Construction Of Yeast Two Hybrid CDNA Library And Expression Analysis Of Translationally Controlled Tumor Protein In The Early Stages Of TcLr19 Under The Stress Of Puccinia Triticina

Part 1: Construction of yeast two hybrid cDNA library in the early stages of TcLr19 under the stress of puccinia triticinaConstruction of yeast two hybrid cDNA library in the early stages of wheat under the stress of Puccinia triticina have been more significant to studying protein-protein, protein-RNA, protein interacted with small molecules in wheat-leaf rust interaction.In this experiment, Wheat near-isogenic line TcLr19 was infected with leaf rust race 366, and then total RNA was extracted from the leaves after infected 4, 8 and 12 hours. The total RNAs were reverse transcribed to cDNA by using Oligo (dT)18 Primer and Random Primer, respectively. Two libraries were constructed by using SMART technique and homologous reorganization method in yeast strain Y187. According to the evaluation on quality, the transformation efficiency were about 1.32×106 transformants/3μg pGADT7-Rec and 1.0×106 transformants/3μg pGADT7-Rec, the library titers would be up to 2.62×108 pfu/ mL and 3.51×108 pfu/ mL and the recombinant rate would be 93% and 100%.The test proved that the two libraries have good diversity, the gene length is more evenly distributed, library titers were≥1. 0×106, can be applied to large-scale screening of protein interactions.We can use it to screen protein interaction, isolate and identify more signal transduction components, study structure and function of disease resistance protein and establish a more complete signal transduction network of leaf rust resistance. It will help us to understand deeply the molecular mechanisms of wheat leaf rust resistance and lay the foundation for the clone of wheat leaf rust resistance gene and the cultivation of new leaf rust resistance varieties. Part 2: Expression analysis of TaTCTP in the early stages of TcLr19 under the stress of puccinia triticinaTranslationally controlled tumor protein (TCTP) is a highly conserved and expressed eukaryotic protein family. It was found that TCTP was ubiquitously present in animals, plants and yeast and has a high degree of homology. Numerous studies show that TCTP is a multi-functioning protein that performs vital roles, particularly in various complicated life processes. TCTP was initially identified as a growth-related protein, recently, some studies suggested that TCTP may have extremely important biological functions in various biological processes, including histamine release function, microtubule binding protein, calcium binding protein, anti-apoptosis, regulation of cell cycle and malignant metastasis, inhibition of Na+, K+-ATPase activity, antioxidant, heat shock protein with molecular chaperone activity and Antimalarial effect, etc.The study of wheat (Triticum aestivum L.) resistance to leaf rust (Puccinia triticina) infection physiological mechanism has long been engaged in our laboratory and has been initially proved that Ca2+, ROS and cytoskeleton , etc., involved in the signal transduction process of wheat resistance leaf rust infection; the differentially expressed library of Wheat Near Isogenic Line TcLr19 under the Stress of leaf rust was constructed using suppression subtractive hybridization technique and it is found that TCTP gene overexpressed at 8h and 16h after inoculation in the library. Refer to the current research progress on TCTP, preliminary conclusions were drawn that TaTCTP may be involved in wheat-leaf rust interaction.In order to further explore the role of TaTCTP, preparation of the specific antibody of TaTCTP is necessary. It is the most effective and specific reagents in localization, quantitation and physiological functions research of TaTCTP. This experiment according to known sequences, using wheat near-isogenic line TcLr19 cDNA as template, amplified gene TaTCTP. Then the recombinant expression vector pET30a(+)-GST-TaTCTP was constructed and transformed into E. coli strain Rosetta(DE3). Using E. coli expression system, TaTCTP fusion protein was inducted by IPTG. The optimal expression conditions of pET30a(+)-GST-TaTCTP: when the cell OD600 reaches 0.5~0.8 in LB medium at 37℃, adding IPTG to a final concentration of 0.1mmol/L, induced at 37℃about 20 h. Further using Ni-NTA, the TaTCTP fusion protein was purified, and SDS-PAGE showed to remove the most unwanted proteins. Protein concentration was about 1.3 mg/mL, consistent with the requirements of the antibody preparation. Purified TaTCTP was used as antigen immuned rabbit, acquire the anti-TaTCTP-specific rabbit antiserum. The study made a foundation of further exploring TaTCTP in wheat-leaf rust interaction of spatial and temporal distribution and function. In this study, the incompatible combination between wheat cultivar TcLr19 and leaf rust race 366 was used as the materials. TaTCTP gene expression at the mRNA and protein levels was detected at different time points by real-time quantitative PCR and Western blotting. The results show that the mRNA expression of TaTCTP began to show up-regulated expression after infected 2 h, and gradually increased with the interaction time and reached maximum at 24h; However, TaTCTP mRNA expression did not change significantly in the control. The results of Western blotting showed that the prepared antibody has good specificity for wheat whole protein, showing a relatively clear main band. However, whether inoculated or control, TaTCTP expression at the protein level had no significant difference. These results suggested that TaTCTP at the transcription level was induced by leaf rust infection, TaTCTP may play a role in the molecular mechanism of wheat resistance to leaf rust, but its specific function remains to be further tests proved.