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Cloning and activity analysis of drought and salt damage induced promoter

An inducible and promoter sequence technology, applied in the field of plant genetic engineering, can solve the problem of few inducible promoters and achieve high-efficiency expression

Inactive Publication Date: 2012-03-21
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few inducible promoters that can be used in transgenic research

Method used

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  • Cloning and activity analysis of drought and salt damage induced promoter
  • Cloning and activity analysis of drought and salt damage induced promoter
  • Cloning and activity analysis of drought and salt damage induced promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the cloning of the promoter of maize dehydrin gene (Zmdhn2)

[0039] The promoter of maize dehydrin gene (Zmdhn2) (the promoter sequence comprises the DNA nucleotide sequence of the -1bp to -1659bp region relative to the transcription initiation site of SEQ ID NO: 1), at the 5' of maize Zmdhn2 gene identified in the region sequence.

[0040] Zmdhn2 is registered in NCBI GenBank (accession number: L35913), and the sequence listing shows the DNA sequences of the maize drought and salt inducible promoter and 5' untranslated region of the above-mentioned gene of the present invention. In SEQ ID NO.1, the translation initiation codon ATG is underlined, using http: / / www.fruitfly.org / seq_tools / promoter.html The website predicts the transcription start site, which is marked with +1. And analyze the site with the promoter ( http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) analyzed the core elements of the promoter.

[0041] Maize genomic DNA was ...

Embodiment 2

[0045] Embodiment 2: the construction of maize dehydrin gene (Zmdhn2) promoter expression vector

[0046] The maize dehydrin gene (Zmdhn2) promoter cloned in Example 1 and the 5' untranslated region Zmdhn2Pro (see sequence listing) of 65bp were inserted into the vector, thereby constructing the maize dehydrin gene (Zmdhn2) promoter expression vector .

[0047] More specifically, the recombinant plasmid pMD18-T::Zmdhn2Pro( Figure 5 ) and the plant expression vector pCAMBIA1301, the target fragments ( Figure 6 ) and pCAMBIA1301 were ligated with T4 ligase to obtain the pCAMBIA 1301::Zmdhn2Pro expression vector, which was used to drive the expression of the GUS gene. The promoter fragment was identified by PCR ( Figure 7 ), with the expected result. Agrobacterium EHA105 was transformed by electric shock method, and PCR detection of bacteria liquid was carried out ( Figure 8 ).

[0048] exist Figure 9 Among them, the gene GUS encoding β-glucuronidase was used as the re...

Embodiment 3

[0049] Example 3: Identification of the activity of the maize drought and salt damage inducible promoter according to the present invention

[0050] In order to identify the stress-induced activity of the promoter, the corn embryos were drought-treated by the method of Jefferson et al. (EMBO J, 1987) and then the activity of GUS was detected.

[0051] 3.1 Preparation of acceptor material

[0052] Soak corn seeds in water for 1 day, let them fully absorb water, put them in an environment of 24°C for 1 day to accelerate germination, and then cut the seeds in half longitudinally for later use.

[0053] 3.2 Preparation of Agrobacterium bacteria solution

[0054] Agrobacterium EHA105 was inoculated on YEP (50mg / L rif, 50mg / L kan) solid medium, and cultured in the dark at 28°C until a single colony grew. Pick a single colony, inoculate it in 5 mL of YEP liquid medium containing the corresponding antibiotic, and cultivate it at 28°C with shaking at 200r / min. When the bacterial sol...

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Abstract

The invention relates to cloning and activity analysis of a drought and salt damage induced promoter, and belongs to the technical field of plant gene engineering. The invention provides a corn drought and salt damage induced promoter sequence containing a DNA nucleotide sequence of -1bp to -1659p areas relative to an initial transcription site of SEQ ID NO: 1, and simultaneously provides a drought and salt damage induced plant expression vector for corn transformation, wherein the plant expression vector contains a corn drought induced promoter sequence and a 5' un-translated region of corn dehydrin genes; and polymerase chain reaction (PCR) primers of SEQ ID NO: 2 and SEQ ID NO: 3 are suitable for amplifying DNA fragments containing the sequence of SEQ ID NO: 1. The cloning and activityanalysis can be used for promoting efficient expression of adversity related genes, are used in drought and salt tolerant transgenic plants, and have significance for solving the problems of food crisis, ecological deterioration and the like in drought and salt damage districts.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to a DNA sequence, which can be used as a promoter to regulate the expression of genes under two adversity stresses of drought and salt damage. Specifically, the invention relates to a dehydration gene derived from maize ( Zmdhn2), and a drought and salt inducible promoter sequence highly expressed in plants. Background technique [0002] Against the backdrop of intensified global climate change, increased pressure on resources and the environment, the impact of the financial crisis, and the deepening downward trend of food supply, ensuring my country's food security has always been an urgent and important strategic task. Adversity stress is one of the most important factors and challenges affecting food production in agricultural production. Every year, crop yield reduction directly caused by adversity stresses such as salinity, drought, and low temperature exceeds...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/11
Inventor 贾承国潘洪玉张世宏刘金亮胡瑞学李桂华
Owner JILIN UNIV
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