Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof

A technology for deleting mutants and promoter sequences, which is applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, etc., and can solve problems such as the lack of inducible promoters

Inactive Publication Date: 2010-02-10
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007]Although inducible promoters have many advantages compared with constitutive promoters, there are few known inducible promoters, and RD29A is the most widely used one There are relatively few classic promoters, and there are very few stress-inducible promoters with independent intellectual property rights in my country. Therefore, it is of great significance to clone and identify new stress-inducible promoter sequences and find out the core functional elements.

Method used

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  • Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof
  • Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof
  • Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Transducing the ZmPIS gene initiated by the TsVP1 promoter to create a drought-tolerant inbred line of maize and its application

[0069] 1) Construct the fusion gene of TsVP1 promoter and ZmPIS

[0070] The TsVP1 promoter was connected with the ZmPIS by a conventional gene recombination method, and recombined into the T-DNA region of the plant expression vector pCUA plasmid after sequencing verification. The pCUA plasmid was constructed in our laboratory and carries the plant selection marker gene als. Then use the freeze-thaw method to introduce into Agrobacterium LBA4404 or AGLO.

[0071] 2) The establishment of the receptor system is based on the corn backbone inbred lines used in my country's production, such as Zheng 58, Ye 478, Ye 515, etc., the inbred seeds are sterilized, and in vitro culture induces shoot tips to produce clustered buds. Genetic transformation was carried out using clustered shoots as recipients. The media used are:

[0072] Seed ...

Embodiment 2

[0086] Example 2: Transducing the T5-ZmPIS gene to create excellent drought-resistant breeding materials for wheat

[0087] 1) Construct the fusion gene of T5 promoter and ZmPIS

[0088] The T5 promoter was connected with the ZmPIS by conventional gene recombination method, and recombined into the T-DNA region of the plant expression vector pCUA-bar plasmid after sequencing verification. The pCUA-bar plasmid was constructed in our laboratory, carrying the plant selection marker gene bar, and then introduced into Agrobacterium LBA4404 or AGLO by freeze-thaw method.

[0089] 2) Obtained sterile wheat seedlings

[0090] Seeds of fine varieties of wheat are soaked with 70% ethanol for 1-3 minutes, then soaked with 0.1% mercury chloride for 8-12 minutes, and then washed with sterile water for 3-5 times. Shake the seeds constantly during sterilization to ensure complete surface sterilization. After sterilization, the seeds are placed in a sterile Erlenmeyer flask to germinate, an...

Embodiment 3

[0103] Example 3, Transducing the betA gene initiated by the T5 promoter to create excellent stress-resistant cotton breeding materials

[0104] 1) Fusion gene and plasmid construction of T5 promoter and betA gene and Agrobacterium culture

[0105] The T5 promoter is connected with the betA gene by a conventional gene recombination method, and recombined into the T-DNA region of the plant expression vector pCUA plasmid after sequencing verification. The pCUA plasmid carrying the plant selection marker gene als was then introduced into Agrobacterium LBA4404 or AGLO by freeze-thaw method. Agrobacterium cultivation and activation were the same as in Example 2.

[0106] 2) Obtain sterile cotton seedlings

[0107] Take the seeds of cotton inbred lines or fine varieties, use concentrated sulfuric acid to remove the surface fluff, soak in 70% ethanol for 1 minute, then soak in 0.1% mercuric chloride for 10-12 minutes, and then wash 3-5 times with sterile water . Shake the seeds c...

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Abstract

The invention discloses a thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and the application of a deletion mutant thereof. A clone carrying a TsVPI promoter is screened from a genomiclibrary of halophyte thellungiella, a 2200 nucleotide sequence positioned on the upstream of a TsVPI coding frame 5' is truncated as an overall-length promoter, promoter snippets with different lengths are obtained through PCR amplification, and then the promoter snippets are respectively blended with gus genes to be recombined into a plant expression vector to be converted into Arabidopsis; the TsVPI promoter and a part of the deletion mutant thereof are determined as salt-stress inducible type promoters by detecting the GUS enzymatic activity of transgenic plants, wherein a T5 promoter not only has a short sequence (667bp) but also has root specificity, and the TsVPI promoter and the T5 promoter are respectively connected with betA genes from colibacillus to be transmitted into tobaccosand corns so as to determine that the TsVPI promoter and the T5 promoter can normally exert functions in the transgenic tobaccos and the transgenic corns, are salt-stress inducible type strong promoters and have important application value in the industrialization development of plant gene engineering.

Description

technical field [0001] The invention belongs to the field of bioengineering breeding of crops and forest trees, and specifically relates to the cloning, transformation and application of a plant gene promoter sequence (sequence of a salt mustard coding tonoplast membrane pyrophosphatase gene TsVP1 promoter). Background technique [0002] Salt stress is one of the main abiotic stresses in nature, high concentrations of Na in soil + Causes great damage to the growth and development of many plants. Soil salinization and secondary salinization have become constraints to the sustainable development of irrigated agriculture in the world. With the growth of population and the scarcity of water resources, improving the utilization efficiency of water resources and fully developing and utilizing saline-alkali land have become important issues related to the national economy and people's livelihood. In recent years, with the in-depth research on Arabidopsis, rice and other model org...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
Inventor 张举仁孙清华李坤朋高强高峰
Owner SHANDONG UNIV
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