Isolation and functional characterization of the promoter of SEPALLATA3 gene in London plane and its application in genetic engineering of sterility

Abstract

In Platanus acerifolia, the SEP3 homolog PlacSEP3 may possess multiple functions, including roles in floral organ identity, development, and vegetative development. This work aimed to characterize the function of PlacSEP3 promoter and analyzed whether PlacSEP3 promoter could be used for genetic engineering flowerless London plane cultivars. The PlacSEP3 promoter was isolated by TAIL-PCR and cis-acting regulatory elements were predicted using the PLACE database and PlantCARE database. Three 5′-deletions of PlacSEP3 promoter were fused to the GUS gene and transformed into tobacco to analyze the functional by Histochemical staining and fluorometric determination. The pPlacSEP3-3::Barnase and pPlacSEP3-3::Barnase-mic35S-Barstar vectors were constructed and transformed into tobacco to test whether pPlacSEP3 could be used for genetic engineering flowerless London plane cultivars. A 1491-bp sequence was isolated, this sequence contained multiple putative cis-regulatory elements related to flower, pollen, and embryo/endosperm development, light responsive. Histochemical staining and fluorometric determination showed that GUS was strongly expressed in reproductive organs and apical buds, and was slightly expressed in vegetative tissues, except the roots of pPlacSEP3-1::GUS and pPlacSEP3-3::GUS transgenic tobacco. However, GUS was not expressed in floral organs and was just slightly expressed in fruits of pPlacSEP3-2::GUS transgenic tobacco. Interestingly, transgenic tobacco of all three constructs showed GUS staining at the bud side of semi-lignified and old stems. The pPlacSEP3-3::Barnase-mic35S-Barstar transgenic tobacco showed various degrees of sterility and vegetative developmental defects. The pPlacSEP3 involved in flower initiation, development and some aspects of vegetative development, such as lateral bud initiation and development. The pPlacSEP3 had the potential application for genetic engineering flowerless cultivars.