Isolation and sequencing analysis on the seed-specific promoter from soybean

Abstract

The low level of foreign genes’ expression in transgenic plants is a key factor that limits plant genetic engineering. Because of the critical regulatory activity of the promoters on gene transcription, they are studied extensively to improve the efficiency of the plant transgenic system. The constitutive promoters, such as CaMV 35S promoter, are usually used in plant genetic engineering. But those constitutive promoters continuously express their downstream genes during the whole life span in all the tissues of the host plants. This is not only wasteful to host plant’s energy, but also harmful to host plants and usually affects their agronomic characteristics. In contrast, the seed-specific promoter only expresses its downstream genes from mid to late stage of seed maturation, and there is no expression or much lower expression in other tissues. So the seed-specific promoters are distinguished for their improvement and what they have brought to plant quality engineering. The aim of this article is to characterize a new seed-specific promoter and improve grain quality. The promoter region of β-conglycinin α-subunit gene was isolated from the genomic DNA of soybean Jilin 43 by PCR method, and successfully extended this fragment by TAIL PCR method and obtained the promoter fragment BCSP666. Sequencing analysis showed that the cloned fragment BCSP666 contained all of the motifs, such as RY repeat element, AG/CCCCA motif, TACACAT motif, ACGT motif, A/T rich motif and E-box etc., which constituted the seed-specific promoter activity. Based on this sequencing analysis, the seed-specific promoter activity of the fragment BCSP666 was predicted. And then the seed-specific expression vector pBI121-666, which contained GUS reporter gene, was constructed with the fragment BCSP666. Transformation of Arabidopsis thaliana plants by Agrobacterium-mediated floral-dip method with the recombined vector pBI121-666 was conducted. The transgenic plants were selected on the kanamycin-resistant MS medium, and confirmed by Southern Blot Analysis. Fluorometric and histochemical analysis of GUS enzyme activity of the transgenic Arabidopsis thaliana plants support our original suggestions. Therefore, the seed-specific promoter BCSP666 is obtained and characterized.