Promoter used for corn low phosphor stress inducible expression, and applications thereof
A technology for inducing expression and promoters, applied in the fields of molecular biology and genetic engineering, can solve problems such as phosphorus poisoning, affecting plant growth and production, and achieve the effect of avoiding energy waste
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Embodiment 1
[0019] Example 1: ZmPHR1 Gene 5' Upstream Sequence P 2205 -ZmPHR1 and the acquisition of missing fragments
[0020] first step: ZmPHR1 Gene 5' Upstream Sequence P 2205 -ZmPHR1 the acquisition
[0021] by ZmPHR1 The sequence of the coding region was used as a probe, and BLAST searched the maize B73 genome sequence database (http: / / maizesequence.com), and the sequence about 2.0 kb upstream of the homologous gene was used as the target region, and the DNA of the maize inbred line 478 was used as a template, and the primer SF1 ( Italics are Sal I restriction sites, SEQ ID NO:6) and SR (italics are Bgl II restriction sites, SEQ ID NO:7) amplified fragments P 2205 -ZmPHR1 Ligated to pMD18-T vector.
[0022] (1) Prepare the PCR reaction solution according to the following components:
[0023] DNA 1 μL
[0024] dNTP Mixture (2.5 mΜ each) 8 μL
[0025] 10×LA PCR Buffer II (Mg 2+ plus) 5 μL
[0026] TaKaRa LA Taq enzyme (5 U / μl) 0.5 μL
[0027] Primer SF1 (10 pmol...
Embodiment 2
[0037] Embodiment 2: Construction of recombinant plant expression vector
[0038] All recombinant plasmids and plant expression vector pCAMBIA1305 were used Sal I and Bgl II double enzyme digestion and ligation, the correct recombinant plant expression vector was obtained after enzyme digestion detection and sequencing verification, and the recombinant plant expression vector contained a GUS reporter gene ( figure 2 , image 3 ), named as P 2205 -ZmPHR1::GUS , P 1763 -ZmPHR1::GUS , P 1502 -ZmPHR1::GUS , P 972 - ZmPHR1::GUS and P 482 -ZmPHR1::GUS . Transform the recombinant vector into Agrobacterium GV3101 middle.
Embodiment 3
[0039] Example 3: Transforming Arabidopsis thaliana with Agrobacterium to obtain pure transgenic lines
[0040] recombinant plant expression vector P 2205 -ZmPHR1::GUS , P 1763 -ZmPHR1::GUS , P 1502 - ZmPHR1::GUS , P 972 -ZmPHR1::GUS and P 482 -ZmPHR1::GUS and pCAMBIA1305 vector (35S::GUS) were transformed into wild-type Arabidopsis Col-0, and the seeds were harvested in the greenhouse. got a T 1 Substitute seeds contained 25 μg mL -1 MS medium screening and PCR detection (SEQ ID NO:12, SEQ ID NO:13) ( Figure 4 ) after transplanting and growing in the greenhouse, self-crossing to get T 2 generation, and then through selfing and hygromycin selection to obtain the transgenic pure line T 3 generation, respectively named as T P 2205 -ZmPHR1 , T P 1763 -ZmPHR1 , T P 1502 - ZmPHR1 , T P 972 -ZmPHR1 , T P 482 -ZmPHR1 and TpCAMBIA1305, for subsequent functional analysis.
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