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Special CAPS marker for identifying wild type or mutant paddy rice salt resistant gene OSRR22, primer, and primer applications

A salt-tolerant gene and wild-type technology, applied in the field of plant biology, can solve problems such as lack of salt tolerance, achieve the effects of reducing costs, improving breeding efficiency, and simple sequencing methods

Active Publication Date: 2018-05-04
SHANGHAI AGROBIOLOGICAL GENE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, OsRR22 is a negative regulatory gene encoding a 696-amino acid B-type response regulatory protein transcription factor involved in cytokinin signal transduction and metabolism, and its loss of function significantly improved salt tolerance

Method used

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  • Special CAPS marker for identifying wild type or mutant paddy rice salt resistant gene OSRR22, primer, and primer applications
  • Special CAPS marker for identifying wild type or mutant paddy rice salt resistant gene OSRR22, primer, and primer applications
  • Special CAPS marker for identifying wild type or mutant paddy rice salt resistant gene OSRR22, primer, and primer applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This example relates to the creation of a rice salt tolerance gene Osrr22 deletion mutant based on CRISPR-Cas9 technology.

[0036] (1) Guide RNA target sequence selection and primer design

[0037] According to the genome sequence of the control rice salt tolerance gene OsRR22 (GenBank: BR000251.1), an sgRNA of the salt tolerance gene OsRR22 was designed. The 20nt nucleotide sgRNA target sequence is designed according to the 5'-N20-NGG-3' sequence, and the target sequence primers RR22-gRT+ and RR22-OsU6aT- are designed at the same time. Paired with the U6a promoter. Compare the designed sgRNA target sequence to the rice genome database to exclude non-specific target cutting sites. The specific target nucleotide sequence is as follows, see figure 1 .

[0038] RR22-gRT+: 5’-AGAGGGATCAATTCCCCGTgttttagagctagaaat-3’

[0039] RR22-OsU6aT-:5'-ACGGGGAATTGATCCCTCTCggcagccaagccagca-3'

[0040] (2) Construction of target sequence sgRNA expression cassette

[0041] Referring t...

Embodiment 2

[0070] This example relates to the establishment of specific CAPS markers for the identification of rice salt tolerance gene OsRR22 wild type and mutant

[0071] (1) Primer design

[0072] The rice salt tolerance gene OsRR22 was amplified and sequenced in the japonica rice line WDR58 and the WDR58-cas-1 mutant, and the analysis found that the WDR58-cas-1 mutant had 1 bp at the 69th nucleotide position of the Osrr22 gene locus (A) The insertion leads to amino acid frameshift mutation, premature translation termination, resulting in loss of OsRR22 gene function and thus improving salt tolerance. Bioinformatic analysis of this region revealed that the 1bp insertion generated a Tail I restriction endonuclease site (5'...ACGT⊥...3'), while the wild type did not contain a Tail I restriction endonuclease site point(5'...CGT...3'), such as figure 2 . The upstream and downstream primers CAPS-TailⅠ containing this site were designed on both sides of the 1bp insertion mutation region...

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Abstract

The invention relates to a special CAPS marker for identifying a wild type or mutant paddy rice salt resistant gene OSRR22, a primer, and primer applications. The marker comprises a marker Cas-Osrr22-1 for detecting a wild type site and the nucleotide sequence of the marker Cas-Osrr22-1 is represented by SEQ ID No.2. The marker also comprises a marker Cas-Osrr-22-2 for detecting a mutant WDR58-cas-1 site, and the nucleotide sequence of the marker Cas-Osrr-22-2 is represented by SEQ ID No.3. Compared with the prior art, the provided maker, primer, and primer applications have the advantages that a molecular marker for differentiating mutant and wild type of WDR58-cas-1 is provided, high efficient identification of salt resistant gene Osrr22 mutant genotype is realized; and WDR58-cas-1 mutant can be applied to breeding of salt resistant species. The CAPS marker can be applied to assistant breeding for improving the salt resistant property of paddy rice, and can be applied to identification for crossbreeding and backcross separation groups, heterozygosis genotype and homozygosis genotype can be accurately differentiated, and the breeding efficiency is improved.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a specific CAPS marker, a primer and the use of the primer that can be used to identify the rice salt-tolerant gene OsRR22 wild type or mutant. Background technique [0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, and is the main ration of one-third of the world's population. Sufficient rice production directly affects global food security. Salt-alkali stress is one of the main abiotic stress factors restricting rice production. [0003] Regarding saline-alkali stress, on the one hand, there is still a large area of ​​saline-alkali land that cannot grow rice. On the other hand, the environment we are facing is deteriorating, and the salinization of the land is increasing year by year. The rice fields affected by salt in the world account for about 20% of the cultivated area. , Improving the salt tolerance of rice has become one of the impor...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 刘毅刘国兰张安宁唐金娟李天菲孔德艳余新桥罗利军
Owner SHANGHAI AGROBIOLOGICAL GENE CENT
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