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Plant transgenic screening vector pcalsm1 and its application

A genetically modified and plant-based technology, applied in the field of agricultural biology, can solve potential safety risks and other problems, and achieve the effects of reducing potential safety risks, enriching screening methods, market value and social benefits

Active Publication Date: 2022-03-25
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 its application
  • Plant transgenic screening vector pcalsm1 and its application
  • Plant transgenic screening vector pcalsm1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Construction of plant genetic transformation screening vector

[0073] 1. Preparation of plant transgene screening expression cassette

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

[0075] Primers 0310-AAU-F / 0310-AAU-Rv1 were designed to amplify the promoter OsALSP fragment from the rice genome; primers 0310-AAU-F2 / 0310-AAU-Rv2 were used to amplify the synthetic rice ALS mutant gene (ALSm1) fragment Amplify the target gene ALSm1 fragment; use the primer 0310-AAU-F3 / 0310-AAU-Rv to amplify the terminator OsUbiT fragment from the rice genome. Among them, the 5' ends of primers 0310-AAU-F and 0310-AAU-Rv both have about 15 nucleotide sequences repeated with the corresponding junction 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-F...

Embodiment 2

[0100] Example 2 Agrobacterium transformation and identification

[0101] The competent Agrobacterium EHA105 cells stored at -80°C were added with 1 μl of the correctly sequenced pCALSm1 plasmid obtained in Example 1, and electroporated at 1.8KV for transformation. Coat on YEP culture plate containing kanamycin, rifampicin and streptomycin, cultivate at 28°C for about 48h, pick a single colony and shake overnight, and use specific primers (0310-F2 and 0310-R2) to carry out bacterial culture. Liquid PCR validation (e.g. figure 2 ), a target fragment of about 900 bp can be obtained by amplification, positive clones (engineering Agrobacterium) are selected, the bacteria are shaken for 36-48 hours, and the bacterial solution is stored for infection.

Embodiment 3

[0102] Embodiment 3 The critical concentration test of bispyribac, imazapyr and imazethapyr at the seedling stage of common rice

[0103] Bispyribac is a herbicide used in paddy fields. Ordinary rice is resistant to it. Therefore, spraying the herbicide with the prescribed concentration on the rice will not cause damage to the rice. Most of the spraying safe concentrations are 30mg / L; A non-selective lethal herbicide, common rice is not resistant to it, most recommended spraying concentration is 95-98mg / L; imidazolinone herbicide, mainly used for leguminous crops to control grass weeds And broad-leaved weeds, it has the advantages of strong selectivity, wide herbicidal spectrum and high activity. The recommended concentration is 75mg / L, and ordinary rice has weak resistance to it.

[0104] The present embodiment analyzes the critical concentration of rice resistance to bispyribac, imazapyr and imazethapyr, through experimental tests, the results are as follows Figure 5 , I...

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Abstract

The invention relates to the field of agricultural biotechnology, in particular to a plant transgene screening vector pCALSm1 and its application. The plant genetic transformation screening vector provided by the present invention uses an expression cassette containing a rice acetolactate synthase mutant gene as a screening marker, and uses pyrimidine salicylates, imidazolinones and other herbicides as screening agents in the callus screening stage. The obtained transgenic plants have high resistance to herbicides such as pyrimidine salicylates and imidazolinones. The vector can be used as a transgenic screening vector with additional functional elements. During the transgenic process, plant endogenous genes are used without introducing exogenous screening marker genes such as bacterial sources. The potential safety risks of transgenic plants and public concerns about transgenic plants are conducive to the commercial application of transgenic plants, with good market value and social benefits.

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 has become more and more extensive. Transgenic technology has many advantages, such as widening available genetic resources and creating new germplasm resources; directional mutation and selection of plant traits; providing new ways for cultivating high-yield, high-quality, and high-resistance varieties. GM crops were approved for commercial cultivation in 1996. As of 2017, a total of 23 GM crops have been approved for commercial production worldwide, involving more than 16 categories of target traits. According to statistics, in 2017, 26 countries and regions around the wo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/54A01H5/00A01H6/46A01H4/00A01H6/54
CPCC12N15/8274C12N9/1022C12Y202/01006A01H4/001
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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