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Gene 03g induced to express by tissue culture and preparation method and application

A transgenic rice, separation technology, applied in the field of plant genetic engineering, to achieve the effect of high-quality stress resistance and rich detection methods

Inactive Publication Date: 2013-09-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still certain uncertainties in the research on transgenic plants at this stage. Transgenic plants need to be strictly managed, field planting should be treated differently, and the flow direction of transgenic plants should be monitored in real time. Therefore, the research on the detection methods of transgenic plants is very important.

Method used

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  • Gene 03g induced to express by tissue culture and preparation method and application
  • Gene 03g induced to express by tissue culture and preparation method and application
  • Gene 03g induced to express by tissue culture and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A method for preparing the expression sequence of the gene 03g induced by tissue culture, the steps of which are:

[0038] To the gene (AK102606, http: / / www.ncbi.nlm.nih.gov / nuccore / 32987815) required for the present invention, mainly by RT-PCR method (referring to: J. Sambrook, EF Fritsch, T Man Written by Niatis, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guide (Third Edition), Beijing, Science Press, 2002 edition) amplified to obtain a specific sequence of 03g gene:

[0039] 1) A, extraction of RNA from various periods in the rice tissue culture process [the buds grown after 7 days of sub-induction (callus has not yet been induced); B, the buds grown after 11 days of seed induction; C, the buds grown after 11 days of seed induction The callus that grows after 2 days; D, the callus that has been subcultured once; E, the callus that is in the differentiation stage (differentiated buds grow out); F, the seedling differentiated from the c...

Embodiment 2

[0045] Application of a gene 03g induced by tissue culture in the detection of DNA methylation in (Zhonghua 11 and Minghui 63 transgenic rice):

[0046] The bisulfate DNA methylation sequencing method was used in 03g expression materials (Zhonghua 11 materials produced by tissue culture, see Genetic Resources Table 1) and non-expressing materials (wild-type Zhonghua 11, see Genetic Resources Table 1 ) to detect its DNA methylation, the specific implementation method is as follows:

[0047] 1) Genomic DNA was extracted from the material using the CTAB method (Zhang et al., genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis, 1992, Theor Appl Genet, 83, 495-499). Genomic DNA of each material was treated with bisulfate, using a kit purchased from QIANGEN (EpiTect Bisulfite Kit, item number: 59104). For specific methods, refer to the instructions for use.

[0048] 2) Using the DNA obtained in 1) as a template, design primers for DNA methylat...

Embodiment 3

[0054] Application of a gene 03g induced by tissue culture in the detection of histone modification (transgenic rice Zhonghua 11 and Minghui 63):

[0055] Using the method of chromatin immunoprecipitation to detect the histone modification status of 03g, the specific operation method is as follows:

[0056] 1) Material: The material with 03g expression is the Zhonghua 11 material produced by tissue culture (see Genetic Resources Table 1), and the material without 03g expression is the wild-type Zhonghua 11 material (see Genetic Resources Table 1).

[0057] 2) Take the seedlings grown for 14 days and submerge in extraction buffer 1 containing 1% (volume / volume) formaldehyde (0.4M sucrose, 10mM Tris-HCl pH8, 10mM MgCl 2 5mM β-mercaptoethanol, protease inhibitor), vacuumize for 30 minutes, then add 2.5ml of 2M glycine, and continue vacuuming for 5 minutes. Then take out the material, wash it twice with distilled water, blot it dry, put it into liquid nitrogen to freeze it quick...

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Abstract

The invention discloses a gene 03g induced to express by tissue culture in rice and an application. Through a known public database, genes specifically expressed by callus are sieved and the gene 03g after callus induction is expressed continuously, that is, after callus differentiation, 03g is still expressed in high quantity when other genes lose expression activity. Through hybridization of 0.3g expressed plant with wild plant, expression of the 03g is not affected by the other parent. Therefore, expression or no expression of 03g or not can be used to detect whether the rice plant is transgenetic or not. Molecular biological experiments show that expression of 03g is regulated by DNA (Deoxyribonucleic Acid) methylation and histone methylation. Expression of 03g provided by the invention is epigenetically regulated, and the expression mode can be applied to detecting transgenetic rice.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to a gene 03g that is induced and expressed by tissue culture, and also relates to the regulation mode of a gene 03g that is induced and expressed by tissue culture, and also relates to the use of a gene 03g that is induced and expressed by tissue culture, and the gene can be used for Detection of Zhonghua 11 and Minghui 63 transgenic rice. technical background [0002] Epigenetic regulation refers to regulating the spatiotemporal expression of genes on the basis of chromatin without changing the DNA sequence, thereby regulating cell activity, individual development and environmental response. DNA methylation and histone modifications are major components of epigenetic regulation. DNA methylation generally refers to the methylation modification of cytosine bases in DNA. Histone modification means that the end (tail) of the histone that constitutes the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12Q1/68
Inventor 周道绣陈香嵩
Owner HUAZHONG AGRI UNIV
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