Molecular Cloning,Expression And Analysis Of ?-Amylase/Subtilisin Inhibitor Gene Of Ligusticum Chuanxiong

Ligusticum chuanxiong Hort is a famous authentic herb in Sichuan Province. It has the effect of expelling wind and relieving pain and promoting blood circulation. Its indexes are ligustrazine and ferulic acid, which are used in the treatment of cardiovascular and cerebrovascular, respiratory, urinary system and other diseases. a-Amylase inhibitors have been isolated from a variety of plants and are used in weight loss, hypolipidemic, treatment of diabetes and pest resistance.In this study, a potential ?-amylase inhibitor gene was screened based on the transcript data of L. chuanxiong. The full-length sequence of the gene was successfully amplified by RACE technique. The sequence was named Ligusticum chuanxiong a-amylase Inhibitor(LASI) and uploaded to the NCBI GenBank database. The accession number is KX580040.The basic analysis of LASI protein showed that the full-length cDNA sequence (860 bp)of LASI contained the poly(A) tail and an ORF. The ORF was composed of 612 bp which was deduced to encode a protein of 203 amino acid residues. Its relative molecular mass is 22570 Da, the theoretical isoelectric point is 5.06. LASI contains a signal peptide of 23 amino acid residues. It has no transmembrane domain and its subcellular localization is in the extracellular. It was found that LASI belongs to the Kuntize type a-amylase inhibitor and has a-amylase inhibitor/subtilisin bifunctional inhibition by comparing with other plant a-amylase inhibitors and trypsin inhibitors. LASI has two conserved Thrss and Glu194 residues, which are key sites for the activity of subtilisin inhibitor activity and amylase inhibitor activity. The molecular phylogenetic tree shows that LASI has high homology with RASI which was found in rice. There was no significant difference in the codon preference between LASI gene and E.coil, which indicated that LASI protein could be expressed by E.coil. The secondary structure predicted that the extension chain, irregular curl, a helix and? rotation were 33.50%, 31.53%, 21.67% and 13.30% respectively. The initial model of LASI three-dimensional structure was obtained by homology modeling with RASI as template, and to minimize energy and molecular dynamics simulation, which laid the foundation for further research.LASI-pET28a recombinant plasmid was constructed and transformed into Rosetta for the first time. A 23 kDa recombinant LASI protein was successfully expressed by IPTG induction. The expression conditions of recombinant protein were optimized. The recombinant LASI protein was purified by NI2+ affinity chromatography column. Mass spectrometry was used to identify LASI protein.The inhibitory activity of LASI protein on porcine, human, Helicoverpa armigera,Plutella xylostella,Spodoptera litura amylase and subtilisin, trypsin and chymotrypsin were detected by iodine-starch coloring method and Erlanger method. LASI is a weak inhibitor of mammalian amylase, but a strong inhibitor of Lepidoptera pest amylase, which also has a strong inhibitory effect on subtilisin, but has no inhibitory activity against trypsin and chymotrypsin. It is speculated that LASI plays an important role in the fight against biological stress, especially from insects and bacteria.The expression of LASI gene in different tissues of Ligusticum chuanxiong was different, and the expression level of rhizome was higher than that of leaves. This provides a basis for studying the function of LASI in different tissues. The expression of LASI in rhizomes and leaves increased under low temperature,drought and salt stress. This indicates that abiotic stress conditions can stimulate the expression of LASI gene and reduce the damage of plants by inhibiting the activity of endogenous amylase.