Functional Analysis Of An Osmotic Stress Responsive Gene AT5G45480 In Arabidopsis Thaliana

AT5G45480 is a chromosome 5 gene in Arabidopsis thaliana, whose function is totally unknown. According to silico analyses, this gene may belong to a gene family containing 8 members, all of which are functional unknown genes. The proteins encoded by these genes contain multi-transmembrane domains and high level identity between conserved domains. Using Columbia ecotype (Col-0, wild-type) as control, we studied how T-DNA insertion loss-of-function mutant CS859633 (obtained from ABRC) respone to osmotic stress, meanwhile we studied the function of AT5G45480 through analysising of differentially expressed genes between wild-type and CS859633 obtained from Digital Gene Expression Tag Profiling (DGE) technology, which was based on Illumina Hiseq2000.The main results are listed as follows:(1) DNA and RNA were extracted from roots of wild-type and CS859633, and homozygotes of CS859633 were identified by PCR and RT-PCR.(2)Wild-type and CS859633 were cultivated in 3 different conditions including 1/2 MS,1/2 MS+300mM Mannitol and 1/2 MS+100mM NaCl. After 4 and 7 days, germination rate and root length of both wild-type and mutant were tested. Comparing with wild-type, germination rate and average root length of mutant CS859633 in 1/2 MS+300mM Mannitol and 1/2 MS+100mM NaCl were dramatically decreased. This result indicates that CS859633 is more sensitive to osmotic stress.(3)RNA was extracted from 4 samples and sequencing libraries were established through high-throughput sequencing system.4584 reads were obtained and Q20 valued up to 97.38%. This score means that the sequencing quality was high enough. After rogue of original reads, we got 35 millions of clean reads and the max bases number came from wild-type cultivated under 1/2 MS condition, which was 5.3 fold of the genome size of Arabidopsis thaliana.(4)The sequencing results were mapped to reference genome and the mapping rate was up to 98.36%, meanwhile 33557 genes and 41621 transcrips were obtained. Pairwise comparison between 4 samples were conducted and the number of differentially expressed genes(or transcrips) were as follows:1/2 MS-WT VS 1/2 MS-mutant:20 (15); 1/2 MS +300mM mannitol-WT VS 1/2 MS +300mM mannitol-mutant:234 (196); 1/2 MS +300mM mannitol-WT VS 1/2 MS-WT:409 (354); 1/2 MS+300mM mannitol-mutant VS 1/2 MS-mutant354 (273).(5) GO (Gene Ontology) enrichment was conducted on differentially expressed genes (or transcrips) to study their functions. Analysis of functional classification of differentially expressed genes (or transcrips) reveals that Arabidopsis thaliana responses to osmotic stress mainly by material systhesis, molecule transportation, expression or activity regulater of proteins, next to which are changing of cellular structure or component.(6) KEGG pathway analysis was conducted on differentially expressed genes (or transcrips) to investigate the relevant biological processes of these target genes. Basing on the results we draw the conclusion that Arabidopsis thaliana responses to osmotic stress by synthesizing of peroxidase to eliminate the damage of peroxidate, by synthesizing and accumulating of osmotic solutes to adapt to hyperosmotic condition, by regulating synthesis of proteins or expression level of genes, by signal transducting to connect multiple processes to resist stresses.According to the above results, we draw a priliminary conclusion that AT5G45480 involves in responses to osmotic stress. It may work as a regulator of peroxidase activity or expression of relevant genes.