Objective Expression of PsWRKY33 promoter of Nane (Prunus salicina lindley) under stress was determined for a study on the mechanism of WRKY gene associated with the growth and stress-resistance of the plant.
Method Unrooted phylogenetic tree representing relationships among protein sequences of PsWRKY33 was constructed using Nane and other plants through by MEGA 6.06 software. The promoter sequence was cloned by chromosome stepping technique. The cis-acting regulatory elements in the upstream promoter region at the 5′ end of PsWRKY33 were predicted according to the PlantCARE database. Transgenic plants were obtained by using the Arabidopsis immersion method. Histochemical staining and GUS enzyme activity were performed on the transgenic seedlings under different stresses.
Result The phylogenetic tree analysis showed that the PsWRKY protein was most closely related to Arabidopsis WRKY33, thus the gene was named PsWRKY33. The 1 872 bp length of 5′ end upstream in PsWRKY33 was obtained, from Nane via genome walking technology. The promoter region contained motifs of hormones (ABRE and ARE) and biotic stresses (LTR, MYB, W-box, and other cis-acting elements). Then, the 3 5′-deletion fragments were constructed. The histochemical staining and GUS activity measurement showed that the full PsWRKY33 promoter fragment mainly expressed in leaves, petals, and peduncles, and its expression decreased with the loss of fragments. Under low temperature, the various fragments of PsWRKY33 promoter were upregulated, but downregulated under SA stress.
Conclusion PsWRKY33 might response to low temperature and exogenous hormone SA stress in Nane.
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