Molecular Cloning And Analyzing Of Agglutinin From Aspidistra Elatior Blume And Ophiopogon Japonicus

The present study was devoted to clone and analyze the agglutininfrom Ophiopogon japonicus, a traditional Chinese herb medicine fromLiliaceae species. Aspidistra elatior Blume, a member of Liliaceae, alsois a traditional Chinese medicinal herb and ornamental plantspecies. Their rhizomes and leaves have diuresis, abirritation anddetumescence effects on certain illnesses, furthermore, it is also usedto cure the injuries from falls, rheumic fever and rheumatism in Chinafor a long time. In recent years, the research was was devoted to cloneand Analyze of Agglutinin from Aspidistra elatior Blume. However, thereis no report on the molecular Cloning from Aspidistra elatior Blume. Thelectins have received a lot of attention in different scientificdisciplines because of their unique and exclusive specificity towardsmannose, their retrovirus inhibitory activity and toxicity to insects.To clone the gene of ojl, degenerate primers were designed in accordingwith the blast of other mannocot mannose-binding lectins. Using RT-PCR,3'RACE (rapid amplification of cDNA ends) technique, a DNA fragmentswas first cloned from Ophiopogon japonicusby performing PCR that usedOphiopogon japonicus cDNA genome as the template. Then another two primers were designed according to the sequence of 3'ends, one was usedto reverse transcript the RNA into cDNA, poly A were added to the 3'endsof cDNA with the function of TdT (Terminal Deoxynucleotidyl Transferase),using the 5'RACE technique the fragment was cloned. The two partiallyoverlapping cDNA fragments were assembled a full-length cDNA sequenceof Ophiopogon japonicus.The full-length cDNA was 704 bp, and the sequence encoded an openreading frame of 170 amino acid. The start codon was at 46-48bp and thestop codon was at 556-558 bp. The sequence also contained5'nontranslated region with 45bp and 3'nontranslated region with 146bp.The result showed that OJL gene encoded a protein precursor with a signalpeptide,mature protein and C-terminal cleavage amino acids sequence bythe analysis in the Blast of Genbank. The mature protein included 109amino acids residues and the molecular weight is 12.1KD and the PI were4.29 respectively. The mature protein sequence showed the identity tothose of Galanthus nivalis agglutinin, Polygonatum cyrtonema agglutinin,Polygonatum multiflorum agglutinin, Aspidistra elatior Blume SUBUNIT1agglutinin, Aspidistra elatior Blume SUBUNIT2 agglutinin respectivelyare 70.4%,81.1%,83.5%,66.4%,73.2%. In the mature protein, therewere 30.28% hydrophobic amino acids, 44.04% hydrophilic amino acids,2.75% basic amino acids and 7.34% acidic amino acids; The protein sequenceshowed the identity to those Liliaceae family lectin, but OJL had onlytwo specific sugar-binding boxes, which was different from most specificmannose-binding agglutinins. The secondary and 3-D structure of OJL wassimilary to GNA.The total RNA of Aspidistra elatior Blume was extracted, and thenreverse transcript into cDNA. A primer was designed based on the conservedregions of other MBL plant's agglutinin through homology alignment. Thefull-length cDNA was cloned by RT-PCR, 3'RACE and 5'RACE. The full-length cDNA had 1162bp, and the sequence encoded an open readingframe of 310 amino acids. The start codon was at 38—40bp and the stopcodon was at 965-967 bp. The sequence also contained 5'nontranslatedregion with 37bp and 3'nontranslated region with 195bp. The resultshowed that AEL encoded a protein precursor with a signal peptide,subunitl, joint peptide, subunit2 and C-terminal cleavage amino acidssequence by the bioinformatic study. The Aspidistra elatior BlumeSUBUNIT1 agglutinin protein sequence showed the identity to those ofGalanthus nivalis agglutinin, Polygonatum cyrtonema agglutinin,Polygonatum multiflorum agglutinin, SCAfet DOM1 agglutinin, SCAfet DOM2agglulitin respectively are 51.8%,60.4%,65.5%,50.0% and 49.1%. AndSUBUNIT2 was 61.6%,74.3%,77.7%,57.8%,55.7%. Blocks' analysis revealedthat the deduced amino acid sequence of AEL had three functional domainsspecific for lectin and three sugar-binding boxes (QDNY). The subunit1included 101 amino acids result and subunit2 included 111 amino acidsresidues and the molecular weight was about 12 KD, and the PI were5.38, 6.04 respectively. In the mature protein subunit1, there were34.9% hydrophobic amino acids, 33.64% hydrophilic amino acids, 8.81%basic amino acids and 10.91% acidic amino acids; In the mature proteinsubunit2, there were 35.71% hydrophobic amino acids, 34.82% hydrophilicamino acids, 6.25% basic amino acids and 7.14% acidic amino acids Thesignal peptide of AEL had 27 amino acids.The cloning of these gene established an important base to the studyof OJL and AEL gene structure, the cleavage mechanism, the mechanism ofexpression and regulation, the relationship of structure and functionof OJL and AEL.