Cold-inducible Related Gene Idendification And Promoter Functional Analysis Of PHD-finger Transcription Factors Family In Rice

Rice is one of the most important crops in the world. The high and stable yield of the rice is related to national security. Although the rice yield and quality are always destroyed by abiotic stresses, such as cold, drought and salinity. The breeding process of oriented cultivation and directed modification will be shortened via modern genetic technology engineering. It is one of the most effective approaches to improve the resistance-capability by using explored candidate functional genes. Transcription factors in plants play a very important role in stress response to adversity stress. It becomes a hotspot to research on TFs and their function in the genetic improvement of plant resistance breeding.PHD-finger transcription factors family is a kind of zinc finger protein widely exists in eukaryotic. The researches of their function often focus on gene transcription and chromatin state regulation. Thus, there were fewer reports about their function involved in stress response. So it’s significant for adversity-resistance breeding in rice by comprehensively identifying the members of PHD-finger in rice, exploring the candidate genes in reponse to abiotic stress and explaining the resistance mechanism. In this study, we performed genome-wide survey of PHD-finger gene members in rice and characterized their gene structure features, phylogenetic relationships and abiotic stress-induced expression patterns. Two genes in response to coldi-induced were screened, and then the function and acting mechanism of their promoters were also analysed. The detailed results were as follows:1. By Blast homologous search, a total of 58 PHD-finger transcription factors were identified in the rice genome. They were found to distributed unevenly on chromosome 1 to 12 except chromosome 10 by chromosomal location. About 1/4 of the family member genes were distributed near the telomeric region. Additionally, there were 6 gene clusters with contribute to the expansion of PHD-finger family. All the genes were further divided into six class (I-VI) based on phylogenetic tree by Neighbor Joining methord. This revealed highly conserved domain exsisted in the same class.2. The expression patterns of all the PHD-finger genes were analysed by transcriptome data in rice based on Genevestigator system, and 9 candidate genes in response to adversity stress include cold, drought and salinity. Furthermore, then-expression patters were identified by qRT-PCR. The results showed that there were two genes simultaneously in response to the three abiotic stresses. Gene OsPHD13 and gene OsPHD52 were obviously up-regulated more than 15 times under cold treatments, whereas there was no gene obviously up-regulated under drought and salt stress.3. The promoters of Gene OsPHD13 and gene OsPHD52 were cloned respectively from rice genome. Then they were constructed to drive GUS report gene in expression vectors for rice transformation. The results of GUS staining showed that the promoters of pOsPHD13 and pOsPHD52 both had cold-inducible activity in different tissues of transgenic plants. And the expression level was higher in stem than other tissues.4. The cis-acting elements in the promoters of pOsPHD13 and pOsPHD52 were predicted by bioinformatics. And a series of truncated versions from 5’flank of promoters was constructed and transformed into tobacco for detailed study. The fragment -856bp～-817bp in pOsPHD13 and the fragment-295bp～-224bp in pOsPHD52 were the key sequences. The function of key sequences were further confirmed in tobacco transient expression system via in vitro synthesis. Each of the key sequenc had a DRE/CRT cis-acting element which was reponse to cold stress. Subsequently the element was important to the promoters by site mutation and block mutation.5. The trans-acting factor in CBF family, specificly combining with DRE/CRT cis-acting element, was identified using yeast one-hybrid system and EMSA. The same cis-acting element in different promoters had different protein-combinding patterns. Two proteins, CBF1 and CBF3 screened out from the CBF family, would combined DRE/CRT cis-acting element in pOsPHD13. And only one gene CBF1 would combined DRE/CRT cis-acting element in pOsPHD52.