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Cloning of the Coffea canephora SERK1 promoter and its molecular analysis during the cell-to-embryo transition

Electronic Journal of Biotechnology. 2018; 
DoribetJiménez-GuillenaDanielPérez-PascualaRamónSouza-PereraaGregorioGodoy-HernándezaJosé JuanZúñiga-Aguilarb
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Gene Synthesis pCcSERK1 binary vectors were constructed by replacing the 35S promoter that controls the expression of the uidA gene in the binary vector pCAMBIA1301 (http://www.cambia.org/daisy/cambia/585.html) with different versions of the CcSERK1 promoter to generate pCcSERK1::GUS-FL (from + 452 to − 1620), pCcSERK1::GUS-1048 (from + 452 to − 1048), pCcSERK1::GUS-792 (from + 452 to − 792), pCcSERK1::GUS-618 (from + 452 to − 618), and pCcSERK1::GUS-aux(−) (DNA segment from + 452 to − 1620, where the four AuxRe were mutated during chemical synthesis, GenScript, https://www.genscript.com/) and were verified by DNA sequencing. KpnI and NcoI restriction enzymes were used to excise the 35S promoter, and then, the different CcSERK1 promoter fragments, previously amplified by PCR using KpnI and NcoI-containing primers (Table 1), were ligated after enzymatic digestion to the KpnI-NcoI linearized pCAMBIA1301 vector. PCR conditions were as follows: 94°C for 2 min, then 94°C for 15 s, 67°C for 30 s, 72°C for 2 min, and a final extension step at 72°C for 10 min. The identity of the different pCcSERK1::GUS and pCcSERK1::GUS-aux(−) constructs was corroborated by KpnI and NcoI enzymatic digestion, PCR amplification under conditions described above (Fig. S4 and Fig. S5), and sequencing of positive clones. Get A Quote

摘要

Background Somatic embryogenesis receptor-like kinase 1 (SERK1) is a cell membrane receptor active in different plant tissues and involved in cell differentiation activities including somatic embryogenesis. The identification of promoter elements responsible for SERK1 expression during the onset of somatic embryogenesis can be useful to understand the molecular regulation of the cell-to embryo transition, and these promoter elements represent biotechnological tools in plant organ tissue culture. Results A − 1,620 bp DNA sequence located upstream of the Coffea canephora SERK1 gene homologue (CcSERK1) was isolated, and then, different segments containing key response elements (REs) for somatic embryogenesis ... More

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