Plant transgenic screening vector pCALSm1 and application thereof

A genetically modified and plant-based technology, applied in the field of agricultural biology, can solve potential safety risks and other issues

Active Publication Date: 2020-08-21
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of potential safety risks caused by the use of exogenous screening markers in the current genetic transformation, and to provide a carrier that uses plant endogenous genes as screening markers for plant genetic transformation screening

Method used

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  • Plant transgenic screening vector pCALSm1 and application thereof
  • Plant transgenic screening vector pCALSm1 and application thereof
  • Plant transgenic screening vector pCALSm1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 Construction of Plant Genetic Transformation Screening Vector

[0073] 1. Preparation of plant transgenic screening expression cassettes

[0074] The construction method of the plant transgenic screening expression cassette OsALSP-ALSm1-OsUbiT of the present invention is as follows:

[0075] The primers 0310-AAU-F / 0310-AAU-Rv1 were designed to amplify the promoter OsALSP fragment from the rice genome; The ALSm1 fragment of the target gene was amplified; the terminator OsUbiT fragment was amplified from the rice genome using primers 0310-AAU-F3 / 0310-AAU-Rv. Among them, the 5' ends of primers 0310-AAU-F and 0310-AAU-Rv have about 15 nucleotide sequences repeated with the corresponding connection positions of the vector; the 5' ends of the upstream and downstream primers of adjacent fragments also have 15 bp repeats (0310- AAU-Rv1 and 0310-AAU-F2, 0310-AAU-Rv2 and 0310-AAU-F3) for subsequent recombination connection using Gibson Assembly.

[0076] The above...

Embodiment 2

[0100] Embodiment 2 Agrobacterium transformation and identification

[0101] Take Agrobacterium EHA105 competent cells stored at -80°C, add 1 μl of the sequenced correct pCALSm1 plasmid obtained in Example 1, and transform by electroporation at 1.8KV. Spread on a YEP culture plate containing kanamycin, rifampicin and streptomycin, culture at 28°C for about 48 hours, pick a single colony and shake it overnight, and use specific primers (0310-F2 and 0310-R2) to inoculate Liquid PCR verification (such as figure 2 ), can amplify to obtain about 900bp target fragment, select the positive clone (engineering Agrobacterium), shake the bacteria for 36-48h, and save the bacterial liquid for infection.

Embodiment 3

[0102] Example 3 Critical Concentration Test of Bispyribac, Imazapyr and Imazethapyr at the Seedling Stage of Common Rice

[0103] Bispyribac is a herbicide used in paddy fields, and ordinary rice is resistant to it, so spraying the herbicide at the specified concentration on the rice will not cause damage to the rice, and the safe concentration of most sprays is 30mg / L; imazapyr is A non-selective killing herbicide, ordinary rice is not resistant to it, most recommended spraying concentration is 95-98mg / L; imazethapyr is an imidazolinone herbicide, mainly used for the control of grass weeds in leguminous crops And broad-leaved weeds, it has the advantages of strong selectivity, broad herbicidal spectrum and high activity. The recommended concentration is 75mg / L, and ordinary rice has weak resistance to it.

[0104] This embodiment analyzes the critical concentration of rice resistance to bispyribac, imazethapyr and imazethapyr, and through experimental testing, the results ar...

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Abstract

The invention relates to the technical field of agricultural biology, in particular to a plant transgenic screening vector pCALSm1 and application thereof. According to the plant genetic transformation screening vector provided by the invention, an expression cassette containing a rice acetolactate synthase mutant gene is used as a screening marker; in the callus screening stage, pyrimidinylsalicylic acid, imidazolinone and other herbicides are used as screening agents, and the obtained transgenic plants have high resistance to pyrimidinylsalicylic acid, imidazolinone and other herbicides. Thevector can be used as a transgenic screening vector to be added with other functional elements; a plant endogenous gene is utilized in a transgenosis process; exogenous selection marker genes such asa bacterial source are not introduced, so that not only are plant transgenic screening methods enriched, but also potential safety risks of transgenic plants caused by exogenous genes and worries ofthe public about the transgenic plants can be effectively reduced, commercial application of the transgenic plants is facilitated, and the market value and social benefits are good.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a plant transgene screening expression cassette, a plant transgene screening vector containing the expression cassette and applications thereof. Background technique [0002] With the rapid development of genetic engineering and molecular biology technology, the application of transgenic technology is becoming more and more extensive. Transgenic technology has many advantages, such as broadening available genetic resources and creating new germplasm resources; directional and fixed-point variation and selection of plant traits; providing new ways to breed high-yielding, high-quality, and high-resistant varieties. Genetically modified crops were approved for commercial planting in 1996. As of 2017, a total of 23 types of genetically modified crops have been approved for commercial production in the world, involving more than 16 categories of target traits. According to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/54A01H5/00A01H6/46A01H4/00
CPCA01H4/001C12N9/1022C12N15/8274C12Y202/01006
Inventor 欧阳超安保光陈思兰赵惠敏李丹龙湍吴永忠黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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