Extraction And Analysis Of A Soybean Late Embryogenesis-rich Protein Gene Fragment

It was for the first time mat late embryogenesis-abundant gene (Lea gene) was identified, which was expressed during seed maturation and embryonic development Late embryogenesis-abundant proteins were expressed in the embryo during the late stages of seed development. However, expression could also be induced in immature seeds and vegetative tissues by abscisic acid (ABA) or by stress conditions, such as drought, salinity, low-temperature stress. Due to the high hydrophilia and heat stability of die protein products, people thought that LEA proteins could protect cells from the affection of dehydration and salinity. Therefore, many scientists devoted to the research.Since the original cloning of a Lea gene from cotton (Gossypium spp.) by Dure in in 1981, the assemblage had been expanded to a superfamily that now includes sequences from many plants, including soybean (Glycine max), wheat (Triticum aestivum), Maize (Zea mays), Arabidopis thaliana and so on. We designed a pair of primer based on conserved sequence of group 3 Lea genes. Then, we cloned a fragment of 365 op sequence from soybean genome DNA by PCR (polymerase chain reaction), which was called pZYTYl by us.Compared with the 665 bp-1019 bp region of pGmPM2 cDNA sequence which was cloned from soybean (Glycine max L. Merrell), variety Williams' 82 (Yue-ie et al. 1992). There was 100% homology in the sequence that was cloned by us. And mat proved that the sequence was possibly the part of pGmPM2 cDNA sequence or the other Lea genes that were high homology in pGmPM2.We analyzed the pZYTYl sequence with the DNAsis softer. The result showed that the sequence was a part of open reading frame and possibly encoded a polypeptide of 121 amino acids whose molecular mass was13kD. Like other LEA proteins, the sequence had a bias in the component of amino acid. Namely, the distribution of all kinds of amino acid was different in quantity and the Polypeptide contained lots of high hydrophilic amino acid residues. In detail, the content of Alanine, Glutamic acid, Lysine and Threonine was large, and no tryptophane, cysteine, Glutamine, Histidine, Isoleucine, Phenylalanine and Proline residues were present in the protein. What's more, there was no intron in the sequence.These hydroplilic amino acid were periodically distributed and formed six a -helix, eight 0 -turns and ?-pleated sheet structure. There were two repeated sequence motifs (TAET ?EAAK ?K) in the C-terminate, which was a typical character of Lea group 3 proteins. Therefore, the fragment, which was derived by us, belonged to Lea group 3 proteins. The N-terminate of 60 amino acid consisted of three 22-amino acid repeated motifs (VNKMGEYKDYAAEKAKEGKDAT). The two kinds of repeated sequences were prone to form an amphiphilic a -helix structure. The a -helix had been hypothesized to reorient cellular water molecules, and bind ions to ameliorate the consequences of water removal during desiccation.We considered that pZYTYl sequence belonged to group 3 Lea gene type I based on the repeated Lea 11-amino acid motif having apolar residues at positions 1,2,4,5 and 9; negatively charged ammo acids at positions 3 and 11; and the +-+ charged arrangement at 6,7 and 8.