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Retrotransposon promoter PCb-RARE induced by high salt and salicylic acid and application of retrotransposon promoter PCb-RARE

A technology of retrotransposon and promoter, applied in retrotransposon promoter PCb-RARE and its application field

Active Publication Date: 2015-03-25
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how to control the timing and quantitative high-efficiency expression of exogenous genes is an important factor limiting the effective application of genetic engineering technology in crop genetic improvement

Method used

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  • Retrotransposon promoter PCb-RARE induced by high salt and salicylic acid and application of retrotransposon promoter PCb-RARE
  • Retrotransposon promoter PCb-RARE induced by high salt and salicylic acid and application of retrotransposon promoter PCb-RARE
  • Retrotransposon promoter PCb-RARE induced by high salt and salicylic acid and application of retrotransposon promoter PCb-RARE

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Extraction of Bamboo Tree DNA and Putative LTR Sequence of Retrotransposon Cb-RARE-1 ​​P Cb-RARE A clone of:

[0025] Total DNA was extracted from bamboo leaves using the improved CTAB method (Doyle and Doyle, 1990). LA PCR using TaKaRa kit TM Kit, amplify the LTR sequence P of Cb-RARE-1 ​​according to the reaction system of its instructions Cb-RARE .

[0026] The primer pairs used were:

[0027] Cb-promoter-F:5'-GAATTCAGAGGTGTCTATTGAGCATATT-3'(SEQ ID NO:2)

[0028] Cb-promoter-R2: 5'-GGTACCCACATCTATAAGCATAAT-3' (SEQ ID NO: 3).

[0029] The recognition sequence of restriction endonuclease EcoR I (5'-GAATTC-3') or KpnI (5'-GGTACC-3') was added to the 5' end of the primer.

[0030] The PCR reaction program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 45 sec, renaturation at 58°C for 45 sec, extension at 72°C for 3 min, 35 cycles; final extension at 72°C for 10 min. The amplified product was recovered (Axygen Biosciences), connected to the pMDT...

Embodiment 2

[0032] Contains P Cb-RARE :: Construction of the recombinant expression vector pBI-pCb-RARE of the GUS expression cassette:

[0033] Expand the positive clone (this clone contains P Cb-RARE sequences of recombinant plasmids). Using the plasmid extraction kit (AXYGEN AXYPrep TM Plasmid Minprep Kit) extracts plasmid DNA, takes this plasmid as template, and following sequence is primer, amplifies P by the PCR reaction procedure among the embodiment 1 Cb-RARE sequence.

[0034] P Cb-RARE -F:5'-AAGCTTAGAGTGTCTATTGAGCATATT-3' (SEQ ID NO:4).

[0035] P Cb-RARE -R:5'-GTCGAC CACATCTATAAGCATAAT-3' (SEQ ID NO:5).

[0036] The resulting PCR reaction product was recovered with an agarose gel recovery kit (AXYGEN AXYPrep TM Plasmid Minprep Kit, Shanghai Shenergy Gaming) recovered. The recovery fragment was connected with T-vector (Takara, T-easy vector), and transformed into Escherichia coli JM109 bacterial strain, obtained containing P Cb-RARE sequence of the recombinant JM109 ...

Embodiment 3

[0040] Contains P Cb-RARE :: Construction of transgenic Arabidopsis with GUS expression cassette:

[0041] Expansion culture containing recombinant vector (pBI-P Cb-RARE ::GUS) Escherichia coli transformants, pBI-P was extracted after collecting the cells Cb-RARE ::GUS recombinant vector plasmid. Take purified pBI-P Cb-RARE :: Transform Agrobacterium EHA105 with the GUS recombinant vector plasmid, select colonies that can grow on LB culture plates containing 60mg / L rifampicin and 50mg / L kanamycin, and perform PCR identification, and select the colonies that can be amplified to P Cb-RARE Positive transformants of the fragment and save the Agrobacterium strain.

[0042] Take the above-mentioned Agrobacterium strains for activation and culture for one day, inoculate at an inoculation ratio of 1% and carry out expansion culture for more than 12 hours, until the OD 600 =0.6~0.8. Centrifuge at room temperature at 4000rpm for 10min, discard the supernatant, and collect the bacte...

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Abstract

The invention discloses a retrotransposon promoter PCb-RARE induced by high salt and salicylic acid and application of the retrotransposon promoter PCb-RARE. The promoter is derived from an LTR region of Carallia brachiata retrotransposon Cb-RARE-1, and is shown as SEQ ID NO: 1. A recombinant expression vector which contains the promoter and GUS gene is converted into Arabidopsis, the promoter is proved to be inducted of NaCl and the salicylic acid, and the promoter can be used for screening of saline-alkali tolerant plants and molecular breeding by using the characteristic. The inducible promoter is used for inducing foreign gene to be expressed in a plant, and the promoter function can be activated or suppressed by stimulation of the high salt or the salicylic acid, so that gene expression can be activated or suppressed by using high-concentration sodium chloride or salicylic acid in specific time or in a growth stage, synthesis of gene products can be controlled, and the growth of the plant can be adjusted on purposes.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a retrotransposon (also known as retrotransposon, retrotransposon) promoter P induced by high salt and salicylic acid Cb-RARE The promoter is derived from the LTR region of the retrotransposon Cb-RARE-1 ​​of the bamboo tree (Carallia brachiata). Background technique [0002] Retrotransposon (RTN) is an element that uses RNA as a medium to proliferate and transpose. Under the action of reverse transcriptase RT and RNaseH, the retrotransposon is reverse transcribed into extrachromosomal DNA, and then inserted into the new position of the chromosome under the action of integrase. The insertion of retrotransposons can cause stable mutations in or near genes. Therefore, the characteristic of retrotransposon jumping can be used for mutation initiation and for germplasm breeding and so on. [0003] Retrotransposons with long terminal repeats (long terminal repeats, LTRs) have a direct al...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12Q1/68
Inventor 唐恬梁山林星钦刘小如区佩如潘婷
Owner SUN YAT SEN UNIV
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