Study On Isolation, Purification, Cloning, Expression Of Trypsin Inhibitor Of Tartary Buckwheat And Resistance To Diseases And Insect Pest

Protein serine PIs are widely distributed in plants, especially in seeds. These are believed to form a wide spectrum defense mechanism against fungal pathogens and invertebrate pests. This study was aimed at investigating the purification and identification of serine protease inhibitors from tartary buckwheat (Fagopyrum tataricum) seeds (FtTI) The FtTI was isolated by anion exchange chromatography, affinity chromatography and centrifugal ultrafiltration. Under reducing and non-reducing condition, SDS-PAGE analysis showed that the isolated protein consists of a single polypeptide chain with molecular mass of approximately 14 kDa. The purified FtTI inhibited bovine trypsin in the stoichiometric ratio of 1:1. Dixon plots revealed a competitive inhibition of trypsin with inhibition constants (Ki) 1.6 nM. The N-amino acid sequence of FtTI was determined to be NH2-VGKQSWPELV, which shows 80% identity to protease inhibitor from buckwheat. N-terminal amino acid sequence of 10 residues showed high homology with other serine proteinase inhibitors belonging to the Fagopyrum subfamily.FtTI retained its inhibitory activity over a wide range of pH (3-10) and temperature (20-80℃). Even after 30 min of heat treatment(80℃) the inhibitor still keeps over 84% of its trypsin inhibitory activity. FtTI was highly stable under conditions ranging from highly acidic to highly alkaline. FtTI showed maximum inhibition at pH 8.0 and maintained over 90% of its inhibitory activity through a pH gradient between pH 3 to 10. FtTI can be rapidly inactived by the combination of high temperature and high pressure. Approximately 3% and 7% of residual trypsin inhibitor activity in FtTI was observed after 20 min of treatment at 0.198/120 MPa/℃and 0.143/110 MPa/℃respectively.The inactivation of FtTI was proportional to the ultrasound amplitudes (i.e. power levels) and sonication durations.According to the conservative tartary buckwheat trypsin inhibitor amino acid sequence designs degenerate primer. A novel gene encoding a serine proteinase inhibitor was isolated from tartary buckwheat. The full-length FtTI obtained from rapid amplification of cDNA ends and genome-walking contains 644 bp. An open reading frame of 264 bp encodes for a protein of 87 amino acids with the typical inhibitory motifs of phytoserine superfamily, namely the central signature motif XENXXV, and WPELVG-like motifs in the N-terminal part, and conserved RCDRV residues in the C-terminal region. Sequence comparison showed that the deduced amino acid sequence was similar to that of serine protease inhibitor from Ricinus communis (67.8%) XM 002520961.1. Sambucus nigra (66.6%)∴46949.1, Jatropha curcas (65.5%) FJ906844.1, Solanum nigrum (64.3%) ADP05S02.1, Zinnia violacea (64.3%) AB091073.1, Salvia miltiorrhiza (60.1%) EF187459.1, Fagopyrum esculentum(59.7%) AAB46906.1 and Solanum tuberosum (54.7%) AAS46024.1. These results indicated that has successfully won the tartary buckwheat FtTI genes.The FtTI was ligated to expression vector pPIC9k. Recombinant plasmid pPIC9k-Ft TI was constructed and transformed to Pichia pastor is GS115. The transformant named GS115-FtTIⅦwas obtained through MD plate screening and inhibiory activity test. The three significant factors were optimized by the single factor experiment and central composite design response surface. The results showed:the optimal fermentation content is 5-15%, the optimal induction time is 2.5-3.5 day, the optimal methanol content is 0.25-0.75%. Proteins were efficiently expressed at levels of 489.6-527.4 U/mg in shake flasks. The rFtTI was purified by affinity chromatography and centrifuge ultrafiltration. The final expression levels were 527.4 U/mg and we got totally 287 mg (527.4 inhibitor unit/mg) of purified FtTI from 1 L of fermentation broth. The purified rFtTI efficiently inhibited the protease activity of trypsin by competitive inhibition with Ki value 1.59 nM. The molecular mass of purified protein is approximately 13.8 kDa. The molecule of FtTI consists of 87 amino acid residues containing two disulfide bonds which connect Cys8 to Cys65 and Cys49 to Cys58. The active site of FtTI contains an Arg67-Val-68 bond. Analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor I family.The FtTI almostly completely inhibited the growth of Mycosphaerella melonis, Alternaria cucumerina, Alternaria solani, Colletotrichum glaeosporioides and Phytophthra capsi between 5μ.g/mL and 30μg/mL. However, the growth of Gibberella zeae, Ascochyta phaseolorum and Fusariumgraminearum was unaffected, even at levels 100 ug/mL. The assays performed on agar plates showed that the FtTI strongly inhibited pathogenic microbial strains, including Mycosphaerella melonis, Alternaria cucumerina, Alternaria solani, Colletotrichumglaeosporioides, Phytophthra capsi. These results indicate that FtTI directly affects the growth of fungal pathogens. Microscopic observations revealed distortion in the hyphal structure with stunted growth, reduced volume and hyphal tip distortion.The Mamestra brassicae L. larvaes fed on purified recombinant FtTIs individually incorporated into artificial diet show no growth and development inhibition. However, the purified rFtTI has a strong poisonous effect on cabbage night moth larvae, the performance of its toxic effect slowly, only after the treatment began 3 days showed high toxic effect, although the cumulative mortality of cabbage night moth larvae feeding different concentrations of rFtTI is 95%, but with the increasing concentration of feeding rFtTI, the shorter the time of death arrives.