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Fixed-point knockout system for Bel gene of paddy rice and application thereof

A technology of targeted knockout and rice, applied in the field of plant genetic engineering, can solve the problems of easy carrying of mutant genes and weak targeting, and achieve the effect of eliminating the concerns of transgenic and saving manpower.

Active Publication Date: 2015-07-08
FRONTIER LAB OF SYST CROP DESIGN CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the poor targeting of physical and chemical mutagenesis methods, a large amount of experimental materials need to be processed to mutate a gene. In order to ensure the mutants of the target gene, a lot of screening work needs to be done, and it is easy to carry mutated genes that you don’t need; DNA recombination technology In the case of exogenous transgenes, there are certain problems in breeding time or biosafety evaluation

Method used

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  • Fixed-point knockout system for Bel gene of paddy rice and application thereof
  • Fixed-point knockout system for Bel gene of paddy rice and application thereof
  • Fixed-point knockout system for Bel gene of paddy rice and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the sequence and analysis of rice gene Bel

[0028] The sequence of the rice gene Bel is shown in sequence 1 of the sequence listing. Sequence analysis shows that the gene includes 2 exons in total, which are No. 41-964 (the first exon) and No. 1691-2308 (the second exon) of Sequence 1 in the sequence listing. This gene encodes a cytochrome P450 protein.

[0029] In the present invention, the sequences on the first exon are used as the target sequences of transcription activator-like effector nucleases (TALENs). ( figure 1 )

Embodiment 2

[0030] Embodiment 2, TALENs design and construction of its recombinant expression vector

[0031] 1. Selection of TALENs target sequences

[0032] The sequence behind the start codon of the first exon of the target sequence targeting rice gene Bel is as follows: 5'-CAGCTTAGCCATGGAT aacgcctacattattgcc ATTCTCTCTGTAGCT-3' (as specifically shown in SEQ ID NO: 10) where the lowercase letters is a spacer sequence, and the uppercase letters on both sides are TALENs module recognition sequences (named L and R, respectively).

[0033] 2. Design and synthesis of TALENs coding genes

[0034] Build by assembling the elements of the talen expression box figure 1 The indicated pOsBel vector. The vector contains 3 expression cassettes, wherein the first expression cassette is a hygromycin resistance gene expression cassette, composed of a constitutive promoter 35S promoter P35S from tobacco mosaic virus, and its nucleotide sequence is shown in the sequence listing Shown in SEQ ID NO: 2,...

Embodiment 3

[0036] Embodiment 3, transfection pOsBel carrier rice plant Bel gene analysis

[0037] The recombinant vector pOsBel obtained in Example 2 was transformed into Agrobacterium strain EHA105 by heat shock, and the recombinant Agrobacterium containing the recombinant vector pOsBel was obtained, named EHA105 / pOsBel; 100 independent single-copy transgenes were obtained by the method of Agrobacterium infecting the callus Plants, using the genomic DNA of transgenic plants as a template, using primers Ben-F: 5'-ACAGAAACACATCACACATTCG-3' (SEQ ID NO: 11) and Ben-R: 5'-TAGTAGTGGAGCAAGAAGAGGATAG-3' (SEQ ID NO: 12) PCR amplification was carried out, and the amplified products were analyzed by HRM, and it was found that the Bel gene melting curves of plants 4, 10, 18, 19, 24, 36, 41, 44, 56, 66, 67, 76, 78 and 81 changed, Specific results such as image 3 As shown, WT indicates the melting peak of the wild-type rice Bel gene, and its materials include wild-type plants and transgenic plants ...

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Abstract

The invention discloses a fixed-point knockout system for the Bel gene of paddy rice and application thereof, specifically a fixed-point knockout method for the bentazone gene of paddy rice and application thereof, belonging to the field of plant genetic engineering. The invention provides a TALEN site-specific mutagenesis system preferably directed at plant genomic DNA. The TALEN site-specific mutagenesis system includes four recognition modules. Compared with conventional bentazone-sensitive plant creating technology, the TALEN site-specific mutagenesis system provided by the invention has the advantages of convenience and rapidness; with the TALEN site-specific mutagenesis system, one scientific research personnel can acquire a plurality of homozygous mutant plants with different mutation sites in one and a half years, so labor and material resources are substantially saved; obtained mutants do not contain transgenic components, so misgivings of people about transgene are eliminated; and the TALEN site-specific mutagenesis system is of great significance to creation of novel germplasm resources through plant genetic engineering.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a fixed-point knockout method of rice bentazolone gene and application thereof. Background technique [0002] There are two main types of selective herbicides used in wild-type rice. One is benzothiadiazole herbicides, such as bentazone, whose active ingredients can be absorbed by the roots and leaves of crops. Most dicotyledonous plants and Cyperaceae weeds except Leguminosae can be killed, and it is harmless to Grasses. The herbicidal mechanism of bentazone is to inhibit the Hill reaction in plant photosynthesis. [0003] TALENs (transcriptional activator-like effector nuclease) is a new technology for gene-directed modification that has emerged in recent years, and has been extensively studied and applied in the fields of animals, plants and humans. Transcription Activator-Like Protein Effector (TALE) is a toxic protein secreted by Xanthomonas pathogens i...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/63C12N1/21C12N5/10C12N15/82A01H5/00
CPCC12N9/22C12N15/8274
Inventor 刘东风常振仪王承旭卢嘉威唐晓艳邓兴旺
Owner FRONTIER LAB OF SYST CROP DESIGN CO LTD
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