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Method for screening transgenic plant and its used bivalent plant expression vector

A technology of transgenic plants and expression vectors, which is applied in the field of bivalent plant expression vectors, can solve the problems of single-action safety of screening markers, and achieve the effect of convenient screening

Inactive Publication Date: 2003-04-23
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the lack of a single role or safety of screening markers in the existing methods for screening transgenic plants, the present invention provides a novel screening method and a plant bivalent expression vector used therefor

Method used

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  • Method for screening transgenic plant and its used bivalent plant expression vector
  • Method for screening transgenic plant and its used bivalent plant expression vector
  • Method for screening transgenic plant and its used bivalent plant expression vector

Examples

Experimental program
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Effect test

Embodiment 2

[0037] The ligation reaction was performed overnight at 16°C. Embodiment 2. Obtaining of Escherichia coli transformants

[0038] Preparation of E.coli XL-1-blue competent cells: ①Put a single colony of XL1-blue into 5mL LB liquid medium (see Table 2.1) without antibiotics, shake overnight at 37°C; ②Press 1% The amount of (v / v) was transferred to fresh LB liquid medium and shaken to OD at 37°C 600 =0.3; ③Add 50-100mL LB liquid culture medium (see Table 2.1 for LB preparation) into two sterile centrifuge tubes, place on ice for 30 minutes; ④Centrifuge at 4°C, 4000rpm (rev / min) for 10 minutes , remove the supernatant, and add 10 mL of pre-cooled 0.1% CaCl to each centrifuge tube 2 Resuspend the cells in the solution, and ice-bath for 30 minutes. ⑤ Centrifuge at 4°C and 4000rpm for 10 minutes, remove the supernatant, and suspend the bacteria in 2 mL of pre-cooled 0.1% CaCl 2 Add 300 μL of sterile glycerol to the solution, mix well, add 100 μL of XLl-blue competent cells t...

Embodiment 3

[0046] After complete dissolution, adjust the pH to 7.2 with 5mol / L NaOH, then add deionized water to a total volume of 1L, and then autoclave. Example 3. Transformation of Agrobacterium tumefaciens

[0047] Preparation of Agrobacterium tumefaciens LBA4404 competent cells: ① Pick LBA4404 colonies from the YEB solid medium (see Table 3.1) plate containing streptomycin (str) and rifampicin (Rif), inoculate into 5ml containing the corresponding antibiotic In the YEB liquid medium (according to Table 3.1, without adding agar), shake overnight at 200 rpm at 28°C. ② Dilute to 50ml with YEB liquid medium, then culture at 28°C and 200rpm for 6-12 hours until OD 600 The value is around 0.6. ③ Place on ice for 5 minutes, then centrifuge at 4°C, 2000rpm for 7 minutes. ④Take 1ml of ice-cold CaCl with a concentration of 20mmol / L 2 Resuspend the precipitate in the solution and mix well. ⑤ Divide into 10 portions, 100 μl each, and store in a -70°C refrigerator for later use.

[004...

Embodiment 4

[0055] The engineering bacterium that embodiment 3 obtains is infected tobacco explant (blade, cut into 0.5 * 0.5cm size) 5 minutes, blot dry with the filter paper through autoclaving, move into MS+6-BA (6-benzoladenine, 6 -benzylpurine) 1 mg / L+NAA (naphthalene acetic acid, naphthalene acetic acid) 0.1 mg / L+3% sucrose+0.7% agar in the dark for 48 hours. Rinse 3 times with sterile water, blot dry with sterile filter paper, and then transfer to MS+6-BA (1mg / L)+NAA (0.1mg / L) differentiation medium containing cephalosporin (400mg / L), Culture at 28±2°C under diffuse light conditions to induce differentiation. Culture for 1 week, and then transfer to the differentiation medium containing the selective agent glyphosate [MS+6-BA (1 mg / L)+NAA (0.1 mg / L)+glyphosate (30 μmol / L)]. Continue to cultivate for a period of time (3 weeks), when the shoots grow to 2-3cm, cut off the resistant shoots and move them into the MS+NAA (0.1mg / L) rooting medium that does not contain glyphosate to induc...

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Abstract

The present invention provides a method for screening plant transformant and its used plant bivalent expression vector. Said method uses glyphosate resistance gene instead of antibiotic resistance gene as marker gene for screening transgenic plant line. The plant bivalent expression vector used in said invented method contains glyphosate resistance gene and plant character gene to be screened. Said glyphosate resistance gene not only can be used as marker gene for screening plant, but also can be used as anti-herbicide gene, and can be linked with other inportant object character gene for transforming plant, so that it can be conveniently used for screening transformed plant and screening pure line breeding variety.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a method for screening transgenic plants using a glyphosate resistance gene as a marker gene, and a bivalent plant expression vector used therein. Background technique [0002] How to screen plant transformants is a key technique for successfully obtaining transgenic plants. There are several types of selectable markers according to the nature of their gene products: antibiotics, biochemicals, fluorescein and herbicide resistance. At present, antibiotic resistance genes are mostly used as marker genes for screening plant transformation, the most commonly used are neomycin phosphotransferase gene (Npt II) and hygromycin phosphotransferase gene (HPT). The former gene encodes an enzyme resistant to kanamycin (km) and neomycin (neo), and the latter gene encodes an enzyme resistant to hygromycin (hyg). Although these ant...

Claims

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Application Information

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IPC IPC(8): A01H1/00C12N15/82C12Q1/68
Inventor 谢龙旭徐培林
Owner SUN YAT SEN UNIV
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