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Multiplex PCR detection kit for rapidly identifying rice GAT plant and application

A detection kit and kit technology, applied in the field of genetic engineering, can solve problems such as infertility of hybrid offspring, changes in seed production, failure, etc., and achieve the effects of high safety in use, accelerated selection, and no biological safety hazard components

Pending Publication Date: 2021-11-09
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The male sterility of the three-line sterile line is caused by the gene interaction between the nucleus and the cytoplasm. Only when the restorer line with a specific restorer gene in the nucleus is crossed with the sterile line can the fertility of the hybrid offspring be restored to produce hybrid rice, and there is no restoration Hybridization of the genetic variety with the sterile line will result in infertility in the offspring of the hybrid and result in a failure of the harvest
Although the two-line CMS can be "one line for two purposes", its fertility conversion is affected by the light and temperature environment, and it is easy to fail due to changes in the light and temperature environment during seed production.

Method used

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  • Multiplex PCR detection kit for rapidly identifying rice GAT plant and application
  • Multiplex PCR detection kit for rapidly identifying rice GAT plant and application
  • Multiplex PCR detection kit for rapidly identifying rice GAT plant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Construction of GAT vector and GAT plants

[0070] The GAT vector is constructed from several of the following five gene expression cassettes to be constructed in a plant dual expression vector by a joint sequence, and the five gene expression boxes are:

[0071] (1) Plant male dystrophic recovery gene expression cassette, is sequentially connected by promoters, male diet recovery gene coding regions and terminator. In this example, the plant and the nucleotide sequence, such as SEQ ID NO.11, the promoter is the upstream 1112bp sequence of rice OSCYP704B2 gene start codon ATG, the coding region is codon optimization. The coding area of ​​the OSCYP704B2 gene, the terminator is the OSCYP704B2 gene terminates the 274bp sequence of the codon TGA. The expression of the expression cassette is to restore the male fertility of the implicit pure mutant oscyp704b2 of the OSCYP704B2 gene. .

[0072] (2) Plant pollen defeat the gene expression cassette, which is sequentially ...

Embodiment 2

[0085] Example 2 Design and screening of specific primers in GAT vector

[0086] The DNA sequence of each gene expression cassette on the GAT carrier is constructed according to Example 1, and the PCR primer design is performed using the Primr Premier 5 software. Detect Killer 5400 (SEQ ID No. 12) and Killer (SEQ ID NO.13) expression cassette, as well as detecting Marker 2 AAU (SEQ ID NO.14) and Marker 2 UAU (SEQ ID NO.15) expression box The primers are designed for two pairs of expression cassettes. Primers for detecting Marker 1 (SEQ ID NO.16) and Marker 3 ZFN (SEQ ID NO.17) are each based on their special sequence design. The detection primers of each expression cassette are all designed, and the length of the amplified fragment is predicted from 70 bp to 206 bp. 4 pairs of optimal primers were screened according to the amplification effect under various PCR conditions. The primers Killer-F and Killer-r used for Killer 5400 or Killer detection have a nucleotide sequence as show...

Embodiment 3

[0088] Example 3 Establishment of multiple PCR detection systems

[0089] 1. Establish a PCR detection system of a single expression cassette

[0090] Each PCR reaction was prepared by 20 μl of the reaction system, including TAQ PCR MIX (Beijing Kang for Century Biotechnology Co., Ltd.), 0.5μm of Various primers, 10% of DMSO, and 1 μl of DMSO Sribin water supplement 20 μL.

[0091] Two negative controls were set, and the three-F-R, MARKER2-F-R, MARKER1-F-R, and Marker3-F-R, MARKER1-F-R, and Marker3-F-R four-to-objects were added simultaneously. The remaining expression cassette was added to the PC0308-mmmaauck5400 plasmid as a template, but the pair of primers in Killer-F-R, Marker2-F-R, Marker1-F-R or Marker3-F-R were added. The detection of the expression cassette is set to two techniques.

[0092] The PCR response procedure is performed as follows:

[0093] (1) 5 min at 95 ° C;

[0094](2) 95 ° C denaturation 30S; 60-55 ° C, each circulation decreased by 0.5 ° C, annealed 30s, ...

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Abstract

The invention provides a multiple PCR detection kit for rapidly identifying a rice GAT plant and application, the rice GAT plant refers to rice containing a GAT vector, and the GAT vector contains a male fertility recovery gene expression cassette, a pollen abortion gene expression cassette, a herbicide-resistant gene expression cassette, a herbicide sensitive gene expression cassette and / or a fluorescent gene expression cassette. A specific primer combination provided by the invention can rapidly detect whether a to-be-detected sample contains the pollen abortion gene expression cassette, the herbicide-resistant gene expression cassette, the herbicide sensitive gene expression cassette and / or the fluorescent gene expression cassette on the GAT vector, the detection method has the advantages of strong specificity, high sensitivity and high sensitivity, meanwhile, the method can be used for detecting the expression quantity and copy number of each expression cassette, and effective guarantee is provided for transgenosis safety detection.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering, particularly, to a rapid identification of rice plants GAT multiplex PCR Kit and pollen sterility gene expression cassettes, gene expression cassettes herbicide resistant, herbicide sensitive gene expression cassette in the detection system and GAT Application of Fluorescent Gene Expression Box. Background technique [0002] Rice is the most important rations of my country, which is of great significance in ensuring my country's food security. Hybrid rice uses rice with rice, which improves the yield, resistance, adaptability of rice varieties, so that my country's rice yield has increased by 15% -20%, which has made important contributions to the national food security. Since the promotion and application of hybrid rice in the 1970s, hybrid rice breeding technology has experienced the development of several generations (Yuan Longping. The third-generation hybrid rice preliminary study was s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/16C12Q2600/13C12Q2600/158C12Q2600/166C12Q2537/143C12Q2545/101C12Q2565/125
Inventor 龙湍唐杰曾翔吴永忠李新鹏安保光黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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