Site-directed knockout system for rice TMS10 gene, and applications thereof

Abstract

The present invention relates to a site-directed knockout system for a rice TMS10 gene, and applications thereof. The site-directed knockdown system comprises a CRISPR / Cas9 system and a sgRNA target, wherein the sgRNA target is the sequence containing PAM or NGG in a rice male sterile gene TMS10, the CRISPR / Cas9 system is CC-TMS10-1, and the CC-TMS10-1 target sequence is a SEQ ID NO.1 sequence from site 2281 to site 2299. The applications comprise carrying out targeted knockout on the TMS10 genes of different japonica rice varieties and different Indica rice varieties by separately using CC-TMS10-1, wherein the experiment results prove that the heterozygous mutation transformation plant produced through the site-directed knockout induction in different rice varieties shows the temperature sensitive male sterility characteristic. According to the present invention, the site-directed knockout system provides the efficient breeding method for the creation of the temperature sensitive male sterile line germplasm resources based on the rice temperature-sensitive male sterile gene TMS10 and the rice hybridization breeding.

Description

technical field

[0001] The invention belongs to the technical field of rice breeding, and in particular relates to a fixed-point knockout system of rice male sterility gene TMS10 and its application in different rice varieties. Background technique

[0002] Male sterile lines provide key breeding materials for rice hybrid seed production technology, and extensive research has been carried out all over the world. Rice Thermo-sensitive male sterile (TMS10) gene encodes a leucine-rich repeat receptor kinase (LRR-RLK) involved in rice anther development, mainly expressed in the early stage of anther development. Mutations in the TMS10 gene can cause temperature-sensitive male sterility in rice, showing the characteristics of male sterility at high average temperature and restoration of fertility at low average temperature; in addition, the F1 generation produced by crossing the TMS10 sterile line and the restorer line JP69 It has heterosis. Therefore, the temperature-sensitive ...

Examples

Embodiment 1

[0036] Example 1, the sequence and analysis of the rice male sterility gene TMS10

[0037] The sequence of the rice male sterility gene TMS10 is shown in SEQ ID NO.1. Sequence analysis shows that the gene includes 11 exons in total, which are respectively the 235th-307th (first exon), the 2241-2373rd (second exon), the 2nd 2487-2558 (third exon), 3271-3414 (fourth exon), 3514-3585 (fifth exon), 3694-3759 (sixth exon) , 3846-3916 (the seventh exon), 4039-4179 (the eighth exon), 4590-4931 (the ninth exon), 5235-5626 (the tenth exon) sub), 5724-6038 (the eleventh exon).

[0038] The present invention uses the 2281st to 2299th nucleotide sequence on the second exon as the target sequence of the CRISPR / Cas9 system.

Embodiment 2

[0039] Example 2, CRISPR / Cas9 system primer design and construction of recombinant expression vector

[0040] 2.1 Selection of CRISPR / Cas9 system target sequence

[0041] The CRISPR / Cas9 system targets the positive-sense strand of the second exon of the rice TMS10 gene, and the target sequence is as follows: 5'-TGGCAATCAGCTTTCGGAT-3' (2281 to 2299 of the sequence of SEQ ID NO.1.

[0042] 2.2 Design and synthesis of target sequence primers for CRISPR / Cas9 system

[0043] Target sequence primers targeting the TMS10 gene were designed based on the CH-CRISPR / Cas9 system. The target sequence primers were as follows: Forward primer: 5'-TGGCGTGGCAATCAGCTTTCGGAT-3' (sequence shown in SEQ ID NO.2, CC-TMS10-1F) and Reverse primer: 5'-AAACATCCGAAAGCTGATTGCCAC-3' (sequence shown in SEQ ID NO.3, CC-TMS10-1R); synthesize the target sequence primer of the CRISPR / Cas9 system.

[0044] 2.3 Construction of CRISPR / Cas9 system recombinant expression vector

[0045] The sequence shown in SEQ ...

Embodiment 3

[0047] Example 3, Activity Detection of Recombinant Expression Vectors

[0048] The recombinant expression vector CC-TMS10-1 in Example 2 was introduced into rice protoplasts through PEG mediation to obtain the transient expression result of the recombinant expression vector CC-TMS10-1; after sequencing verification, the recombinant expression vector CC-TMS10-1 was obtained Peak map of site-directed mutation induced in rice protoplasts ( figure 1 ).

technical field

[0001] The invention belongs to the technical field of rice breeding, and in particular relates to a fixed-point knockout system of rice male sterility gene TMS10 and its application in different rice varieties. Background technique

[0002] Male sterile lines provide key breeding materials for rice hybrid seed production technology, and extensive research has been carried out all over the world. Rice Thermo-sensitive male sterile (TMS10) gene encodes a leucine-rich repeat receptor kinase (LRR-RLK) involved in rice anther development, mainly expressed in the early stage of anther development. Mutations in the TMS10 gene can cause temperature-sensitive male sterility in rice, showing the characteristics of male sterility at high average temperature and restoration of fertility at low average temperature; in addition, the F1 generation produced by crossing the TMS10 sterile line and the restorer line JP69 It has heterosis. Therefore, the temperature-sensitive ...

Examples

Embodiment 1

[0036] Example 1, the sequence and analysis of the rice male sterility gene TMS10

[0037] The sequence of the rice male sterility gene TMS10 is shown in SEQ ID NO.1. Sequence analysis shows that the gene includes 11 exons in total, which are respectively the 235th-307th (first exon), the 2241-2373rd (second exon), the 2nd 2487-2558 (third exon), 3271-3414 (fourth exon), 3514-3585 (fifth exon), 3694-3759 (sixth exon) , 3846-3916 (the seventh exon), 4039-4179 (the eighth exon), 4590-4931 (the ninth exon), 5235-5626 (the tenth exon) sub), 5724-6038 (the eleventh exon).

[0038] The present invention uses the 2281st to 2299th nucleotide sequence on the second exon as the target sequence of the CRISPR / Cas9 system.

Embodiment 2

[0039] Example 2, CRISPR / Cas9 system primer design and construction of recombinant expression vector

[0040] 2.1 Selection of CRISPR / Cas9 system target sequence

[0041] The CRISPR / Cas9 system targets the positive-sense strand of the second exon of the rice TMS10 gene, and the target sequence is as follows: 5'-TGGCAATCAGCTTTCGGAT-3' (2281 to 2299 of the sequence of SEQ ID NO.1.

[0042] 2.2 Design and synthesis of target sequence primers for CRISPR / Cas9 system

[0043] Target sequence primers targeting the TMS10 gene were designed based on the CH-CRISPR / Cas9 system. The target sequence primers were as follows: Forward primer: 5'-TGGCGTGGCAATCAGCTTTCGGAT-3' (sequence shown in SEQ ID NO.2, CC-TMS10-1F) and Reverse primer: 5'-AAACATCCGAAAGCTGATTGCCAC-3' (sequence shown in SEQ ID NO.3, CC-TMS10-1R); synthesize the target sequence primer of the CRISPR / Cas9 system.

[0044] 2.3 Construction of CRISPR / Cas9 system recombinant expression vector

[0045] The sequence shown in SEQ ...

Embodiment 3

[0047] Example 3, Activity Detection of Recombinant Expression Vectors

[0048] The recombinant expression vector CC-TMS10-1 in Example 2 was introduced into rice protoplasts through PEG mediation to obtain the transient expression result of the recombinant expression vector CC-TMS10-1; after sequencing verification, the recombinant expression vector CC-TMS10-1 was obtained Peak map of site-directed mutation induced in rice protoplasts ( figure 1 ).

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