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High-salt and dehydration-inducible promoter ipdhn-pro and its application

A promoter and inducible technology, applied in the field of plant genetic engineering, can solve problems such as unfavorable quality and yield improvement, poisoning, plant death, etc., and achieve the effect of reducing negative effects

Active Publication Date: 2019-01-29
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of promoter has high activity, but because it leads to long-term overexpression of foreign genes in all parts of the transgenic plants throughout the growth period, affecting the growth and normal physiological metabolism of plants, some may produce toxic effects, which is not conducive to quality and yield increases, and can even lead to plant death

Method used

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  • High-salt and dehydration-inducible promoter ipdhn-pro and its application
  • High-salt and dehydration-inducible promoter ipdhn-pro and its application
  • High-salt and dehydration-inducible promoter ipdhn-pro and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Cloning and sequence analysis of the promoter IpDHN-PRO of the Achillea dehydrin IpDHN gene

[0039] The rattan material used in the present invention is the seedling of the rattan seedling germinated in this laboratory, and the seeds were collected in Zhuhai Beach (22°16′25.37″N, 113°34′18.00″E). Take about 100 plump vine seeds, soak them in 10% sulfuric acid for 12 hours, wash them 20 times with tap water, germinate the vine seeds with vermiculite (28°C, 16 hours of light / 8 hours of darkness per day), about a month later Grow into seedlings. Take 0.1 g of leaves of healthy growing vine seedlings, put them into a mortar and add liquid nitrogen to grind them to powder, and use the plant genomic DNA extraction kit One-Tube Plant DNAOUT (article number: 60705) from Beijing Tianenze Gene Technology Co., Ltd. Genomic DNA from vine leaves. Using electrophoresis detection and ultraviolet spectrophotometer to detect the purity and concentration of the thick rattan...

Embodiment 2

[0043] Example 2: The expression of the Achillea dehydrin IpDHN gene is induced by high salt / dehydration stress

[0044] The present invention discloses for the first time the expression of the IpDHN gene in the Achilles vine under the regulation of its own promoter, and the detection method adopted is Real time RT-PCR technology. Real time RT-PCR primers were designed online through the cDNA sequence of IpDHN gene obtained by cloning in our laboratory and the website NCBI (http: / / www.dtd.nlm.nih.gov / ). The primers used to detect the expression pattern of IpDHN gene were IpDHN-RTF:5'-CCTGGGTACCACCCAAAGAC-3'(SEQ ID NO.4) and IpDHN-RTR:5'-GCACATAAAGTACTTCACAGCAAACC-3'(SEQ ID NO.5). The internal reference gene is IpUBQ, the ubiquitin protein gene of Achilles vine, and the primers are IpUBQ-RTF:5'-TCGACAATGTGAAGGCAAAG-3'(SEQ ID NO.6) and IpUBQ-RTR:5'-CTTGATCTTCTTCGGCTTGG-3'(SEQ ID NO.7) . Refer to BIO-RAD iTaq TM Prepare the real time RT-PCR reaction system (operated on ice) ac...

Embodiment 3

[0049] Example 3: Construction of the GUS transgenic Arabidopsis material under the regulation of the promoter IpDHN-PRO of the pachyderm dehydrin IpDHN gene

[0050] With the IpDHN-PRO-pGEMT plasmid DNA inserted with the Acanthus IpDHN gene promoter as a template, the following primers IpDHNProF: 5'-CGACTCTAGAGGATCCCAGTGGGGCTTCTTCTCCT-3' (SEQ ID NO.8) and IpDHNProR: 5'-ACCTACCCGGGGATCCGCTCCTCCGCAGGCTTCTG-3' were designed ( SEQ ID NO.9) performs PCR amplification on the promoter IpDHN-PRO of the Achilles dehydrin IpDHN gene. The PCR products were recovered by agarose gel electrophoresis according to the instructions of HiPure Gel Pure DNAKits of Magen Company. At the same time, the Arabidopsis transgene binary expression vector pBI101.2 was treated with BamHI single enzyme digestion, and the linearized plasmid was recovered. The concentration of recovered IpDHN-PRO promoter PCR fragment and linearized pBI101.2 plasmid was measured by UV spectrophotometer of Nanodrop Company, ...

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Abstract

The invention relates to a promoter of ipomoea pescaprae Dehydrin protein genes (IpDHN-PRO for short). The promoter has a nucleotide sequence shown in SEQ ID No. 1. Expression results of detection ofGUS provided in the invention show that the polynucleotide has promoter activity, and the high salt and drought stress induced activity is enhanced, so that an effect of enabling LEA protein gens to respond to a high salt / dehydration stress in the ipomoea pescaprae is shown. The promoter disclosed by the invention provides a theoretical basis for further researching and being applied to regulatingexpressions of ipomoea pescaprae related salt-tolerant genes, increasing and improving salt tolerance of the ipomoea pescaprae and other plants and expressing salt / drought stress related target proteins by taking plants as a bio-reactor.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a thick rattan high-salt, dehydration and ABA-inducible promoter and application thereof. Background technique [0002] High salinity and drought are the most common adversity stresses in the process of plant growth, which usually cause water loss in plants, resulting in an imbalance of intracellular water balance, which in turn affects the growth and development of plants. For crops, poor crop growth due to high salinity and drought, resulting in reduced yield and quality of crops is a common problem in the agricultural production process. Therefore, cultivating and promoting high-salt / drought stress-resistant varieties is an effective way to ensure stable and high yield of crops. Utilizing the stress-resistant genes of plants themselves, new stress-resistant varieties can be bred through conventional cross-breeding and molecular marker-assisted breeding meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82A01H5/10A01H6/20
CPCC12N15/8223C12N15/8273
Inventor 张会郑洁旋张美简曙光夏快飞
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI