Cloning And Functional Analysis Of Trypsin Inhibitor Gene CpKTI From Chimonanthus Praecox (L.) Link

Trypsin inhibitor (TI) which is a kind of defense protein is distributed widely in animals, plants, microorganisms and viruses. In recent years, TI has become a major type in insect-resistant gene resources. According to the sequence homology of amino acid, trypsin inhibitors are divided into two categories:Bowman-Birk type inhibitor (BBI) and Kunitz inhibitors (KTI). KTI with a molecular mass of20to25kD have typical amino-terminal Kunitz motif domain and a signal peptide with20amino acid residues. One active center in protein steric structure can bind to trypsin specifically. Kunitz trypsin inhibitor can resist against pests on plants. The combination of Kunitz trypsin inhibitor and the digestive enzyme in the insect can form enzyme-inhibitor complex (EI). El causes indigestion and malnutrition of the insects. Then the growth and development of insects are affected. Finally the insects die of hunger.Based on the cDNA library constructed from Chimonanthus praecox flowers and EST analysis, a new member of trypsin inhibitor (TI) was cloned by randomly cloning and sequencing, named as CpKTI (GenBank accession No. DW223520), and the bio-informatics, eukaryotic expression, prokaryotic expression and real-time quantitative PCR were used to analyze its potential biological functions. The main results are as follows:1. Molecular characteristics of CpKTIThe full length of CpKTI cDNA sequence is845bp, with an open reading frame (ORF) of594bp encoding a putative polypeptide of197amino acid residues. Se-quence alignments and phylogenetic analysis shows that CpKTI has typical amino-terminal Kunitz motif domain and a signal peptide with20amino acid residues. The amino acid sequence of the protein has high homology with those of the other species, such as Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Arachis hypogaea, Populus tremula Linn. The secondary structure is composed of a-helix (8.63%), extended strand(34.52%), beta turn(5.08%), random coil(51.78%), with6cysteine residues. And C61-C106forms a disulfide bond. There are9protein phos-phorylation sites. Tertiary structure is typical β shamrocks folded conformation.2. Transcriptional Expression of CpKTIAccording to expression analysis by real-time quantitative PCR, CpKTI was con-stitutively expressed in vegetative and reproductive organs at defined developmental stages under normal conditions, which was highly expressed in stems than other organs. CpKTI transcripts were detected during flower opening and more abundant in stamens than that in petals and pistils. CpKTI was highly expressed in wither period. In the light of seed development period of Chimonanthus praecox, we can speculate that CpKTI may play a significant role in the formation of seed storage protein. Also speculated that CpKTI which was highly expressed in stems may promote the formation of flow-ering branch.3. Eukaryotic Expression of CpKTIAfter being inserted into a eukaryotic expression vector pC2301G, CpKTI gene was transformed into tobaccos and overexpressed in plants. Identification of resistance on transgenic tobacco lines was carried out by biological stress with Prodenia litura (Fabricius) as test insects. Use detached leaf feeding method and covering cage method outdoor to determine resistance indexes:insects’ food consumption, body weight, molting situation and leaf damage degree. The results showed very significant differences between some of transgenic tobaccos lines and wild tobaccos. Transgenic tobaccos lines’ insect resistance was higher than the wild tobaccos. Prodenia litura (Fabricius) larvae in a gluttony period ate the control of tobaccos and avoided trans-genic tobaccos. Part of the insects had died in third instar. The insect resistance of transgenic tobaccos lines showed that trypsin inhibitor of Chimonanthus praecox had insect-resistant activity.4. Prokaryotic expression of CpKTIAfter being inserted into an prokaryotic expression vector pET32a (+), CpKTI was transformed into E.coli BL21(DE3) and overexpressed. The result of SDS-PAGE was indicated that the molecular weight of recombinant protein by IPTG induced was21kD which coincided with our expectation. The recombinant protein was existed as inclusion bodies.