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A promoter and gene technology, applied in the field of Populus euphratica NAC1 gene promoter
Inactive Publication Date: 2011-01-26
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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In the study on the upstream regulation of stress-induced NAC t...
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Embodiment 1
[0024] Embodiment 1, Populus euphratica PeNAC1 gene promoter isolation
[0025] The promoter fragment of Populus euphratica PeNAC1 gene was isolated from the genomic DNA of Populus euphratica by PCR Walking method, and the specific operation method was carried out according to the instructions of the Genome Walking Kit method (TaKaRa, Japan).
[0026] The extraction of Populus euphratica DNA adopts CTAB method, and its steps are as follows:
[0027] (1) Add 800 μL of 2×CTAB extraction buffer [2g / 100ml CTAB, 1.4mol / L NaCl, 20mmol / L EDTA, 100mmol / L Tris·Cl (pH8.0)], 8μL mercaptoethanol in a 2mL centrifuge tube (1%, v / v), preheated at 65°C.
[0028] (2) Take about 0.15 g of leaves, grind them into frozen powder in liquid nitrogen.
[0029] (3) Transfer the frozen powder into a centrifuge tube, keep warm at 65°C for about 30 minutes, and shake gently twice during this time.
[0030] (4) Take out the centrifuge tube and cool to room temperature. Add an equal volume of chlorofor...
Embodiment 2
[0036] Example 2, Analysis of the promoter activity of Populus euphratica PeNAC1 gene
[0037] The promoter activity of the 1217bp 5' flanking sequence of Populus euphratica PeNAC1 gene was detected by transient expression experiment of onion epidermal cells.
[0038] Replace the 35S promoter in the plant expression vector pCAMBIA-1304 with the 1217bp 5' flanking sequence of the obtained Populus euphratica PeNAC1 gene, and insert it into the upstream of the reporter gene CUS and GFP to obtain a new plant expression vector pCAMBIA:PeNAC1P-GFP plasmid, which was transformed into In Agrobacterium LBA4404, the onion epidermal cells were infected by Agrobacterium, and the 1217bp 5'flanking sequence of Populus euphratica PeNAC1 gene obtained by transient expression experiment was detected whether it had promoter activity.
[0039] Using the enzyme cutting site analysis system (http: / / tools.neb.com / NEBcutter2 / index.php) on the NEB website to analyze all the enzyme cutting sites conta...
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Abstract
The invention discloses a subsequence of Ammopiptanthus mongolicus PeNAC1 gene 1217bp promoter, which is named as PeNAC1P. A PLACE database can be used for analyzing and predicting cis acting elements which are contained in PeNAC1P and are relevant to the induction of drought, high salts, ABA, GA, JA, SA, MeJA and other hormones, elements relevant to bacterial and salts, cis elements responding to calcium ions and cis elements participating in the sugar signal transfer. Analyzed by a transient expression experiment, the activity of the PeNAC1P promoter is induced by the salts, GA and IAA. The Ammopiptanthus mongolicus PeNAC1P promoter provided by the research belongs to an induction type promoter and provides an alternative promoter for generating a normal tolerant transgene plant on the basis of researching the function.
Description
technical field [0001] The invention relates to the isolation and identification of the NAC1 gene promoter PeNAC1P of Populus euphratica, sequence analysis, induction activity analysis, construction of its full-length and missing fragment plant expression vectors and application of the promoter. Background technique [0002] NAC transcription factor is a newly discovered plant-specific transcriptional regulator in the past decade, which plays an important role in plant growth and development, organ building, hormone regulation and defense against various biotic and abiotic stresses. However, the research on NAC transcription factors is still in its infancy, and neither their upstream regulators nor their downstream target genes are very clear. At present, only AP3 / PI (MADS box) is reported to regulate the expression of NAP (Sablowski et al., 1998), STM (KNOX) and MP (ARF) positively regulate the expression of CUC1 / CUC2, but they are also regulated by the MYB transcription fa...
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IPC IPC(8): C12N15/11C12N15/82C12N1/00C12N1/21
Inventor 王俊英尹伟伦夏新莉
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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