Isolation And Functional Characterization Of A GRAS Family Member BnLAS In Brassica Napus

GRAS proteins as a unique transcription factor family to plants were involved in the growth and development and signal transduction pathways. So far there have been very few studies on the functions of GRAS proteins in Brassicasea. Arabidopsis LAS, a member of the GRAS family, has an important role in the initiation of axillary meristems. To study the function of GRAS family in Brassica napus, an Arabidopsis LAS homologous gene was cloned in three Brassica species. The BnLAS gene function was analyzed through overexpressing of BnLAS in Arabidopsis, focusing on the increased drought tolerance. The main results are as follows.Based on the sequence information of Arabidopsis LAS, BnLAS was amplified from the genomic DNA of Brassica napus cv. Huashuang.5. The full ORF sequence with 1326bp of BnLAS was finally cloned by TAIL-PCR. The upstream region of 1944bp from the start codon of BnLAS was also isolated using TAIL-PCR. Bioinformatic analyses showed that BnLAS gene was one of the members of GRAS family. Southern blotting indicated that there are likely two copies of the gene in Brassica napus genome.The expression pattern of BnLAS was showed highest levels in roots, and lower in bud, flower organs and shoot tips based on qRT-PCR analysis and GUS staining. Lateral meristems, stems and leaves were observed lowest levels in qRT-PCR analysis, but not detectable with GUS staining.In order to research the function of BnLAS, the vector of overexpression BnLAS was constructed under the CaMV 35S promoter and transformed in Arabidopsis. Compared with wild type Arabidopsis plants,35S::BnLAS transgenic plants exhibited various morphological alterations as well as growth and developmental modifications, such as growth retarded, chlorophyll content increased, flower time delayed, fertility reduced.Dehydration treatment was conducted with controlled amount of biomass for wild type and transgenic plants. After 15 days, wild type plants were more severe withering than transgenic plants. In a different experiment, three types of plants (wild type, las mutant, transgenic plants from OL14 and OL18 lines) were grown in the same pot and subject to dehydration treatment. After re-watering,71.9%(OL14) and 91.6%(OL18) of transgenic plants recovered, whereas only 21.9% wild type and 11% las mutant plants did. Then water loss rate of leaves was slower in transgenic lines OL14 and OL18, compared to wild type.The root development in transgenic plants was not significantly different from the wild type. However, the water loss rate in leaves of plants was reduced, indicating that drought resistance of transgenic plants may have a direct relationship to the leaves structural changed. The results of scanning electron microscope (SEM) and light microscopy showed that the leaf stomatal density in line OL18 increased by 1.7-1.9 times compared with the wild type plants. But the number of open stomata was no significant difference between wild type and transgenic plants. However, the stomatal apertures on the leaves of line OL18 reduced to about 18%-40% of wild type plants.The SEM observations showed a thicker wax in leaf surface, of transgenic plants. Several key wax biosynthetic genes were analyzed by quantitative PCR. The transcription levels of four genes(CER1, CER2, KCS1, KCS2) showed a dramatic increase in transgenic plants, indicating that ovexexpressing BnLAS in Arabidopsis has an important impact on wax biosynthesis. By chlorophyll leaching experiments, the results of thicker wax led to altered cuticular permeability were further confirmed.In summary, BnLAS play an important role on plant growth and development. The overexpression of the gene resulted in an increased drought tolerance by ovexpressed in Arabidopsis. The increased drought tolerance is mainly caused by the changed leaves structures.