Cloning, Expression Analysis And Genetic Transformation Of BdASR Gene Family In Brachypodium Distachyon

Plants are constantly confronted with multiple adverse stresses, such as extreme temperatures, drought, high salinity and pathogen attact, which adervsely affect crops' productivity and quality. Therefore, the functional investigation on the abiotic stress- related genes is of great significance to further explore the molecular mechanisms of plant in response to stress and molecular breeding for stress tolerant crops. Abscisic acid, stress and ripening- induced proteins(ASRs), identified originally as a component in plants' response to ABA and fruit maturation, play an important role in various stress responses. Although ASRs have been studied in many plants, there is no such report on the new model plant Brachypodium distachyon.In this paper, based on bioinformatic analysis, all five members of ASR gene family in B. distachyon were cloned. Then the expression patterns of these five BdASRs in different organs, and in response to abiotic stresses and signal molecules treatments were analyzed by qRT-PCR. Further, the tolerance of yeast transformed with BdASRs under stresses was analyzed. The transcriptional activation activity of Bd ASRs was examined by yeast one-hybrid assay. In order to investigate the function of BdASR1, subcellular localization of BdASR1 in onion epidermis was analyzed and the BdASR1 was transformed into tobacco. The main results of present study are as follows:1) The full- length c DNA sequences of the five BdASRs were cloned using specific primer pairs by means of RT-PCR, and were designated as BdASR1, BdASR2, BdASR3, BdASR4 and BdASR5, respectively.2) The expression patterns of BdASRs in different organs and under stress treatments were analyzed by qRT-PCR. Our data showed that the expression of different BdASR varied in different organs, and the expression of different BdASRs in the same organ were also different. It also showed that BdASRs had differential expression changes under various abiotic stresses(osmotic stress, high salt, low temperature) and signaling molecules(ABA, ETH and H2O2).3) The yeast expression vectors pYES2-BdASRs were constructed and were transformed into yeast. Results showed that yeast cells transformed only with BdASR1, BdASR2 and BdASR3 respectively had increased tolerance to C uSO4 treatment among different stress treatments including H2O2, CuSO4, sorbitol, PEG, 50? and-20?.4) The pGBKT7- BdASRs recombinant plasmids were constructed, which were used to analyze the transcriptional activation activity of Bd ASRs by yeast one-hybrid assay. It was found that only the Bd ASR1 protein had the transcriptional activation activity, indicating that BdASR1 may function as a transcription factor.5) In order to investigate the subcellular localization of Bd ASR1, the pCAMBIA1304-BdASR1 recombinant plasmid was constructed and transformed into onion epidermis. The result showed that BdASR1 was lo calized in nucleolus, which again confirmed BdASR1's function as a transcription factor.6) The pC AMBIA1304-BdASR1 recombinant plasmid was, therefore, transformed into Agrobacterium tumefaciens, and transgenic tobaccos with overexpression of BdASR1 were obtained by Agrobacterium-mediated transformation method.In conclusion, our study provided useful information and experimental materials for further research on BdASR1 in response to various stresses.