The Research On Interaction And Related Functions Of Potato Virus Y HC-Pro Protein With Processing Tomato Protein Cab-1A-like

Potato virus Y(PVY),which belongs to the Potyvirus genus, is one of the main diseases of Processing Tomato and seriously affected the yield and quality of Processing Tomato. While the helper component proteinase HC-Pro encoded by PVY is a multifunctional protein, it not only has the function of protease, but also participates in various processes, such as aphid-mediated non-persistent spread of PVY, symptom expression, the movement between cell-to-cell, RNA silencing in plants as well as trans-activating other viruses and so on. These functions of HC-Pro are more or less achieved by interaction ith host protein factors. It involved in the self-interaction of HC-Pro, plant protein translation initiation factor eIF(iso)4E, ethylene induceing transcription factor RAV2, chloroplast division protein NtMinD and so on. Using HC-Pro as bait protein, a encoded chlorophyll a/b-binding protein 1A(Cab-1A) homologous gene from processing tomato cDNA library was screened by the way of yeast two-hybrid, and named this gene as LeCab-1A-like.Objective: This study would further validate whether or not the HC-Pro protein interacts with the Processing Tomato protein Cab-1A-like in plants through bimolecular fluorescence complementation, in order to in-depth probe the interactions mechanisms between HC-Pro and Cab-1A-like in tomato, and to lay the foundation for the cultivation of antiviral processing tomato.Method: 1 Cab-1A gene is amplified from processed tomato cDNA library of full-length sequence, YFP N- terminal and C- terminal fusions of BiFC binary vector pSPYNE-35S-Cab-1A, pSPYCE-35S-Cab-1A are constructed. And construct HC-Pro BiFC expression vector pSPYNE-35S-HC-Pro and pSPYCE-35S-HC-Pro; 2. The Sequence is analyzed by bioinformatics, respectively, including a homologues multi-alignment analysis, as well as prediction analysis of Cab-1A-like protein in primary, secondary and tertiary structure, prediction of molecular weight and cellular localization, isoelectric point of Cab-1A, and a transmembrane prediction; 3. N- terminal and Cterminal of YFP fused BiFC binary vectors and N-terminal, C-terminal fused GFP subcellular localization vectors pSPYNE-35S-Cab-1A, pSPYCE-35S-Cab-1A, pSPYNE-35S-HC-Pro, pSPYCE-35S-HC-Pro, pCBGFP-Cab-1A, pSPGFP-Cab-1A; 4.Respectively transform these two localization vectors into agrobacterium and then instantaneously transform tobacco, observe the distribution of green fluorescence by laser scanning confocal microscope;5. mix equal volume infection liquid which contained YFP-N terminal BiFC plasmid and the corresponding YFP-C terminal BiFC vector,respectively, transiently transform tobacco epidermal cells, observe the distribution of yellow fluorescence by laser scanning confocal microscope; 6.Cultivate processing tomato seedlings and transformed BiFC interaction vector by leaf disc method, then observe the distribution of corresponding fluorescence by laser scanning confocal microscope.Results and analysis: 1.cloned cab-1a-like was 798 bp by gene sequencing, and we predicted that it can encode a polypeptide sequence of 265 amino acids, processing tomato cab-1a gene has a chlorophyll a/b binding protein family domain, belonging to the family, and there is a potential N-terminal chloroplast transit peptide, and Cab-1A-like protein molecular weights about 28.07 KDa, pI(isoelectric point) is 5.51, and the 92.1% sequence was conserved. The secondary structure prediction of Cab-1A-like protein was that it has three transmembrane helices, and Cab-1A-like protein also consists of a 35.09% α- helix, 8.68% β-sheet, and 56.2% random coil structure; 2. In the transiently transformed pSPYCE-35S-HC-Pro/pSPYNE-35S-Cab-1A vectors tobacco cells, strongly yellow fluorescent was observed along the nucleus, it was indicated that the HC-Pro protein possiblely interacted with Cab-1A-like protein; 3. In transiently transformed tobacco cells with the C-terminal fused GFP vector pSPGFP-Cab-1A,2d later, the fluorescence was observed in the cytoplasm;4. Geneticly transformed BiFC vectors by leaf discs, got one transgenic plants with pSPYNE-Cab-1A, and two transgenetic plants with pSPYCE-Cab-1A, and one plant with pSPYCE-HC-Pro.