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Plant chlorenchyma specific expression promoter pGreen and application thereof

A green tissue and plant expression carrier technology, applied in the field of promoters driving gene expression, can solve problems such as failure to reach transgenes, damage to the physiological activities of transgenic plants, and violation of the natural physiological activities of plants.

Active Publication Date: 2014-01-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these genes mostly use constitutive promoters to drive their expression in transgenic plants. Because this expression method violates the natural physiological activity rules of plants, it may not only fail to achieve the purpose of transgenics, but even cause damage to the physiological activities of the transgenic plants themselves. a great deal of damage [10-12]

Method used

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  • Plant chlorenchyma specific expression promoter pGreen and application thereof
  • Plant chlorenchyma specific expression promoter pGreen and application thereof
  • Plant chlorenchyma specific expression promoter pGreen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Plant expression binary vector construction

[0066] The cry1Ac (published gene, GenBank: AY225453.1) insect-resistant gene was selected as the research object, and the green tissue-specific promoter pGreen of the present invention (see SEQ ID NO: 1) and the constitutive promoter ZmUbi (Zea mays Ubiquitin -1) (as a control) to drive the expression of an insect-resistant gene, and use the glyphosate-resistant gene G6 (patent application number: 200610052573.4) as a screening marker gene to construct a binary expression vector.

[0067] details as follows:

[0068] 1), for pCambia1300 (Cambia, Australia; figure 1 .a) The carrier is transformed:

[0069] Replace the original hygromycin-resistant gene with glyphosate-resistant gene G6, and the modified vector can use Hind Kpn was digested with enzyme I, and it was used as the basic vector pCambia1300-p35S-G6 for constructing T-DNA.

[0070] The specific steps are as follows: first, the maize ubi gene and the...

Embodiment 2

[0081] Example 2: Agrobacterium-mediated transformation of rice

[0082] Agrobacterium-mediated transformation of rice was based on the experimental methods of Hiei et al. and Nishimura et al. (Hiei et al., 1994; Nishimura et al., 2007). The glyphosate-resistant gene G6 was used as a selection marker. Infect the induced fresh callus with Agrobacterium LBA4404 carrying pGreen-Cry1Ac and pUbi-Cry1Ac respectively (one Agrobacterium carrying pGreen-Cry1Ac, one Agrobacterium carrying pUbi-Cry1Ac), and after three days of co-cultivation, use sterile Repeatedly rinse the callus with water, shake it at 120rpm with 200mg / L Timentin in sterile water, shake at 28°C for one hour, then blot the moisture on the surface of the callus with sterilized filter paper, and transfer to the solution containing 2mM glyphosate (Sigma, USA) and 100mg / L Timentin selection medium for selection, the selection cycle was three weeks, and a total of two screenings were performed. It can be observed that f...

Embodiment 3

[0084] Example 3: T 0 Transgenic rice analysis

[0085] 1. Detection of resistance of transgenic rice seedlings to cotton bollworm

[0086] 226 and 159 transgenic lines were obtained by infecting calluses with Agrobacterium carrying specific promoters pGreen and ZmUbi promoters, respectively. Using the "Xiu Shui-134" non-transgenic plants as a control, the first-instar cotton bollworm (H. armigera) larvae were used to conduct bioassays on the leaves of non-transgenic and all transgenic rice line seedlings. When the growth period of the plant to be tested is 3-5 cotyledons, take 1-2 leaves and place them on a 75mm petri dish covered with a layer of filter paper, add 200ul of sterile water on the filter paper to moisturize, cultivate in a greenhouse, and observe the results after 48 hours. Compared with the control of non-transgenic plants, the transgenic lines had good resistance to cotton bollworm ( figure 2 ). Among them, 156 of the strains containing the specific promot...

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Abstract

The invention discloses a plant green tissue-specific expression promoter pGreen. A nucleotide sequence of the promoter is shown as SEQ ID NO:1. The plant green tissue-specific expression promoter pGreen comprises an enhancer sequence of a 35S promoter of a Cauliflower mosaic virus of tobacco and a promoter sequence which is separated from a maize PD450 gene. The invention also discloses application of the plant green tissue-specific expression promoter pGreen to the preparation of transgenic plants with insect resistance, and application of the plant green tissue-specific expression promoter pGreen to the improvement on the anti-insect activity of plants.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to a promoter for driving gene expression in plant green tissue and its application. Background technique [0002] As the world's population continues to increase, people's demand for agricultural products is also increasing, and food as a life energy supply is particularly important, such as rice and corn. However, in agricultural production, the food and economic crops that human beings rely on for survival are extremely vulnerable to insect pests, which can reduce production or degrade the quality of agricultural products, and even produce toxic and harmful substances to the human body, causing greater losses. In modern agriculture, the use of chemical pesticides is an important method to reduce insect pests. However, the long-term and large-scale use of chemical pesticides not only increases production costs, but also brings a series of hazards to the ecological environ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/84A01H5/00
Inventor 秦毅方军张先文沈志成
Owner ZHEJIANG UNIV
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