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Plant genetic transformation screening carrier and application thereof

A genetic transformation and plant technology, applied in the field of plant genetic transformation screening vectors, can solve problems such as safety risks of exogenous selection markers, and achieve commercial applications, reduce concerns about safety risks, and have good market value and social benefits. Effect

Active Publication Date: 2019-11-05
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of safety risks of exogenous screening markers used in the current gene transformation work, and to provide a vector that uses plant endogenous genes as screening markers and is applied to plant genetic transformation screening

Method used

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  • Plant genetic transformation screening carrier and application thereof
  • Plant genetic transformation screening carrier and application thereof
  • Plant genetic transformation screening carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Construction of Plant Genetic Transformation Screening Vector

[0053] 1. Preparation of plant transgenic screening expression cassettes

[0054] The sequence of the plant transgene screening expression cassette ZmUbiP-p450-OsUbiT of the present invention is cloned from the constructed plasmid pC1300-ZmUbiP-p450-OsUbiT by designing primers. The plasmid pC1300-ZmUbiP-p450-OsUbiT was amplified by the Gibson Assembly method to amplify the promoter ZmUbiP fragment; amplify the target gene p450 fragment from rice cDNA; amplify the terminator OsUbiT fragment from the rice genome. The 3 amplified fragments were ligated and inserted between the EcoRI and BamHI double restriction sites of the pC1300 vector, in which about 15 nucleotide sequences were repeated at the 5' ends of the upstream and downstream primers of the above fragments and the corresponding connection positions of the vector, so that the Gibson Assembly recombination is sequentially connected to for...

Embodiment 2

[0074] Embodiment 2 Agrobacterium transformation and identification

[0075]Agrobacterium EHA105 stored at -70°C was streaked on a YEP plate containing rifampicin (25 μg / ml) + streptomycin (50 μg / ml), and cultured at 28°C. Pick a single colony and inoculate it in 5 ml of YEP liquid medium containing the above-mentioned antibiotics, culture at 220 rpm and shake at 28° C. for 12-16 hr. Take 2ml of the bacterial liquid and transfer it to 100ml of YEP liquid medium containing the above-mentioned antibiotics, culture at 28°C with shaking at 220rpm until OD600=0.5. Precool on ice for 10 minutes, centrifuge at 5000 rpm for 10 minutes at 4°C (refrigerated centrifuge is precooled to 4°C). Wash twice with sterile deionized water (10 ml each time), wash once with 10% sterile glycerol, centrifuge at 5000 rpm for 10 min at 4° C., and resuspend the bacteria in 3 ml of 10% sterile glycerol. Take 100 μl of competent cells and add 1 μl of the sequenced correct pCBA6 plasmid obtained in Examp...

Embodiment 3

[0076] Example 3 Screening stress test of bentazon and bensulfuron-methyl at the seedling stage of common rice

[0077] Bentazone and bensulfuron-methyl are herbicides used in paddy fields. Ordinary rice is resistant to both. Therefore, spraying herbicides at specified concentrations on rice will not cause damage to rice. Most of the safe spraying concentrations are 1 -3g / L bentazon and 30-40mg / L bensulfuron-methyl (Zhang Jiwen (2010) Research progress on rice bentazon-sensitive mutations. Chinese Rice Science 24(5):551-558). This embodiment studies the upper limit of resistance of rice to bentazone and bensulfuron-methyl, and through experimental testing, the results are as follows image 3 and 4 shown. For bentazon, both the japonica rice Zhonghua 11 (ZH11) and the indica rice 9311 can resist 10g / L at the seedling stage, and the resistance of the indica rice 9311 exceeds 20g / L; for bensulfuron-methyl, both ZH11 and 9311 Can resist more than 1200mg / L. These results show t...

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Abstract

The invention provides a plant genetic transformation screening carrier and an application thereof. The carrier uses an expression box containing a rice P450 gene(CYP81A6) as a screening label, bentazon or bensulfuron methyl is used as a screening agent for screening in the callus tissue stage, and the obtained plant is detected to be a genetically modified positive plant and has high resistance to herbicide. The plant genetic transformation carrier can be used as the genetically modified screening carrier, and through cooperation with other functional elements, in the genetically modified process, rice autogene is used, and exogenous screening label genes of bacterium sources and the like are not introduced, so that the genetically modified screening method for plants is enriched, the concern about security risks of genetically modified plants can also be reduced by public, promotion of business application of the genetically modified plants is facilitated, and the plant genetic transformation screening carrier has favorable market value and social benefits.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a plant genetic transformation screening carrier and its application. Background technique [0002] Due to its many advantages, transgenic technology can broaden the available genetic resources and create new germplasm resources; it can conduct directional and fixed-point mutation and selection of plant traits; The scope is getting wider and wider. Since GM crops were approved for commercial planting in 1996, in the past 21 years, a total of 23 GM crops have been approved for commercial production in the world, involving more than 16 diseases including insect resistance, herbicide resistance, disease resistance, fertility change, and quality improvement. Class target traits. According to statistics, in 2016, 26 countries and regions around the world planted herbicide-resistant and insect-resistant soybeans, corn, cotton, rape and other genetically modified ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/65A01H4/00A01H5/00A01H6/46
CPCA01H4/00C12N15/65C12N15/82C12N15/821
Inventor 黄培劲吴永忠安保光陈思兰欧阳超龙湍李新鹏曾翔
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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