Tea Yeast Two Hybrid CRNA Library And The Interactive Protein Screening Of CsCOR1

Tea plants (Camellia sinensis L.) is an economic woody crop in China. Due to the sudden cold or "cold spell in later spring" climates in late autumn and early spring of each year, tea plants suffered frequently cold damage. Therefore, cold damage is the major problems in tea production. In order to reduce freeze injury in tea plants, the research on the cold-damage mechanism was studied extensively in recent years. A cold induced gene from tea plants, CsCOR1,was cloned in our laboratory. Our results showed that the protein encoded by CsCOR1 may be a extracellular signal molecules. To further analyze the mechanism on CsCOR1 function, we carried out a series of research. The main research results are as follows:1. Construction of yeast two hybrid cDNA Library of tea plantAfter the total RNA of tea plants was extracted, the first strand cDNA was synthetised using the reverse transcriptase and Biotin-attB2-Oligo(dT) primer (InvitrogenTM company). After the double strand cDNA was synthetised,5’-end of the cDNA was connected a joints with recombinant sequences. Then, the cDNA was connected to the pDONR222 vector by BP reaction of site-specific recombination technology, and the primary library was constructed by transforming the pDONR222-cDNA into E.coli. DH10B. The yeast two hybrid cDNA library of tea plants was constructed finally by transformed pDEST22TM-cDNA into MaV203 after the cDNA of the pDONR222 vector was linked into pDEST22TM vector via LR recombination reaction.2. Construction of the bait vector with CsCOR1 gene for yeast two hybridThe CsCOR1 sequence encoding mature protein (without coding the signal peptide sequence) was amplified via PCR according to the full-length CsCOR1 sequence (Accession No. EU563236) submitted previously by our laboratory. The pDONR222-CsCOR1 carrier was then obtained through a BP reaction. After confirmation via sequencing, CsCOR1 of pDONR222 vector was recombined to pDEST32 vector by a LR reaction. The bait vector, DEST32-CsCOR1, was transformed into E. coli. DH5α for amplifying.3. Screening of CsCOR1 interaction protein genesFirst of all, we transformed the pDEST32(vector) and bait plasmid vector pDEST32-CsCOR1 into yeast MaV203; the different concentration of 3-AT (3--1,2,4-three amino triazole) screening assay, the appropriate concentration of 3-AT on the inhibition of vector self activation were investigated. The results showed that the 3-AT can effectively inhibit the carrier at 50mM. Then, we use the method of co-transformation to transform the pDEST32-CsCOR1 bait expression plasmid and cDNA library expression plasmid into the yeast MaV203. The transformed bacterial solution was coated on the SD-Leu-Trp-His+50 mM 3-AT defect plates, and screened the positive clones which contained the bait vector and cDNA library. Then, we transferred these positive clones to SD-Leu-Trp-Ura defect type, and screened the clones which were related to the presence of CsCORl protein. Finally, we will be use SD-Leu-Trp-Ura defect type flat plate to screen positive clones; and then transfer to YPDA solid plate containing X-a-gal (20 mg/mL). After the lacZ gene expression was initiated by the interaction of the substrate, the substrate was blue, to existence and protein CsCORl interaction candidate cloning for further screening. After removal of the false positive clones, we finally screened 43 candidate clones interacting with CsCORl protein.4. Sequencing and sequence analysis of positive clonesThrough the above experiments, we selected 43 positive clones; which were screened by plasmid and transformed into E.coli DH5a. By ampicillin screening, cloning plasmid library was obtained with pDEST22TM-cDNA, and sent to DNA sequencing subsequently. After sequencing, we obtained several complete read cDNA sequence frames, to get the sequence after the database search of related protein encoding gene and CsCORl interaction of annotation analysis. Here, we report three of these interacting proteins, the three EST sequences encoding unknown functional proteins, novel short chain dehydrogenase/reductase and ribosome inactivation proteins.