Molecular Cloning, Expression And Analysis Of Kunitz Trypsin Inhibitor Gene (CoTI2) Of Cassia Obtusifolia

Cassia obtusifolia L., which was well-know as both medicine and food in China, belong to Leguminosae annual plant. The pharmaceutical part of C. obtusifolia was mature seed. Preliminary study has shown that a trypsin inhibitor was isolated from C. obtusifolia which could be used for Pierisi rapae control. In this article, we analyzed the transcriptome data of C. obtusifolia at first, and screened a candidate gene of Kunitz type trypsin inhibitor. Then, the candidate gene was cloned, expressed, and analyzed its bioinformatics and expression pattern. The results of this thesis made a contribution to identify the function and study molecular mechanism of this candidate gene. The main results were as follows:1) To Clone a new Kunitz type trypsin inhibitor gene in C. obtusifolia (CoTI2) for the first time using RACE techniques. The full-length of CoTI2 was composed of 792bp, which has a complete open reading frame encoding 248 amino acids with calculated molecular mass of 26.68 KDa and isoelectric point of 5.03. The CoTI2 peptide, which was composed of 11.26% acidic,7.1% basic,36.94% hydrophobic and 27.48% polar amino acids, showed strong hydrophobicity.2) Bioinformatics analysis was carried out to initially predict the basic physicochemical properties and structure in amino acids sequence encoding by CoTI2:the deduced amino acid sequence contained N-terminal signal sequence of 26 amino acids. It main located to outside and vacuole, N-terminal 1-11 amino acid in inside and 35-247 amino acid in outside, so it was shown to be a transmembrane protein. Restriction enzyme cutting site was confirmed with BamHI and EcoRI for prokaryotic expression, and BamHI and SnaBI for eukaryotic expression respectively by Restriction Enzyme cutting site analysis. CoTI2 showed strong bias toward the codons with A or U at the third codon position, establishing a massy groundwork for CoTI2 gene selecting superior exogenous expression system. The 66 α-helices,28 β-turns and 154 random were included in its secondary structure, suggesting that space configuration of the protein is mainly in this position. The analysis of its homology showed that Lys108 was the key residue of CoTI2, the Cys84, Cys135, Cys187, Cys200 might be involved in the formation of disulfide bond. The CoTI2 and its homologene sequence were used to analyze evolutionary relationship, revealing that CoTI2 had closer relationship with Arabidopsis thaliana, which was the superior eukaryotic expressing system.3) Real-time fluorescent quantitative PCR results showed that the expression level of CoTI2 gene was different in various organs and under various treatments. The high expression level in immaturate seed/pod/callus/hypocotyl, among this tissues the immaturate seed expression highest, but lowest expression in flower. The expression level of CoTI2 was up-regulated under drought stress/mechanical damage/ABA/Mej a/cold treatment, but salt stress does not affect expression of CoTI2, which indicated that the above condition might stimulate CoTI2 gene expression in order to inhibit trypsin-like degradation in the body. Such result provided a theoretical basis for study the function of CoTI2 in different organizations as well as metabolic pathway under various stress.4) The prokaryotic expression recombinant plasmid CoTI2-pET28a was successfully constructed and transformed into E. coli Rosetta (DE3). The CoTI2 protein was successfully expressed at 1.6 mM IPTG,27℃ and 7 h. This work lay the foundation for the further function research and molecular mechanism of anti-insects.5) Moreover, the eukaryotic expression recombinant plasmid ZCoTI2-pBI121 was successfully constructed and transformed into Arabidopsis thaliana, which could be used for the futural genetic and physiology function research in plant.