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Rice ALS mutant gene, plant transgenic screening vector pCALSm3 which contains gene and application of plant transgenic screening vector pCALSm3

A mutated gene and transgenic technology, applied in the field of plant transgenic screening expression cassettes and plant transgenic screening vectors, to achieve the effect of high resistance

Active Publication Date: 2020-08-28
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[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 pCALSm3 which contains gene and application of plant transgenic screening vector pCALSm3
  • Rice ALS mutant gene, plant transgenic screening vector pCALSm3 which contains gene and application of plant transgenic screening vector pCALSm3
  • Rice ALS mutant gene, plant transgenic screening vector pCALSm3 which contains gene and application of plant transgenic screening vector pCALSm3

Examples

Experimental program
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Effect test

Embodiment 1

[0088] Embodiment 1 Contains the construction of the plant genetic transformation screening vector of rice ALS mutein

[0089] 1. Obtaining rice ALS mutant protein

[0090] 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 ALS muteins with amino acid mutations at positions Ala), 96 (Ala is mutated to Thr), 171 (Pro is mutated to Arg), 548 (Trp is mutated to Leu), and 627 (Ser is mutated to Ile) As shown in SEQ ID NO.1, the nucleotide sequence of the coding gene is shown in SEQ ID NO.2), the mutant protein is compared with the single point mutations at positions 95, 96, 171, 548 and 627 Mutants and other site combination mutants have higher resistance to ALS inhibitors, and solve the problem that each single point mutant has a sing...

Embodiment 2

[0117] Embodiment 2 Agrobacterium transformation and identification

[0118] Take Agrobacterium EHA105 competent cells stored at -80°C, add 1 μl of the sequenced correct pCALSm3 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

[0119] Example 3 Critical Concentration Test of Bispyribac, Imazapyr and Imazethapyr at the Seedling Stage of Common Rice

[0120] 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.

[0121] 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 rice ALS mutant gene, a plant transgenic screening vector pCALSm3 which contains the gene and application of the plant transgenic screening vector pCALSm3. The amino acid sequence of the rice ALS mutant protein is shown in SEQ ID No. 1. According to the plant genetic transformation screening vector, an expression cassette of the rice ALS mutant gene is used as a screening marker, pyrimidinylsalicylic acid, imidazolinone and other herbicide are used as screening agents in a callus screening stage, and obtained transgenic plants have high resistance to pyrimidinylsalicylic acid, imidazolinone and other herbicide. The vector can be used as a transgenic screening vector which is matched with other functional elements, and plant endogenous genes are utilized in the transgenosis process, no bacterium sources and other exogenous screening marker genes are introduced, so that a plant transgenosis screeningmethod is enriched, potential safety risks, caused by the exogenous genes, of the transgenic plants are reduced effectively, and commercial application of the transgenic plants is facilitated.

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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IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/00A01H6/46
CPCC12N9/1022C12Y202/01006C12N15/8274
Inventor 安保光欧阳超陈思兰赵惠敏陈欣妍曾翔吴永忠黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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