Isolation and Characterization of a Novel Seed-Specific Promoter from Malaytea Scurfpea
Xiaoyan Fang & Dan Kang & Chengjian Xie &
Xingyong Yang & Anping Sui
# Springer Science+Business Media New York 2014
Abstract Psc-AFP, an antifungal protein with trypsin inhibitor activity, was isolated from a traditional Chinese herb, malaytea scurfpea (Psoralea corylifolia L.); the protein was found to be preferentially expressed in seeds. A 737-bp upstream sequence from the ATG initiation codon site of the gene encoding this protein was isolated using a chromosomal walking method and identified as pPsc-AFP. The analysis of the cis-acting elements in PlantCARE database indicated that pPsc-AFP contains RY elements and GCN4 motifs, a kind of seed-specific elements, and a Skn-1 motif. The features of this fragment were studied by fusing it into the upstream region of the β-glucuronidase (GUS) reporter gene and transforming the recombinant expression vector pBI121-pPsc-AFP::GUS into tobacco by using the leaf disk method. The expression pattern was determined using GUS histochemical staining. The fragment showed a strong GUS activity in seeds, whereas its expression level was considerably lower in other organs such as the roots, stems, and reproductive organs, including
Xiaoyan Fang and Dan Kang contributed equally.
Electronic supplementary material The online version of this article
(doi:10.1007/s11105-014-0785-2) contains supplementary material, which is available to authorized users.
X. Fang : D. Kang : A. Sui (*)
The Key Laboratory of Eco-Environments in Three Gorges
Reservoir Region, Ministry of Education, and the Library, Southwest
University, Chongqing 400715, China e-mail: suianp@swu.edu.cn
C. Xie : X. Yang (*)
The Chongqing Key Laboratory of Molecular Biology of Plant
Environmental Adaptations, Chongqing Normal University,
Chongqing 401331, China e-mail: yangxy94@swu.edu.cn
C. Xie : X. Yang
The College of Life Science, Chongqing Normal University,
Chongqing 401331, China
stamens and pistil, indicating that pPsc-AFP was able to direct
GUS expression in a seed-specific manner in transgenic tobacco. Keywords pPsc-AFP . Seed-specific promoter .
Chromosomal walking . hiTAIL-PCR
Introduction
With the advancement of genetic and molecular biology techniques, transgenic engineering of plants has facilitated a thorough understanding of gene expression, offering the possibility of introducing desirable genes from foreign species into target plants, more specifically, into target tissues or cells. In most cases, the foreign genes are driven by a powerful constitutive promoter, such as the cauliflower mosaic virus 35S
(CaMV 35S) (Covey et al. 1981) and its derivatives (Rushton et al. 2002). However, excessive accumulation of heterologous proteins and metabolic wastes, including toxins, might lead to undesirable pleiotropic effects in transgenic plants
(Kasuga et al. 1999; Hsieh et al. 2002). Hence, identification of tissue-specific promoters that particularly regulate expression in certain organs or tissues is crucial for the development of new genetically modified (GM) plants (Zavallo et al. 2010).
Thus, a critical and prominent problem is to determine whether foreign genes introduced into target plants or receptor organs can show controlled and stabilized expression without affecting the normal growth, development, and differentiation of plants. Promoters regulate the interactions between ciselements and trans-factors at all growth and developmental stages and play a crucial role in the temporal and spatial expression of genes. Improving the genetic characters of crops requires that foreign genes are expressed in a specific site at a precise time in order to coordinate plant growth and development. Tissue-specific promoters involve certain elements that
Plant Mol Biol Rep
direct the expression of downstream genes in specific tissues or cell types only. At present,
