Functional Characterization Of OsHsfAs And OsCH2 Genes In Rice

Rice (Oryza Sativa L.) is one of the most important crops worldwide. Abiotic stresses such as high temperature, drought and high salinity are major limiting factors that greatly affect rice growth, development and yield performance. Therefore, it is very significant to find important 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) can improve plant tolerance to stresses and are mainly regulated by heat shock transcription factors (Hsfs). Studies showed that over-expression of P-carotene hydroxylase gene in transgenic Arabidopsis plants increased resistance against stresses. In this experiment, twelve OsHsfA genes in rice(Oryza sativa L.) were studied for their expression levels in different tissues and under high temperature, high salinity, PEG and low temperature stresses. The functions of heat shock transcription factor gene OsHsfAla, OsHsfA7 andβ-carotene hydroxylase gene OsCH2 in rice development and their responses to stresses were analyzed by over-expression and RNAi technology. The main results are as following:1. The OsHsfA genes exhibited tissue-specific expressions under normal condition. OsHsfAla, A2d and A9 were predominantly expressed in young spike. Most OsHsfA genes responded quickly to heat stress and accumulated to higher level at 45 min to 3 h. In particularly, the induction of OsHsfA2a expression in response to heat stress was highest among the heat shock factors examined. However, most of the increased OsHsfAs expression responses to salt, PEG and cold treatments primarily occurred during the later stages (3 to 24 h) of stress exposure. Furthermore, several OsHsfA gene expressions were little affected under cold stress,2. Over-expression of OsHsfA7 gene affected rice growth and development. OsHsfA7-OE transgenic plant exhibited reduced plant heights and tiller numbers. Scanning electron microscopy observation of stem structure showed that the numbers of columnar cells were significantly reduced but the width was increased in the first internode of OsHsfA7-OE rice plants. The change of columnar cell may be related with the dwarf character of OsHsfA7-OE plants. Meanwhile, OsHsfA7-OE transgenic plants exhibited stout and long young roots (including axial root and adventitious root) but short and less lateral roots. The roots of OsHsfA7-OE rice at the tillering stage were thick, sparse, wide distributed and almost no lateral roots on the adventitious root compared with that of wild type. The OsHsfA7 over-expression transgenic plants didn't improve tolerance to heat stress. Nevertheless, under 200mM salt treatment, OsHsfA7-OE transgenic plants showed delayed appearance of damage symptoms and elevated survival rate, the electrical conductivity and MDA content of OsHsfA7-OE plants were lower than those of wild type. These observed phenotype, physiological and biochemical index indicated that the over-expression of OsHsfA7 gene in rice seedlings increased tolerance to salt stress. There was obvious phenotypic difference between OsHsfA7-OE transgenic rice and wild type seedlings after drought and re-watering treatment, suggesting that over-expression of OsHsfA7 gene might enhance resistance to drought stress. However, OsHsfA7-RNAi transgenic plants did not show sensitivity to high temperature, high salt and drought stress.3. Plant expression vectors for OsHsfAla including over-expression, RNAi inhibition and promoter vectors were constructed. RNAi transgenic rice plants were obtained via Agrobacterium-mediated transformation and confirmed at DNA and mRNA level by PCR. OsHsfAla-RNAi transgenic plants showed low pollen fertility as detected by I2-KI stain method and significantly reduced seed setting rate. Pollen development was observed by paraffin section in the OsHsfAla-RNAi plants and we found that the pollen abortion appeared from single nuclear pollen grains phase to uninucleate eccentric stage. In addition, OsHsfAla-RNAi transgenic plants were not sensitive to high temperature.4.OsCH2 gene exhibited diverse expression in different organs and at higher level in leaves, and responded to high temperature and high light stress. Visible color changes appeared in young leaf, stem, panicle and seed of OsCH2-OE transgenic plants. Chloroplasts in yellow spotted leaves of OsCH2-OE transgenic rice seedlings degenerated and OsCH2-OE seedlings exhibited poor tolerance to low light stress. The total content of photosynthetic pigments, leaf SPAD readings and Fv/Fm value were reduced, thus photosynthetic efficiency decreased in the OsCH2-OE transgenic plants. The content of (3-carotene decreased in OsCH2-OE transgenic rice. The expression of OsZEP gene involved in xanthophyll cycle was up-regulated in favor of zeaxanthin into violaxanthin, while OsVDE gene expression was little affected, therefore, violaxanthin content in OsCH2-OE plants increased. In addition, ABA biosynthesis related gene aba2 expression in transgenic plants was enhanced, over-expression of OsCH2 gene resulted in the delay of seed germination. Besides, OsCH2-OE transgenic rice seedlings were treated by different stresses, results showed that over-expression of OsCH2 gene didn't improve the high temperature, high light and UV stress tolerance of the seedling stage.