Molecular Cloning And Analyzing Of Agglutinin From Lycoris Chinensis; Transformation And Expression Of ZGA Gene In Tobacco

Lycoris chinensis Agglutinin (LCA) from Amaryllidaceae species is a mannose-binding protein with various biological activities. It belongs to the monocot mannose-binding lectin (MBL). The lectins have received a lot of attention in different scientific disciplines because of their unique and exclusive specificity towards mannose, their retrovirus inhibitory activity and toxicity to insects. The total RNA of Lycoris chinensis was extracted, and then reverse transcript into cDNA. A primer was designed based on the conserved regions of other MBL plant's agglutinin through homology alignment. Using the RT-PCR, 3'RACE (rapid amplification of cDNA ends) technique, a DNA fragments was first cloned from Lycoris chinensis by performing PCR that used Lycoris chinensis cDNA genome as the template. It has 516 nucleotides (accession number in Genbank: AY690318). Then another two primers were designed according to the sequence of 3'ends, one was used to reverse transcript the RNA into cDNA, poly A were added to the 3'ends of cDNA with the function of TdT (Terminal Deoxynucleotidyl Transferase), using the 5'RACE technique the fragment of 411 nucleotides was cloned (accession number in GenBank: AY763111). The two partially overlapping cDNA fragments were assembled a full-length cDNA sequence of Lycoris chinensis (accession number in Genbank: AY763112).The full-length cDNA had 683bp, and the sequence encoded an open reading frame of 162 amino acids. The start codon was at 37-39 bp and the stop codon wasat 523-525 bp. The sequence also contained 5'nontranslated region with 36bp and 3' nontranslated region with 141bp. The result showed that LCA gene encoded a protein precursor with a signal peptide^ mature protein and C-terminal cleavage amino acids sequence by the analysis in the Blast of Genbank. The mature protein included 109 amino acids residues and the molecular weight is 12.1 KD. The mature protein sequence showed the identity to those of Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin, Lycoris radiate agglutinin, Clivia miniata agglutinin, 7ephranth.es grandiflora agglutinin, Amaryllis vittata agglutinin, Zephyranthes Candida Herb agglutinin respectively are 82.6% > 83.5% ^ 77.3% > 83.5% ? 80.9%, 79.^ 72.7 % Blocks'analysis revealed that the deduced amino acid sequence of LCA had three functional domains specific for lectin and three sugar-binding boxes (QDNY). In the mature protein, there were 34.9% hydrophobic amino acids, 46.8% hydrophilic amino acids, 8.3% basic amino acids and 10.1% acidic amino acids. The signal peptide of LCA had 23 amino acids, included a hydrophobic core of 9 amino acids. The cloning of this gene established an important base to the study of LCA gene structure, the cleavage mechanism, the mechanism of expression and regulation, the relationship of structure and function of LCA.