Isolation And Functional Analysis Of A Cell Wall Expansin Gene From Triticum Aestivum L

Wheat (Triticum aesitivum) is the second important crop worldwide and its planting area only less than that of rice. The growth and yield of wheat directly affects the food supply of people. And the research of wheat molecular biology falls behind other crops, especially rice, maize and arabidopsis, etc. Since elongation of plant cell is restricted by plant wall, the growth of plant cell is close correlated with extension of plant cell wall. So, it is very important to understand the molecular biological mechanism of regulating cell wall extension during wheat growth and development. In recent years, it had been reported that expansin played an important role in relaxing plant cell wall. In this study, total RNA was isolated from wheat coleoptiles. Based on the sequences corresponding to conserved regions ofβ-expansin proteins from different plant species, two degenerate oligonucleotides were designed and used for RT-PCR, and a sequence of cDNA was isolated combining with 3'- and 5'-RACE strategies. An identification of nucleotide sequence of PCR products suggested that it was a deducedβ-expansin gene full-length cDNA, named Tri-EXPB1 (Triticum aesitivumβ-EXPANSIN 1). In order to determine the function of endogenous expansin Tri-EXPB1 in growth and development of plants cell, a construct containing full-length cDNA of Tri-EXPB1 gene in sense orientation driven by the constitutive cauliflower mosaic virus 35S promoter was assembled and introduced into tobacco plants. An expression vector carrying full-length cDNA of Tri-EXPB1 gene in sense orientation with vector PET-30a(+) was constructed and introduced into E.coli. The strong induced expression fusion proteins from E.coli loading Tri-EXPB1 gene were collected into PBS solution and immuned to rabbits for obtaining antiserum. The Tri-EXPB1 gene expression pattern and its response to drought stress of Tri-EXPB1 transcripts were characterized by RT-PCR with wheat coleoptile as material, and the biological function of Tri-EXPB1 gene was studied with transgenic Tri-EXPB1 gene tobacco plants. The main results are as follows:...