Preliminary Functional Characterization Of OsHsp17.0, OsDREBA5-1 And OsRAV1-1 Genes In Rice

Rice is an important crop that more than half of the world people live on. Abiotic stresses such as high temperature, drought, and high salinity affect rice growth and development resulting in serious influence on rice yield and quality performance. Therefore, it is very important to find key regulatory or functional genes responsive to abiotic stresses, to reveal the molecular mechanisms of stress tolerance and to improve rice resistance by genetic engineering techniques. Heat shock proteins (Hsps) are important proteins whose expression is increased when the cells are exposed to elevated temperatures or other stresses. Increased expression of these proteins can protect the organism against stress-induced damage. Small Heat shock proteins (sHsps) are more abundant in plant. The AP2/EREBP transcription factors are known to be unique in plants, and they are involved in growth, development and environmental stress responses. DREB and RAV are two major subfamilies of AP2/EREBP. To investigate the gene function of OsHsp17.0,OsDREBA5-1 and OsRAVl-1, OsHspl7.0-overexpression rice plants have been exposed to different abiotic stresses including salt stress, drought stress, high temperature and low temperature treatment and the stress-tolerance has been analysised. Using semiquantitative RT-PCR method and real-time RT-PCR, the expression profile of OsDREBA5-1 and OsRAVl-1 genes in different rice tissues and under different stress have been analysised. In addition, over-expression vector and RNAi vector of OsDREBA5-1 and OsRAVl-1 genes have been constructed successfully. The main results are as following:1. OsHspl7.0-OE transgenic rice plants were obtained via Agrobacterium-mediated transformation and confirmed at transcriptional level. The results of salt and drought stress treatment showed that OsHspl7.0-OE transgenic plants exhibited less damage symptoms and higher survival rate than WT. After stress treatments, the proline content increased more significantly in transgenic plants than in wild type plants. By contrast, the MDA content and ion leakage increased more significantly in wild type plants than in transgenic plants. These results indicated that the over-expression of OsHsp17.0 gene in rice seedlings increased tolerance to salt stress and drought stress.2. After high and cold temperature treatments, both OsHsp17.0-OE transgenic and WT plants can keep on growing, and no significant phenotypic differences have been found. After stress treatments, the proline content increased more significantly in transgenic plants than in wild type plants. Oppositely, the MDA content increased more significantly in wild type plants than in transgenic plants.3. After stress treatment with heavy metal ions (Cadmium), the growth of both OsHsp17.0-OE transgenic and WT seedlings were inhibited, but no significant phenotypic difference have been found.4. The two AP2/EREBP family gene OsDREBA5-1 and OsRAVl-1 exhibited tissue-specific expressions under normal condition.OsDREBA5-1 gene is predominantly expressed in roots; while OsRAVl-1 has higher expression in leaves, suggesting that these two genes have different function emphasis.5. OsDREBA5-1 and OsRAVl-1 are up-regulated by several stresses including high temperature, PEG stress, salt stress and ABA, however, differential expression profiles have been observed. It is interesting that, under streses, the induced expression of OsDREBA5-1 is much stronger than OsRAVl-1 in rice roots; by contrast, the induced expression of OsRAVl-1 is much stronger than OsDREBA5-1 in rice shoots. It is suggest that OsDREBA5-1 mainly function in the roots and OsRAVl-1 mainly function in the shoots.6. Plant over-expression and RNAi inhibition vectors for the two AP2/EREBP gene OsDREBA5-1 and OsRAVl-1 were constructed. Transgenic rice plants were-obtained via Agrobacterium-mediated transformation and confirmed at transcriptional level.