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A plant genetic transformation screening vector and its application

A technology of genetic transformation and screening medium, which is applied in the fields of application, plant regeneration, plant products, etc., and can solve the problems of safety risks of exogenous screening markers, etc.

Active Publication Date: 2021-05-28
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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  • A plant genetic transformation screening vector and its application
  • A plant genetic transformation screening vector and its application
  • A plant genetic transformation screening vector and its application

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 with 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 ...

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Abstract

The invention provides a plant genetic transformation screening vector and its application. The vector utilizes an expression cassette containing the rice P450 gene (CYP81A6) as a screening marker, and uses bentazon or bensulfuron-methyl as a screening agent to carry out the screening in the callus stage. After screening, the obtained plants were detected as transgenic positive plants and possessed high resistance to herbicides. The plant genetic transformation carrier of the present invention can be used as a transgenic screening carrier with additional functional elements. During the transgenic process, the rice's own genes are used without introducing exogenous screening marker genes such as bacterial sources, which not only enriches the plant transgenic screening method, but also reduces public Concerns about the safety risks of genetically modified plants are conducive to promoting the commercial application of genetically modified plants, which have good 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 Patents(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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