Method for vreeding transgenic antifungal rape with specificities of dual genes being expresses

An anti-fungal and transgenic technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of low efficiency of transgenic technology, weak disease resistance of single-gene transformation of rapeseed, and large energy consumption, etc. To achieve the effect of reducing physiological disturbance, reducing energy consumption and improving efficiency

Inactive Publication Date: 2004-08-11
HUAZHONG AGRI UNIV
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Benefits of technology

This patented describes methods used by research scientists to combine both enzyme types involved in plant growth processes called chitins (chymer) lytic proteins or oxalates into one type that shows greater ability to fight various forms of bacterial pests like root rot caused by phytopathogenic molds such as Fusarium wiltum sporozoides). By combining these two factors together it becomes possible to develop novel crop germplasm without causing damage from harmful microorganisms during production. Additionally, this approach allows us to improve the effectiveness of certain chemical treatments on rice seeds while reducing their impact upon health issues associated therewith. Overall, this technique provides technical benefits over existing techniques but also makes it easier than current approaches due to its potential applications across multiple industries including agriculture, medicine, food processing, environmental science, etc..

Problems solved by technology

This patented technology describes how specific parts of rice called seed knots act like an invading bacterium for attacking other crops during their growing process. These seeds cause damage when they germinate from harmful microorganisms. To prevent this problem, researchers developed methods involving introducing foreign DNA sequences containing various types of polynucleotides coding for chitinosome lyseranol degradative enzymes, including monooxylicacids derived from lignocytosine hydrolysis, and oxazoline quaternary ammoniums linked through a peptide linkage between them. Overall, this new method allows scientists to improve crop health while reducing nutrients lost due to root rotations.

Method used

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  • Method for vreeding transgenic antifungal rape with specificities of dual genes being expresses

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Embodiment 1

[0028] Example 1: Construction of organ and development-specific expression double-gene vector ApSCO

[0029] 1.1 with SAG 12 The construction of the intermediate vector pSPCHiN of promoter, chitinase gene and cell membrane guiding peptide gene:

[0030] Double digestion of Arabidopsis leaf senescence promoter SAG with EcoRV and NcoI 12 After the plasmid vector pSG449, the 1.3kb fragment was recovered by electrophoresis, filled in with the Klenow fragment, and inserted into the EcoRV site of the barley oxalate oxidase gene vector pHvOxOa to obtain the recombinant plasmid pSAGOxO (see attached figure 1 A). The plasmid vector pHGC39 containing chitinase gene and glucanase gene was digested with BamHI, and the 1.4kb fragment containing chitinase gene and nos terminator was recovered and inserted into the BamHI site of pSAGOxO to obtain a recombinant plasmid pSOCN (see attached figure 1 B). The vector pSOCHiN containing the oxalate oxidase gene was digested with EcoRI, and th...

Embodiment 2

[0035] Embodiment 2: Transform Brassica napus with living transformation method (in planta)

[0036] Take 100 μl of Agrobacterium containing the carrier ApSCO cryopreserved in glycerol, inoculate it in 10 ml of fresh LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH7.0) culture medium, shake at 150 rpm at 28°C overnight . Inoculate 10ml of the above culture into 1L containing 50mg·L -1 In the LB medium of kanamycin, shake culture at 150 rpm at 28°C for more than 24 hours until OD 660 = 1-1.5.

[0037] The cultured Agrobacterium suspension was centrifuged at 8000 g for 8 minutes, and the precipitated Agrobacterium was resuspended with 1 liter of fresh LB medium. In the experiment, the rapeseed inflorescences were directly dipped with the Agrobacterium suspension. Before dipping, make sure that there is no dew and soil on the flowers, and then select those branches whose flowers are about to open, and cut off the flowers that have already opened on the branches. When dipping, ...

Embodiment 3

[0040] Example 3: Screening and Molecular Biological Identification of Transgenic Rapeseed Plants

[0041] 3.1 Kanamycin selection in vivo transformation method (in planta) to treat seeds

[0042] Select the plump ones from the seeds treated with in planta, soak them in 0.1% mercuric chloride for 12 to 15 minutes, wash them with sterile water three times, and sprinkle them evenly on seeds containing kanamycin 270 mg·L -1 MS solid medium (see Murashige T and Skoog F. A revised medium for rapid growth and bioassays with tobacco issue culture., 1962, Physiologia Plantarum 15: 473-497). Four to five days later, the germinated seedlings with the exogenous gene transferred had kanamycin resistance and could survive normally; the seedlings without the exogenous gene did not have kanamycin resistance, and the edges of the cotyledons were browned, White spots appear on the surface of the cotyledon and the first true leaf, the petiole turns purple and red, and eventually dies. The sur...

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Abstract

A process for culturing the antifungal transgenic rape with dual-gene specific expression includes using RCR or restriction endonuclease to separate the amino acid coding sequences of chitinase gene and oxalate oxidase inplant and the promoter sequence specifically expressing the senile leaf, configuring the recombinant dual-gene expression carrier, introducing the exogenous chitinase gene and oxalate oxidase gene to rape receptor to obtain transgenic plant, and molecular auxiliary selection while conventional culturing.

Description

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Claims

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Application Information

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Owner HUAZHONG AGRI UNIV
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