Screening Of Proteins Interacting With Glutamyl-tRNA Synthetase In Arabidopsis Thaliana And Researching On Abiotic Resistance Of Transgenic Plants

Abscisic acid (ABA) is a kind of plant hormone which is widely distributed within some fungi and many organs of most plants. It plays a very important role in the process of plant growth and modulates seed's dormancy and germination by participating in the regulation of specific genes' transcription and translation. It has been found that ABA accumulated at the roots and leaves when the plant was under abiotic stress. At the same time, it has also been found that ABA can regulate the statue of stomatal (opening or closing) by adjusting ion channels and the infiltration. These results demonstrated that ABA has some effect on plant resistance. By yeast two-hybrid screening, some proteins interacting with the ABI2 bait protein were separated from Arabidopsis thaliana cDNA library. A fragment including the N-terminal 262 amino acid residues domain of the glutamyl-tRNA synthetase had been obtained. It presumes that glutamyl-tRNA synthetase is an element of ABA signaling pathway and has very close relationship with resistance of plant under abotic stress.Glutamyl-tRNA synthetase (GluRS) which belongs to the class I aminoacyl-tRNA synthetase plays the primary role in the strictly matching process that amino acids combine aminoacyl-tRNAs. Therefore, it guarantees the accuracy of protein synthesis. Almost each kind of amino acids has its corresponding aminoacyl-tRNA; and this specific catalysis is critical for the transmission of genetic information correctly. Meanwhile, many other aminoacyl-tRNA synthetases have been found other founctions which are also quite important for plant to maintain nomal life activities. It was never reported before that glutamyl-tRNA synthetase took part in the resisting mechanism to the abiotic stress. One part of my experiment in this thesis was treating six lines of transgenetic plants by various abiotic stresses to investigate further explore the effect of glutamyl- tRNA synthetase on seed germination and root growth and to work out the function of glutamyl- tRNA synthetase in plant resistance.In this experiment, competent host strain AH109 was prepared by using PEG/LiAc, according to the method of Clontech. The sequence of glutamyl-tRNA synthetase from Arabidopsis thaliana (AtGluRS) was inserted to BD vector to construct the bait plasmids (pGBKT7-GTS). Then total RNA was extracted from the seedling of Arabidopsis thaliana (RLD) which were cultivated at 22℃with a photophase of 16h/d for 2-3 weeks and also acted as the template of the first strand cDNA synthesis.Then the cDNAs were used to be the template for amplifying dscDNA which built the AD fusion library of prey proteins. BD-vectors,dscDNA and AD vectors were subsequently cotransformed into the yeast AH109 competent cells. After being cultured for 1h at 30℃, transformed cells were spread onto SD/-Leu/-Trp/-His agar plates. The plates were incubated for 4—10 days at 28℃, until discrete colonies appeared. These colones were supposed to be positive clones and need further testings. Spread these putative positive clones onto SD/-Leu/-Trp/-His and SD/-Leu/-Trp agar plates for galactosidase fliter assay which is a colorimetric assay for the transcription of LacZ reporter gene productβ-glactosidase. By detecting LacZ gene expression, most of the false positive clones were removed. Total yeast DNA has been extracted to gain the BD and AD vectors with interaction proteins and transformed these plasmids into E. coil JM109 competent cells. The cells which only include AD plasmid can survive on LB (Amp) ager plates and those which include BD plasmid can survive on LB (Kan) ager plates. Therefore, BD and AD vectors can be obtained, respectiviely. Moreover, specific primers of AD vector were used to amplify sequences in the recombinated AD plasmid by PCR. Furthermore, these recombinant AD plasmids were extracted for non-restriction enzymes treatment to remove duplications of interacting proteins. These final positive clones need to be screened again using yeast two-hybrid system, with transforming two recombinant plasmids AD and BD together into yeast AH109 and tested again by His report gene and LacZ gene.