Isolation And Function Identification Of A Kunitz Trypsin Inibitor Gene In Tobacco

Phytopathogens attack represents a major problem for agriculture in general and has caused devastating famines in human history. Fungal pathogens are responsible for significant crop losses worldwide, resulting from both infection of growing plants and destruction of harvested crops. Tobacco is an important economic crop worldwide. The cultivation area and production of tobacco in our country occupy an important position in the world. However, there was a wide range of tobacco diseases and in particular the most serious fungal diseases. Rhizoctonia solani is one genus of the Deuteromycotina, with a wide range of host and widely spreading. The soil-borne fungal pathogen comprised several groups which are pathogenic to more than 27 families in both monocots and dicots. Through long time study, there was little research progress in molecular plant pathology. In recent years, molecular biology can help to reveal the mechanism of plant-pathogen interaction. Introducing antifungal proteins to crops now is an effective instrument to increase the resistance to phytopathogens and has achieved important progress in crop improvement.In this experiment, a fungi-induced cDNA library from tobacco seedlings was constructed, and a new Kunitz trypsin inhibitor gene NtKTI1 related to biotic stress was identified by differential hybridization screening. Our studies are mainly focused on the sequence and expression analysis, function identification of the tobacco gene NtKTI1. The main results are as follows:1. A fungi-induced cDNA library of tobacco was constructed with Clontech SmartTM cDNA Library Construction Kit. We screened a strongly repressed gene NtKTI1 by differential hybridization from the cDNA library. The full length cDNA was further isolated by 3'-RACE. The clone contains 840 nucleotides, encodes 209 amino acid residues with the predicted molecular mass of 23.1 kDa and a calculated pI of 9.36. The GenBank accession number of NtKTI1 is FJ494920.2. Sequence and homology comparison revealed that the deduced NtKTI1 showed similarity to a number of putative proteins from other plant species, including Lemir from tomato, two proteins with predicted KTI function in Arabidopsis At1g17860 and At1g73260 and miraculin precursor MIR. It contained one reactive site and four Cys residues that form two conserved intramolecular disulfide bonds. Secondary and tertiary structure predictions using SWISSMODEL, JPred, ESyPred3D, and CPHmodels exhibited that NtKTI1 had similar three-dimensional structure with KTI3. Meanwhile, molecular model of the 3-D structure of NtKTI1 was simulated using SWISS-MODEL and the 3D model was drawn using RasMol software.3. Multiple BLAST searches of several databases of Arabidopsis and Rice obtained 7 and unique homologs of NtKTI1 respectively. Phylogenetic analysis indicated that two Arabidopsis KTI proteins and OsKTI were most closely related to NtKTI and significantly clustered together into a single branch.4. NtKTI1 was constitutively expressed in all organs of tobacco preferentially expressed in roots and stems. Northern blot assays combined with further RT-PCR analysis showed the expression of NtKTI1 was repressed in response to Rhizoctonia solani inoculation, wounding and SA treatment, but wasn't affected by MeJA, ABA and NaCl treatment. Upon the expression pattern of PR1c, we speculated that the signal transduction pathway of tobacco after R. solani inoculation, which was similar to wounding treatment, was SA dependent.5. The 1471bp fragment upstream of the NtKTI1 gene was isolated by high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). Further computer prediction by PLACE database and PlantCARE found that the promoter region contained several motifs probably relate to biotic stress and phytohormones. 6. We produced an N-terminally His-tagged protein without the predicted signal peptide in E. coli BL21 (DE3 pLysS) and recombinant NtKTI1 proteins were affinity purified under native conditions. It exhibited similar inhibitory effect on bovine trypsin activity as soybean TI. In vitro assays show that NtKTI1 exerts prominent antifungal activity toward Rhizoctonia solani and moderate activity against Rhizopus nigricans and Phytophthora parasitica var.nicotianae, but had no action against bacteria, such as E. coli DH5α.7. NtKTI1 sense and antisense lines under the control of the 35S promoter were generated and confirmed with PCR and northern blot analyses. Bioassays in transgenic tobacco demonstrate that overexpression of NtKTI1 significantly enhances the resistance of tobacco against Rhizoctonia solani, and the antisense lines exhibited higher susceptibility towards the phytopathogen than the control lines.