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Rice als mutant gene, plant transgenic screening vector pcalsm2 containing the gene and application thereof

A technology for mutating genes and transgenics, applied in the fields of application, plant regeneration, plant products, etc., to achieve the effect of enriching screening methods, facilitating commercial applications, and reducing potential safety risks

Active Publication Date: 2022-03-04
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] One of the purposes of the present invention is to provide rice ALS mutant protein and its coding gene; another 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 gene genetic transformation, to provide a method using Plant endogenous gene as a screening marker applied to the carrier of plant genetic transformation screening

Method used

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  • Rice als mutant gene, plant transgenic screening vector pcalsm2 containing the gene and application thereof
  • Rice als mutant gene, plant transgenic screening vector pcalsm2 containing the gene and application thereof
  • Rice als mutant gene, plant transgenic screening vector pcalsm2 containing the gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0089] Embodiment 1 Contains the construction of the plant genetic transformation screening vector of rice ALS mutein

[0090] 1. Obtaining rice ALS mutant protein

[0091] The present invention has carried out a large number of analyzes and combination comparisons on the existing plant ALS herbicide resistance mutation sites, and screened from different site combinations for the rice ALS protein, relative to the rice wild-type ALS protein at position 95 (Gly mutation Ala), 548 (Trp is mutated to Leu) and 627 (Ser is mutated to Ile) ALS mutant protein with amino acid mutation (amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.2), the mutant protein has higher resistance to ALS inhibitors than the single point mutants at positions 95, 548 and 627, and other site combination mutants, and solves the problem of each single point mutation The problem of a relatively single type of herbicide resistance, it has high res...

Embodiment 2

[0118] Embodiment 2 Agrobacterium transformation and identification

[0119] Take Agrobacterium EHA105 competent cells stored at -80°C, add 1 μl of the sequenced correct pCALSm2 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

[0120] Example 3 Critical Concentration Test of Bispyribac, Imazapyr and Imazethapyr at the Seedling Stage of Common Rice

[0121] 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.

[0122] 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 field of agricultural biotechnology, in particular to a rice ALS mutant gene, a plant transgene screening vector pCALSm2 containing the gene and applications thereof. The amino acid sequence of the rice ALS mutant protein of the present invention is shown in SEQ ID NO.1. The plant genetic transformation screening vector of the present invention uses the expression cassette of the rice ALS mutant gene as a screening marker, and pyrimidine salicylic acid is used in the callus screening stage Herbicides such as salicylates and imidazolinones were used as screening agents, and the transgenic plants obtained had 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 are beneficial to the commercial application of transgenic plants.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a rice ALS mutant gene, a plant transgene screening expression cassette containing the gene, 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/00A01H6/46A01H4/00
CPCC12N9/1022C12N15/821C12N15/8274C12N15/8278A01H4/001A01H4/008C12Y202/01006
Inventor 安保光陈思兰欧阳超陈欣妍赵惠敏龙湍吴永忠黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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