Functional Characterization Of A Homolog Of Human SKI-Interactin Protein In Rice

Rice(Oryza Sativa L.) is one of the most important crops worldwide.Abiotic stresses such as drought and high salinity are major limiting factors for growth, development and productivity of crop plants.Rice production consumes huge amount of water,but the availability of fresh water resource is continuously decreasing in China, which is one of the bottleneck to the development of agriculture.Therefore,improvement of stress resistance of crops is a very urgent task.Great effort has been spent on the discovery of potentially useful genes in genetic improvement of stress resistance of crops in the past decade,and a number of studies suggest that over-expression of some important regulatory or functional genes in the transgenic plants can significantly improve stress resistance.In this study,eight stress-responsive genes in rice were studied for their expression levels under various stress conditions.These genes were transformed into rice to test their effectiveness on improving stress resistance.Based on the evaluation of stress resistance of these genes,we found that OsSKIPa showed very significant effect in improving stress resistance in rice,and thus,the function of this gene was investigated.The main results are as follows:1.Northern blot analysis revealed that the selected genes were induced by treatments of stresses(such as drought,salinity,and cold stresses) and/or phytohormones such as abscisic acid(ABA),jasmonic acid(JA),and salicylic acid(SA).2.The over-expression and RNAi constructs of eight stress-responsive genes were transformed into japonica rice Zhonghua 11.The salinity testing at seedling stage and drought testing at reproductive stage were conducted.Compared to the wild type, transgenic rice of three candidate genes including OsSKIPa showed increased stress tolerance.According to the results,we chose OsSKIPa for function analysis.3.The genes encoding SKIP proteins are present throughout eukaryotic phylla.The intron/exon structures of SKIP genes are quite diverse among the eukaryotic species,but SKIP proteins are highly conserved.Besides SNW domain,four additional putative domains and two conserved motifs were identified in OsSKIPa.The phylogenetic tree based on the SKIP homolog sequences agrees well with the evolutionary relationship of these species.4.OsSKIPa is expressed in all the tissues or organs investigated.The expression level of OsSKIPa is induced by various abiotic stresses and stress-related phytohormone treatments.5.OsSKIPa can functionally complement the lethal defect of the knock-out mutant of the SKIP homolog in yeast.6.The seeds from OsSKIPa-suppressed plants had a significantly lower germination rate than the WT.The growth of OsSKIPa-suppressed plants was severely retarded throughout the entire life cycle.The growth arrest mainly resulted from the reduced cell viability in the active growth regions.7.Transgenic rice over-expressing OsSKIPa exhibited significantly improved growth performance in the medium containing stress agents(ABA,NaCl,or mannitol). The improved growth performance was partially due to the enhanced cell viability of transgenic plants under the stress conditions.8.Transgenic rice over-expressing OsSKIPa exhibited significantly improved drought resistance at both the seedling and reproductive stage.The OsSKIPa-overexpressing rice showed significantly increased ROS-scavenging ability and transcript levels of many stress-related genes,including SNAC1 and rice homologs of CBF2,PP2C,and RD22 under drought stress conditions.9.OsSKIPa can affect the transcript levels of a large number of genes and the transcript level changes are due to the change of transcription rate.Gene ontology analysis revealed that genes in 3 categories of biological processes were significantly over-represented:response to stimulus(biotic,abiotic and endogenous stimulus), metabolism and cell communication.Among these genes,a great many genes are responsive to drought,salt,and cold stresses.Bioinformatics analysis of the cis-elements in the promoters of these genes suggested that several stress and ABA specific cis-elements were enriched.10.OsSKIPa could interact with 35 proteins with diverse functions,which suggested that OsSKIPa may be involved in diverse cellular processes.Most of the OsSKIPa-interacting proteins(SIPs) could be induced by several stresses.Interestingly, more than 90%of the SIPs have no match with the SKIP-interacting proteins identified in yeast,animals and human. 11.Preliminary function of another stress-responsive gene,RAMY,was investigated. Three alleles of RAMY were identified from several rice varieties.RAMY-1(Minghui 63) was induced by drought,salt,wounding,and ABA treatments.Transgenic rice over-expressing RAMY-1 growing in agar medium showed compact curly and helix-like roots.In conclusion,the main finding of this work is that OsSKIPa has evolved a novel function in positive modulation of stress resistance through transcriptional regulation of diverse stress-related genes in rice.