Molecular Cloning of Promoter of <i>AP3</i> Gene from <i>Arabidopsis thaliana</i> Ecotype (Col) and Construction of the Plant Expression Vector

Abstract

Belonging to MADS-Box gene family, APETALA3 ( AP3 ) gene of Arabidopsis floral organ class B is expressed in petals and stamens specifically. AP3 gene which encodes a transcription factor controls the development of petals and stamens of dicotyledons by cooperation with class A and class C genes, respectively. The results showed that the promoter of AP3 was the flower-specific expression promoter. Therefore, the cloning and functional identification of AP3 gene promoter from Arabidopsis thaliana will play significant effects on the oriented improvement of the commercial traits related to the flowers of ornamental plants. Here, a pair of specific PCR primers was designed according to the promoter sequence of AP3 gene of Arabidopsis thaliana ecotype (Ler) reported in GenBank (U30729). A length of 1 767 bp of AP3 gene promoter was obtained from Arabidopsis thaliana ecotype (Col) by using PCR technique with high-fidelity DNA polymerase KOD-plus, named as pAtAP3 , the GenBank accession No. is FJ619533. Online Bl2seq analysis indicated that the sequence similarity between pAtAP3 and U30729 was 98%. It also showed that the sequence similarity between pAtAP3 and the BAC clone T12E18 (AL132971) of Arabidopsis thaliana ecotype (Col) from 9 264 to 11 030 was up to 100%, and its downstream sequence encoded AP3 protein (CAB81799), which approved that the sequence we cloned was the promoter of AP3 gene of Arabidopsis thaliana ecotype (Col). Online PLACE analysis displayed that pAtAP3 contained the basic cis -elements of promoter such as TATA-box and CAAT-box. It also comprised several cis -elements related to flower-specific expression, such as CArG1, CArG2, CArG3 and anther-box. Moreover, plant expression vector of pAtAP3 :: GUS was successfully constructed in this research, which would lay the foundation for functional identification of the AP3 promoter of Arabidopsis thaliana ecotype (Col).