Construction Of The Expression Vector Containing A Fused Pollen And Seed-Specific Promoter

Since the first transgenic plant appeared in the early 1980s, its biosafety related to environment, health and transgene transfer arouse growing concerning. The environmental safety of transgenic plants, which means having no toxic affection on other species and incapable of horizontal gene transfer, is one of the prerequisites for official approval of field release and application. Practical technical approaches such as pollen sterilization, seed failure or deletion of foreign DNA segments in certain developmental stage of pollen and seeds, are under intensively research. Such techniques are out of question for application in asexual species but not applicable for the sexual ones (especially in F1 combination). One of the solutions for this problem is to create inducible, tightly controlled and specific expression system, with which the generation of pollen and seeds with fertilization or containment of foreign genes and the production of materials with homozygous genotype may be achieved. Here an binary expression vector containing pollen-seed specific promoter was constructed. In this study pollen specific promoter of LAT52 was amplified through PCR from tomato genome DNA and fused with the regulatory element on a seed specific promoter from pea, and with the GUS gene an expression vector was constructed and transformed into tobacco and Arabidopsis thaliana to test the function of fusion promoter. The main results are as follows:1. Cloning of tomato pollen specific promoter LAT52An 618bp fragment was amplified from genome DNA by means of semi-nested PCR and inserted into vector containing seed specific promoter to generating pMD-LAT52. Sequencing result showed that, compared with DAN data in Genbank the similarity is 98%, with an addition of 7 nucleotides, simple sequence repeat AAAAAAT (SSR) in the non-key region.2. Constructed with the seed-pollen expression of fusion promoter LAT52Sd and GUS reporter gene in binary vectorAn fusion promoter was produced after combination pollen specific promoter of LAT52 with a key regulatory element from a pea seed specific promoter. With the reporter gene gus an expression vector LAT52Sd-GUS was constructed.3. Transformation of LAT52Sd-GUS into tobacco and Arabidopsis thalianaBy means of leaf disc method gus was transferred into tobacco and Arabidopsis thaliana medicated by Agrobacterium tumefaciens C58. Under selectable pressure of 150 mg/L Kanamycin positive seedle of tabacco and Arabidopsis thaliana were obtained.