Ginger flower floral specific and damage and pest induced TPS2 (Trehalose-6-Phosphate Synthase 2) promoter and application thereof

A promoter and specific technology, applied in the field of genetic engineering, can solve the problems of lack of space-time specificity, abnormal changes in plant physiology and morphology, affecting the normal growth and development of plants, etc., and achieve the effect of shortening the breeding time.

Active Publication Date: 2015-05-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the choice of promoters in transgenic technology mainly adopts constitutive promoters. Under the control of constitutive expression promoters, foreign genes can be expressed in all parts of transgenic plants and at different developmental stages, and can be expressed continuously and efficiently. No space-time specificity, resulting in energy loss, affecting the survival rate of positive plants, causing abnormal changes in plant physiology and morphology, thus affecting the normal growth and development of plants

Method used

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  • Ginger flower floral specific and damage and pest induced TPS2 (Trehalose-6-Phosphate Synthase 2) promoter and application thereof
  • Ginger flower floral specific and damage and pest induced TPS2 (Trehalose-6-Phosphate Synthase 2) promoter and application thereof
  • Ginger flower floral specific and damage and pest induced TPS2 (Trehalose-6-Phosphate Synthase 2) promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Obtaining of TPS2 promoter sequence

[0037] S1. Extraction of total DNA from Gingerflower leaves: 0.1-0.2 g of Gingerflower plant leaf powder ground with liquid nitrogen was transferred to a 1.5 mL centrifuge tube. Add 1 mL of CTAB solution, bathe in water at 65°C for 30 minutes; invert once every 10 minutes. Then, add 1 mL of phenol:chloroform (1:1) to the centrifuge tube, invert several times, centrifuge at 10,000 rpm for 10 minutes, transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1) , mix well, and centrifuge at 10,000 rpm for 10 minutes. Transfer the supernatant to a new centrifuge tube. Add an equal volume of isopropanol, mix upside down, centrifuge at 10,000rpm for 10 minutes, remove the supernatant, wash once with 70% ethanol, vacuum dry, dissolve in 30 μL sterile water, and use for PCR amplification. The composition of the CTAB solution is: Tris 100mM, NaCl 1.4M, 20mM EDTA, CTAB 2%, mercaptoethanol 0.1...

Embodiment 2

[0054] Fusion expression of TPS2 promoter and GUS to transform tobacco

[0055] S1. According to the sequence of the TPS2 promoter obtained in Example 1, primers for amplifying the three 5'-terminal deletion sequences p-320, p-780 and p-19283 containing HindIII and BamHI restriction sites were designed. The primer sequences are as follows:

[0056] Primers for amplifying p-320:

[0057] p-320-F: 5'-GGCCAAGCTTATACGTGCATGTTACAAGTATC-3' (SEQ ID NO: 14)

[0058] Primers for amplifying p-780:

[0059] p-780-F: 5'-GGCCAAGCTTCTGTTTCAAAACATCCAATTCCTC-3' (SEQ ID NO: 15)

[0060] Primers for amplifying p-1928

[0061] p-1928-F: 5'-GGCCAAGCTTACTGATAAGATAAGTTGC-3' (SEQ ID NO: 16)

[0062] Downstream primers are:

[0063] R: 5'-GGCGGATCCAATTTCAACTTCAAGTCCTGAC-3' (SEQ ID NO: 17)

[0064] S2. The pCAMBIA1300G plant expression vector was double-digested with HindⅢ and BamHI restriction endonucleases, and the large fragment was recovered on 1% agarose gel. Directly insert the target fr...

Embodiment 3

[0070] Transformation of Tobacco by Fusion Expression of TPS2 Promoter and Floral Aroma Gene HcTPS1

[0071] The HcTPS1 cloned recombinant plasmid was used as a template, and PCR amplification was carried out with specific primers introduced into SpeI and BamHI restriction sites. The PCR product was recovered with a TaKaRa recovery kit, and the recovered product was directly digested with SpeI and BamHI restriction endonucleases, and the target fragment was recovered on 1% agarose gel. The above constructed p-780 plant expression vector containing the HcTPS2 promoter was double-digested with SacI and BamHI restriction enzymes, and the large fragment was recovered from 1% agarose gel. The digested products were ligated and transformed into Escherichia coli DH5α. After enzyme digestion and sequencing identification, the recombinant plant expression vector is obtained. Agrobacterium tumefaciens EHA105 competent cells were prepared, and the above-mentioned recombinant plant expr...

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Abstract

The invention belongs to the technical field of gene engineering and particularly discloses a ginger flower floral specific and damage and pest induced TPS2 (Trehalose-6-Phosphate Synthase 2) promoter and an application thereof. The promoter has a nucleotide sequence as shown in SEQ ID NO: 2. A target gene controlled by the TPS2 promoter disclosed by the invention is only expressed in a flower tissue of a transgenic tobacco, and the expression is relevant to a flower development process and is induced by damage and pests. In addition, the TPS2 promoter disclosed by the invention can be introduced to a cell of a ginger flower or other plants after being connected to any one plant transformation vector to obtain promoter-containing transgenic fragrance and resistance to be used for production; or a specific molecular marker can also be generated according to the sequence information of the promoter disclosed by the invention and is used for authenticating the fragrance and resistance genotypes of the ginger flower or other plants and realizing the molecular-marker-assisted selective breeding, so that the breeding selection efficiency is increased.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a TPS2 promoter which is specific to the flower part of ginger flower and can be induced by damage and pests and its application. Background technique [0002] The promoter is a DNA sequence located in the upstream region of the 5' end of the structural gene that can recognize and activate RNA polymerase to accurately combine with the template DNA to ensure accurate and efficient initiation of transcription. It is the center of gene transcription regulation in plants, and is the key to understanding gene transcription regulation mechanism and expression pattern. [0003] According to the transcription mode of promoters, they can be divided into: constitutive promoters, tissue or organ-specific promoters and inducible promoters. Among them, tissue-specific promoters are also called organ-specific promoters, including fruit-specific promoters, root-specific promoters, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N1/21C12N1/15C12N1/19
Inventor 范燕萍李昕悦余让才吴永琼岳跃冲玉云祎
Owner SOUTH CHINA AGRI UNIV
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