypic analysis.

[0032] The CRISPR / Cas9-generated OsJAZ12 mutants OsJAZ12-1 and OsJAZ12-2, as well as the wild-type Zhonghua 11 (WT) rice variety, were subjected to panicle germination. The specific method was as follows: Healthy, plump, fresh panicles were selected for each replication, placed in a germination box, filled with distilled water to a depth of 2.5 cm, and incubated at 25°C for 5 and 7 days with 12 hours of light and 12 hours of darkness, respectively. Germination rates were then calculated, and the experiment was repeated three times. The results showed that compared to the wild-type, the mutant OsJAZ12 gene significantly reduced seed germination rates ( Figure 1 ). It can be seen that this gene mutation plays an important role in reducing rice panicle germination.

[0033] 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 merely illustrate the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of the rice JAZ protein gene OsJAZ12 in screening or breeding rice varieties resistant to panicle sprouting. The nucleotide sequence of the rice OsJAZ12 gene is shown in the sequence listing as SEQ ID NO.1, and the amino acid sequence is shown in the sequence listing as SEQ ID NO.

2.

2. Application of a rice JAZ protein gene OsJAZ12 mutant in promoting seed dormancy and improving rice panicle germination resistance. The nucleotide sequence of the rice OsJAZ12 gene is shown in the sequence listing as SEQ ID NO.1, and the amino acid sequence is shown in the sequence listing as SEQ ID NO.2.

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