Constitutive promoters and their uses
A constitutive promoter, plant tissue technology, used in applications, recombinant DNA technology, biochemical equipment and methods, etc.
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no. 1 example
[0060] The first embodiment, the synthesis of prOsRUBQ1 promoter sequence
[0061] Obtain the constitutive promoter prOsRUBQ1 of the present invention, as shown in SEQIDNO: 1 in the sequence listing; the prOsRUBQ1 promoter sequence (as shown in SEQIDNO: 1 in the sequence listing) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; the synthesized The 5' end of the prOsRUBQ1 promoter sequence (SEQ ID NO: 1) is also connected with a KpnII restriction site, and the 3' end of the prOsRUBQ1 promoter sequence (SEQ ID NO: 1) is also connected with a HindIII restriction site.
no. 2 example
[0062] The second embodiment, construction of recombinant expression vector p100024 containing prOsRUBQ1 promoter sequence and transformation of recombinant expression vector into Agrobacterium
[0063] 1. Construction of the recombinant cloning vector pT-prOsRUBQ1 containing the prOsRUBQ1 promoter sequence
[0064] The prOsRUBQ1 promoter sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector pT-prOsRUBQ1. The construction process is as follows: figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; prOsRUBQ1 is the rice variety Nipponbare Ubiqutin (ubiquitin) 1 gene promoter (SEQ ID NO: 1); MCS is a multiple clon...
no. 3 example
[0075] The third embodiment, the acquisition of maize plants transferred to the prOsRUBQ1 promoter sequence
[0076] According to the routinely used Agrobacterium infection method, the immature embryos of the aseptically cultivated corn variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 4 in the second example, so as to infect 2 and 3 in the second example. The T-DNA (including the promoter sequence of maize Ubiquitin gene, prOsRUBQ1 promoter sequence, control sequence, PAT gene, PMI gene and Nos terminator sequence) in the constructed recombinant expression vector p100024 and p100079 (control sequence) was transferred into maize In the genome, maize plants transformed with the prOsRUBQ1 promoter sequence and maize plants transformed with the control sequence (positive control) were obtained; meanwhile, wild-type maize plants were used as negative controls.
[0077] For Agrobacterium-mediated maize transformation, briefly, immature immature embryos are ...
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