title: Isolation, structure modeling and function characterization of a trypsin inhibitor from Cassia obtusifolia.
abstract: A trypsin inhibitor gene (CoTI1) from Cassia obtusifolia was isolated and the deduced amino acid sequence was attributed to the Kunitz-type trypsin inhibitor. The recombined CoTI1, expressed in E. coli, exhibited strong inhibitory effect on bovine trypsin and trypsin-like proteases from Helicoverpa armigera, Spodoptera exigua, and Spodoptera litura. CoTI1 thus presents insecticidal properties that may be useful for the genetic engineering of plants. Leu84, Arg86 and Thr88 were predicted as three key residues by molecular modeling in which Arg86, inserted into the substrate pocket of trypsin, interacted directly with residue Asp189 of trypsin causing the specific inhibition against trypsin. The predicted results were confirmed by site-directed mutagenesis with L84A, R86A and T88A, respectively. The substantial changing expression level of CoTI1 under salt, drought and abscisic acid treatment suggested that CoTI1 might play important role in the resistance against abiotic stress.
authors.affiliation: School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, 610031, China.
authors.forename: Zubi
authors.initials: Z
authors.lastname: Liu
authors.forename: Qiankun
authors.initials: Q
authors.lastname: Zhu
authors.forename: Juanjuan
authors.initials: J
authors.lastname: Li
authors.forename: Gan
authors.initials: G
authors.lastname: Zhang
authors.forename: Aerguli
authors.initials: A
authors.lastname: Jiamahate
authors.forename: Jiayu
authors.initials: J
authors.lastname: Zhou
authors.forename: Hai
authors.initials: H
authors.lastname: Liao
mesh_terms: D002366:Cassia / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D018744:DNA, Plant; D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors