Cloning and activity analysis of corn adversity inducing promoter
An inducible promoter sequence technology, applied in the field of plant genetic engineering, can solve the problem of few inducible promoters
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Embodiment 1
[0036] Embodiment 1: the cloning of the promoter of maize lipoxygenase gene (ZmLOX10)
[0037] The promoter of maize lipoxygenase gene (ZmLOX10) (promoter sequence comprises the DNA nucleotide sequence of -1bp to-1273bp region relative to the transcription initiation site of SEQ ID NO:1), in maize lipid oxygenation The enzyme gene (ZmLOX10) was identified in the 5' untranslated region sequence.
[0038] ZmLOX10 is registered in NCBI GenBank (accession number: NM_001112510), and the sequence listing shows the DNA sequence of the maize stress-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 prom...
Embodiment 2
[0043] Embodiment 2: Construction of maize lipoxygenase gene (ZmLOX10) promoter expression vector
[0044] The maize lipoxygenase gene (ZmLOX10) promoter cloned in Example 1 and the 5' untranslated region ZmLOX10Pro (see sequence listing) of 170bp were inserted into the vector, thereby constructing the maize lipoxygenase gene (ZmLOX10) Promoter expression vector.
[0045] More specifically, the recombinant plasmid pMD18-T::ZmLOX10Pro and the plant expression vector pCAMBIA1301( Figure 4 ), recover the target fragment and pCAMBIA1301 respectively, and connect them with T4 ligase to obtain the pCAMBIA1301::ZmLOX10Pro expression vector, which is used to drive the expression of the GUS gene. identified by PCR ( Figure 5 ) The obtained promoter fragment was the same as the expected result. Agrobacterium EHA105 was transformed by electric shock method, and PCR detection of bacteria liquid was carried out ( Image 6 ).
[0046] exist Figure 7 Among them, the gene GUS encodin...
Embodiment 3
[0047] Example 3: Identification of the activity of the maize stress-inducible promoter of the present invention
[0048] In order to identify the stress-induced activity of the promoter, the embryos of maize were drought-treated by the method of Jefferson et al. (EMBO J, 1987), and then the activity of GUS was detected.
[0049] 3.1 Preparation of acceptor material
[0050] 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.
[0051] 3.2 Preparation of Agrobacterium bacteria solution
[0052] 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 solution grows to OD 600...
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