Hybrid crop transgenic safety control method and gene deletion system for implementing same

A safety control and transgenic technology, applied in the field of plant genetic engineering, can solve problems such as unsolved problems

Inactive Publication Date: 2013-04-24
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no effective

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  • Hybrid crop transgenic safety control method and gene deletion system for implementing same
  • Hybrid crop transgenic safety control method and gene deletion system for implementing same
  • Hybrid crop transgenic safety control method and gene deletion system for implementing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] [Example 1] Extraction of DNA and amplification of target fragment sequence

[0033] 1. Extraction of DNA

[0034] Select 0.3-0.5 g of young leaves of tobacco (Nicotiana tabacum, Xanthin), quickly grind them into a white powder in liquid nitrogen, transfer to a 10 mL centrifuge tube, add 3 mL of 65°C preheated DNA extraction buffer, shake and mix quickly. Water bath at 65°C for 45 minutes, during which time mixing 2-3 times. Then add 1mL 5mol / L KAc, ice bath for 20min. Extract once with an equal volume (4 mL) of chloroform:isoamyl alcohol (24:1) (10,000 r / min, centrifuge at 25°C for 10 min). Take the supernatant, add 2 / 3 times the volume of -20°C pre-cooled isopropanol, mix well, and let stand at room temperature for about 30 minutes. Pick out the flocculent precipitate, rinse twice with 75% ethanol, and rinse once with absolute ethanol. Blow dry at room temperature and resuspend in 600 μL TE. Add 1 μL RNaseA (10 mg / mL), and treat at 37°C for 1 hour to remove RNA i...

Embodiment 2

[0044] [Example 2] Construction of the Trigger plant expression vector of the flower primordium cell-specific promoter controlling the transcription activator gene

[0045] 1. Acquisition of plant flower primordia cell-specific promoters

[0046] According to the CaMV 35S minimal core promoter sequence on the plasmid pER8 (GenBank accession number: AF309825.2), primers (SEQ ID NO.1 and 2) were designed, and a sequence of about 600 bp was obtained by PCR amplification using the plasmid pER8 as a template. Sequencing results showed that the sequence was 602bp in length and contained a 58bp CaMV 35S (-46to+12) core promoter (minP for short), a multiple cloning site MCS and pea ribulose-1,5-bisphosphate carboxylase small The polyA sequence of subunit rbcS-3A gene (referred to as "T3A").

[0047] According to the second intron sequence of tobacco flower development AGAMOUS (AG) homologous gene 1 (GenBank accession number: GU143404.1), primers (SEQ ID NO.3, 4) were designed, and a ...

Embodiment 3

[0058] 【Example 3】Agrobacterium-mediated genetic transformation of tobacco

[0059] See Table 1 for the medium used for the genetic transformation of tobacco by the method mediated by Agrobacterium tumefaciens

[0060] Table 1: Medium for genetic transformation of tobacco mediated by Agrobacterium tumefaciens

[0061]

[0062] MS: Murashige & Skoog, 1962

[0063] B5: Gamborg, 1986

[0064] Agrobacterium-mediated introduction of leaf discs into tobacco. The specific method is as follows:

[0065] Tobacco seeds were sterilized with 1% sodium hypochlorite on solid medium MSB 0 The culture conditions were 25°C, 16hr light / 8hr dark photoperiod. After about one month, the sterile seedlings that grow vigorously can be used as transformed explants.

[0066] Select robust leaves, cut them into leaf discs of about 0.5 cm × 0.5 cm on a clean bench, and keep them moist for later use. Pick a single colony of Agrobacterium for transformation cultured on the YEB plate with a toothp...

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Abstract

The invention provides a hybrid crop transgenic safety control method. An transgenic plant automatic deletion double-unit system, comprising a recombinase system, a transcription activation system and an exogenous gene expression control system, is established; and a plant flower primordium cell specific promoter is utilized as a promoter in the transgenic plant automatic deletion double-unit system to control the transcription activation system, and the transcription activation system controls the start of the recombinase system, so that the exogenous gene introduced into the plant stably exists in the F1-generation plant hybrid as well as root, stem, leaf and other non-deletion tissues, but is deleted in the pollen and seed of the F1-generation plant, thereby implementing the hybrid crop transgenic safety control.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a method for safety control of transgenic hybrid crops and a system for realizing the method. Background technique [0002] Plant transgenic technology has set off a new green revolution in agricultural production. It is estimated that by 2015, the number of farmers planting genetically modified crops in the world will reach more than 20 million in 40 countries, and the planting area will reach 200 million hectares (James, 2011). On the other hand, the insect resistance, disease resistance, herbicide resistance and antibiotic resistance genes introduced into transgenic crops have aroused public concern that the existence of these genes may bring potential harm to the ecological environment and human health. even have catastrophic consequences. Such fears and concerns are increasingly exacerbating the public's hesitation in purchasing GMO products, and when informed, the...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H1/02A01H5/00
CPCC12N15/8216C12N15/8265
Inventor 裴炎邹修平刘若尘宋水清侯磊
Owner SOUTHWEST UNIVERSITY
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