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Method for appraising albino seeding gene type wound curing

A technology of albino seedlings and genotypes, applied in the field of plant genetic engineering, can solve problems such as difficulties, lethality, and damage to gene functions, and achieve the effects of quick and convenient identification, simple methods, and accurate and reliable results

Inactive Publication Date: 2009-06-17
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Because the insertion of T-DNA destroys the function of the gene, resulting in the inactivation of the homozygous recessive gene, some mutants such as albino seedlings, lethality, premature aging, and infertility that cannot be harvested for seedlings are produced, so it is recommended to use mature embryos to guide healing in complementation experiments. injury caused some difficulties

Method used

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  • Method for appraising albino seeding gene type wound curing
  • Method for appraising albino seeding gene type wound curing
  • Method for appraising albino seeding gene type wound curing

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1: Detection of albino seedlings is due to T-DNA insertion

[0023] The T-DNA vector pFX-E24.2-R15 (Wu et al., Development of enhancer trap lines for functional analysis of the rice genome. Plant J. 2003, 35: 418-27.) inserted into the middle flower using the enhancer trap system Seeds of the T0 generation of japonica rice variety 11 (Oryza sativa L.subsp.japonica cv.Zhonghua11) were soaked at 25°C for 4 days, germinated at 37°C for 3 days, and sown in the field. After two weeks of field investigation, the albino mutant phenotype strain EV57 was obtained. . The mutant exhibited albino traits when seedlings, such as figure 1 As shown, the mutant plants gradually died after growing for about one month. 20 green seedlings were planted in the field for harvesting seeds for further research.

[0024]The total DNA of the albino seedling mutant EV57 was extracted using the CTAB method (Liu et al., A genome-wide analysis of wide compatibility in rice and precise loca...

Embodiment 2

[0026] Embodiment 2, screening heterozygous single plant harvesting seed

[0027] Design PCR primers according to the T-DNA flanking genome sequence. For this example, a represents the primer designed upstream of the T-DNA insertion site, b represents the primer designed downstream of the T-DNA insertion site, and c represents the primer designed according to the T-DNA end Primers for sequence design. In the T1 generation plants, if it is a single plant homozygous for the T-DNA insertion site, since the T-DNA fragment is more than 10kb, the general PCR reaction cannot amplify a fragment of more than 10kb, so the PCR amplification of the pairing of a and b is negative , but the pairing of a and c can be amplified to obtain a 0.8kb product. If the amplified site is a wild-type single plant without T-DNA insertion, since there is no c primer binding site, only a and b can be paired to obtain a 1.0kb product. As for the heterozygous single strain of the T-DNA insertion site, the...

Embodiment 3

[0028] Embodiment 3, inducing callus, extracting callus DNA, identifying callus genotype

[0029] Select the seeds harvested from plants of heterozygous genotype, peel off the chaff, soak in 75% ethanol for 1 minute, soak in 0.15% mercuric chloride for 15 minutes, then wash 5-6 times with sterilized water, and put the rice grains in the induced culture Callus was induced substantively (see section Induction of Callus in Major Steps of Agrobacterium-mediated Genetic Transformation below). Place it in a dark room at 27°C for about 50 days, and a callus will grow. Pick one callus from each grain of rice and use the CTAB method (Liu et al., A genome-wide analysis of wide compatibility in rice and precise location of s5 locus in the molecular map. Theor Appl Genet, 1997, 95: 809-814) to extract DNA. Use a+b, a+c two pairs of PCR primers to screen out the T-DNA insertion homozygous genotype according to the principle of Example 1, that is, the callus phenotyped as albino seedlings ...

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Abstract

The invention discloses a method of identifying albino genotype callus. First, inducing callus by heterozygous genotype seeds, stripping rice husks and immersing in alcohol and Hg in a certain time, setting on inducing culture medium after washing by bactericidal soultion, inducing callus in illumination culture room; second, extracting callus DNA, stripping inducing callus on a subculture medium, selecting one granule of induced callus to extract callus overall DNA by means of CTAB, adding water to dissolve; third, amplifying callus overall DNA through PCR except a+b in albino genotype, amplifing a+c, detecting callus genotype by PCR products of TBE gel electrophoresis; four, selceting albino homozygenotype callus and seeting on differentiation culture medium in illumination culture room to differentiate certification. The method can identify homozyrecessivegene inactivation caused by insertion of T-DNA to cause seed mutant homozygenotype callus of death, sterility, presenility and so on for function complementation experiment.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. The present invention relates to a method for identifying albino seedling genotype callus; more specifically, the present invention relates to a method for identifying rice albino mutants caused by T-DNA insertion, screening albino seedling homozygous genotype callus for use in Methods for functional complementarity experiments. Background technique [0002] Rice is one of the most important food crops, and more than half of the world's population depends on it as a staple food. Rice is also a model plant of monocotyledonous plants, and it is of great economic and scientific value to carry out research on its gene function. [0003] In recent years, with the completion of the international rice genome sequencing project, it has become an urgent task to study and analyze rice genes and finally elucidate their functions. In the process of gene cloning, when a series of candidate genes ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 吴昌银郭冬张启发
Owner HUAZHONG AGRI UNIV
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