A Preliminary Study On Arabidopsis Function-unknown Gene AtCS82

On the basis of the SSH libraries constructed from the key development period of seeds of Brassica napus during assimilation product converting, after comparing the EST sequences with the genome of Arabidopsis thaliana, we screened a highly homologous gene, named AtCS82. We investigated the gene by means of bioinformatics, molecular biology and gene engineering techniques, the results were as follows:1. By using bioinformatics approaches, the amino acid sequence of the AtCS82was deduced, and the hydrophobicity/hydrophilicity, signal peptide, secondary structure and transmenbrane structure of the AtCS82were predicted. The results showed that the AtCS82gene sequence composed of792base pairs, and the CDS composed of432base pairs, coding a protein of143amino acids. AtCS82was a hydrophilic secreted protein, encompassed a signal peptide and a transmembrane sequence, with a molecular weigh of14323.3Da, and a isoelectric point of11.09.2. The amino acid sequence of AtCS82was compared with those derived from cruciferous plants deposited in public databases. The results showed that the sequence was highly homologous with sequnences of Arabidopsis lyrata, Thellungiella halophila, Capsella rubella and Brassica rapa, respectively, and that those proteins had a similar conservative domain with unknown function.3. The result from the semi-RT-PCR showed that the AICS82gene expressed in varying degrees in stems, leaves, flowers and siliques of Arabidopsis thaliana. The highest expression level was detected in the siliques after flowering4days and7days, followed by those in stems and flowers, and the lowest was in leaves.4. By means of the double-primer assay as well as the RT-PCR verify method, AtCS82gene-silencing T-DNA insertion mutant homozygous plants were screened.5. After the AtCS82gene overexpression vector pCAMBIA1300-35S::CS82was constructed and then was transformed into the Col ecotype of Arabidopsis thaliana by floral dipping via Agrobacterium tumefaciens mediating, AtCS82gene-overexpression transgenic plants were successfully screened.