High-resolution-ratio dissolution curve detection method for mutation of OsNramp5 gene specific site

A melting curve and high-resolution technology, which is applied in the field of high-resolution melting curve detection, can solve the problems of time-consuming and labor-intensive detection, and achieve the effects of fast detection time, cost saving and efficiency improvement

Pending Publication Date: 2019-09-20
江西省超级水稻研究发展中心
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Problems solved by technology

[0005] Aiming at the time-consuming and labor-intensive shortcomings of using sequencing methods to detect target site mutations after targeted gene knockout, the present invention provides a method for detecting specific gene editing sites when gene editing is performed on OsNramp5, an important control gene for low-cadmium traits. The high-resolution melting curve method

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  • High-resolution-ratio dissolution curve detection method for mutation of OsNramp5 gene specific site
  • High-resolution-ratio dissolution curve detection method for mutation of OsNramp5 gene specific site
  • High-resolution-ratio dissolution curve detection method for mutation of OsNramp5 gene specific site

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Embodiment 1

[0038] In the following examples, the experimental methods that do not indicate specific conditions are generally in accordance with conventional conditions, such as the book "Molecular Cloning Experiment Guide (3rd Edition)" (J. Sambrook, written by D.W. Russell; Huang Peitang et al. translation; Science Press Society, 2008), or in accordance with the conditions recommended by manufacturers of reagents or equipment manufacturers.

[0039] Embodiment 1, the creation of a specific site mutation of OsNramp5 gene

[0040] The specific site of the OsNramp5 gene was mutagenized by the CRISPR / Cas9 system, and the steps were as follows:

[0041] 1. CRISPR vector construction

[0042] CRISPR / Cas9 vector construction. The CRISPR / Cas9 vector used in this study was self-constructed, and the vector was the binary vector pCUbi1390 (obtained by adding the corn Ubiquitin3 promoter to the pCAMBIA1390 backbone) [Peng H, ZhangQ, Li Y, et al. Aputative leucine-rich repeat receptor kinase , Os...

Embodiment 2

[0057] Example 2. Using the high-resolution melting curve detection method to detect mutations at specific sites in the OsNramp5 gene

[0058] Using the high-resolution melting curve method provided by the present invention, the transformed seedling T0 generation plants obtained by the method of Example 1 were analyzed, and compared with the sequencing results, it was found that the method provided by the present invention can identify almost all heterozygous or diisozygous For the genotype of the allele mutation, see typical test results image 3 (A).

[0059] For the mutant T1 generation plants obtained in Example 1, after using the leaf segment hygromycin soaking method to screen out individual plants that are not resistant to hygromycin, that is, do not contain transgenic components such as resistance markers, use the high-resolution method described in the present invention Homozygous identification of mutant individual plants was carried out by the rate melting curve de...

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Abstract

The invention discloses a high-resolution-ratio dissolution curve detection method for mutation of an OsNramp5 gene specific site. By knocking out an OsNramp5 gene, low-cadmium paddy rice strains can be quickly created. In conventional CRISPR / Cas9-mediated gene knockout, if identification of current-generation mutants of transformed plants and screening of homozygous mutant genotypes of offsprings with markers removed are detected by a conventional sequencing method, time and labor are wasted and the cost is high. For overcoming the shortcomings, the invention provides the high-resolution-ratio dissolution curve detection method for rapidly detecting mutations in current generations of the transformed plants and rapidly identifying homozygous mutant individuals in the mutant isolated offsprings in the work of knocking out the OsNramp5 gene by CRISPR / Cas9. The method provided by the invention has the advantages of large flux, fast speed and low cost.

Description

technical field [0001] The invention belongs to the technical field of rice molecular breeding, and relates to a high-resolution melting curve detection method for a specific site mutation of OsNramp5 gene. Background technique [0002] Rice is a crop that is easy to accumulate cadmium, but the acidification or pollution of cultivated land increases the content of active cadmium in the soil, which intensifies the accumulation of cadmium in rice, resulting in frequent incidents of rice cadmium exceeding the standard. Excessive intake of cadmium seriously endangers health. In some areas in southern my country, excessive cadmium in rice has become a food safety issue that has attracted widespread attention from the society. Planting rice varieties with low cadmium accumulation is the best way to quickly solve the problem of cadmium pollution in rice at low cost. Studies have shown that the OsNramp5 gene function loss mutation in rice can greatly reduce the cadmium content in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6895
CPCC12Q1/6858C12Q1/6895C12Q2600/156C12Q2600/166C12Q2600/13C12Q2531/113C12Q2527/107C12Q2545/101
Inventor 龙起樟黄永兰万建林唐秀英王会民芦明
Owner 江西省超级水稻研究发展中心
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