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Low-temperature induction-type promoter of tea tree

A low-temperature induction and promoter technology, applied in the field of genetic engineering, can solve the problems of identification, isolation and expression research, and achieve significant economic benefits, improve cold resistance, and broad market prospects

Inactive Publication Date: 2014-12-24
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As one of the most common adversity stresses, low temperature has a profound impact on the growth and development of plants, especially tea trees, but there are few studies on the identification, isolation and expression of related promoters

Method used

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  • Low-temperature induction-type promoter of tea tree
  • Low-temperature induction-type promoter of tea tree
  • Low-temperature induction-type promoter of tea tree

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Acquisition of plant adversity stress-induced expression promoter CsPE1α

[0023] 1. Primer Design

[0024] According to the cDNA sequence of the tea tree pollen CsE1α gene, primers were designed to amplify its promoter, and the specific primer sequences were as follows:

[0025] Specific primer sequence:

[0026] GSP1: as shown in SEQ ID NO.2;

[0027] GSP2: as shown in SEQ ID NO.3;

[0028] GSP3: as shown in SEQ ID NO.4;

[0029] Random primers:

[0030] AD1: as shown in SEQ ID NO.5: 5'-tgwgnagwan casaga-3', wherein, w=a or t; n=a or g or c or t; s=g or c;

[0031] AD2: as shown in SEQ ID NO.6: 5'-cawcgicnga iasgaa-3', wherein, w=a or t; i=a or g or c or t; n=a or g or c or t; s= g or c;

[0032] AD3: as shown in SEQ ID NO.7: 5'-ntcgastwts gwgtt-3', wherein, n=a or g or c or t; s=g or c; w=a or t;

[0033] AD4: as shown in SEQ ID NO.8: 5'-ntcgastwts gwgtt-3', wherein, n=a or g or c or t; s=g or c; w=a or t;

[0034] AD5: as shown in SEQ ID NO.9: 5'...

Embodiment 2

[0051] Example 2, construction of recombinant expression vector (pBI121-CsPE1α-GFP)

[0052] 1. Use the following primers: pE1-ScaI-R PBI121-GFP and pE1-HindIII-F PBI121-GFP to perform PCR amplification on the Escherichia coli DH5α recombinant bacterial liquid, and introduce restriction sites Sca I and Hind III. The PCR amplification process is the same as the third round in Example 1.

[0053] pE1-ScaI-R PBI121-GFP is shown in SEQ ID NO.11

[0054] pE1-HindIII-F PBI121-GFP as shown in SEQ ID NO.12

[0055] 2. Digest the vector pBI121-GFP with restriction endonucleases Sca I and Hind III, cut off the CaMV35S region, and recover the vector skeleton of about 11 kb.

[0056] 3. Digest the plasmid extracted from the DH5α bacterial liquid obtained in Example 1 with restriction endonucleases Sca I and Hind III to obtain the digested product.

[0057] 4. Combine the vector backbone of step 1 and the digestion product of step 2 at T 4 Under the action of DNA ligase, the recombinan...

Embodiment 3

[0058] Example 3, Transient expression analysis of plant adversity stress-induced expression promoter CsPE1α

[0059] According to the PDS-1000 / HE Biolistic gene gun bombardment operation method of BioRad Company, the recombinant vector pBI121-CsPE1α-GFP gene gun method was used to transform the onion inner epidermis cultured by hyperosmosis. After 1 day of dark culture, the transient expression was observed with fluorescent stereomicroscope and laser confocal microscope respectively. Some results are as follows: Figure 4 , 5 shown.

[0060] The results showed that under the excitation light of GFP, the onion inner epidermal cells transfected with pBI121-CsPE1α-GFP could emit fluorescence; CsPE1α could drive the expression of downstream genes.

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[0066]

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Abstract

The invention belongs to the field of gene engineering, and provides an induction-type promoter CsPE1 alpha from a tea tree and expression of the induction-type promoter CsPE1 alpha. The induction-type promoter provided by the invention is any one DNA molecule as shown in 1) and 2), wherein 1) the induction-type promoter is a DNA molecule shown as a sequence 1 in a sequence table; or 2) the induction-type promoter is a DNA molecule which is hybridized with a DNA sequence limited in the 1) under a stringent condition and is coded so as to achieve a promoter function. The promoter provided by the invention can be used for driving the expression of target genes in a plant, thereby providing a meaningful way for people to research the growing development of the tea trees and offering assistance for genetic modification of plants and especially for genetic modification of theaceae. The promoter provided by the invention is capable of inducing and starting cold-resistant genes under the low-temperature stress so as to effectively prevent and control cold damage, and simultaneously ensures that the cold endurance of the tea tree is enhanced and the acclimatization capacity of the tea tree is improved. Thus, tea leaves can be produced in regions in higher latitudes.

Description

1. Technical field [0001] The invention belongs to the field of genetic engineering, and relates to a low-temperature inducible promoter, including a structure and a vector containing the promoter, as well as a host cell and a transgenic plant containing the promoter or the structure or the vector. 2. Technical background [0002] In the process of cell metabolism, the pyruvate dehydrogenase complex (PDC) occupies a key metabolic position and has a significant impact on energy metabolism. The PDC-E1α gene is the gate connecting the energy metabolism pathway, if the gene is abnormal, it will definitely affect the entire energy metabolism pathway. [0003] Promoters commonly used in plant genetic engineering can be divided into three categories according to their mode of action and function: constitutive promoters, tissue-specific promoters and inducible promoters. The promoter CsPE1α provided by the present invention is an inducible promoter. Inducible promoters have always...

Claims

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Application Information

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IPC IPC(8): C12N15/113
Inventor 黎星辉杜昱林王玉花
Owner NANJING AGRICULTURAL UNIVERSITY
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