The Mechanism Of Zinc Finger Protein OsZFP207 Regulating Plant Growth And Development In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is not only one of the world’s most important food crops which feeds more than one third of the people on the earth,but also a model plant for research on molecular biology in monocotyledons.Plant height is one of the most important agronomic traits in rice.The research on the molecular mechanism of plant height have contributed to high yield rice breeding and will contribute to super rice breeding with ideal architecture.Many studies showed that the rice plant height is influenced by various hormone pathways and complex environmental factors but the detailed molecular regulatory network needs to be further studied to be clarified.It was previously found that a C2H2 type single zinc finger protein 207(OsZFP207)played important role in plant growth and development.Here we further analyzed the mechanism of OsZFP207 regulating plant growth and development,especially pant height in rice.1)It is predicted that OsZFP207 has only one C2H2 zinc finger protein domain,which belongs to C2H2 single zinc finger protein through SMART.It was found that OsZFP207 only existed in monocotyledonous plants,including all kinds of wild rice and cultivated rice and was highly conserved in different species through ENSEMBL.The expression of OsZFP207 was predicted to be low in most rice tissues but high in callus and stems through ENSEMBL.The expression of OsZFP207 was found to be high in root,leaf sheath and increase gradually during seed development by temporal and spatial expression analysis through RAP-DB database.It was induced by salt,drought and low temperature by real-time quantitative PCR analysis.The protein OsZFP207 was localized in the nucleus by sub-cellular localization analysis using tobacco leaves.2)The OsZFP207 RNA interference and over-expression transgenic lines were obtained by using transgenic technology.The plant height changing rate of OsZFP207 over-expression transgenic lines was lower than wild type.Whereas the plant height changing rate of OsZFP207 RNA interference transgenic lines was slightly higher or similar to the wild type at seedling stage in green house and all growth stages after transplanted to the field.Compared with the wild type,the three over-expression transgenic lines of OsZFP207 had shorter plant height,less tillers,shorter panicle length,smaller seeds,less 1000-grain weight and enclosed panicle phenotype.While only the number of tillers and primary branches from the three OsZFP207 RNA interference transgenic lines were all increased.The OsZFP207 over-expression transgenic line OE1 showed significant lower germination index and rate but the OsZFP207 RNA interference transgenic line R10 showed slightly higher germination index and rate compared to the wild type ZH11 under normal conditions.The width and length of lemma epidermal cells of OsZFP207 over-expression transgenic line OE1 became smaller than the wild type,while only the length of lemma epidermal cells of OsZFP207 RNA interference transgenic line R10 became longer than the wild type by using electron microscope scanning.The results of internode length analysis showed that the dwarfing of OsZFP207 over-expression transgenic lines was mainly caused by the decrease of the length of the first and second internodes.The size of seedling leaf epidermal cell in OE1 was found smaller than WT but R10 was bigger than WT by checking the leaf epidermal cells after applying a whole clearing technique under a differential interference contrast microscope.The SNP analysis of OsZFP207 showed that there were four SNP mutation sites in the exon of this gene by using the database from http://www.rmbreeding.cn/snp3k.The haloptypes analysis of OsZFP207 showed that this gene had the highest correlation with plant height,panicle length and ligule length of rice by using the four main haplotypes which with a sample number≥50.It means there are OsZFP207 allelic variations which modulate plant height in natural rice populations.3)The content of GAs was lower in OsZFP207 over-expression transgenic lines but higher in OsZFP207 RNA interference transgenic lines than the wild type.The expression of several gibberellin synthesis related genes,including the "green revolution" gene OsGA20ox2/SD1,was found to be down regulated in OsZFP207 over-expression transgenic lines when compared with the wild type by Affymetrix microarray analysis.Yeast one-hybrid and EMSA analyses showed that zinc finger protein OsZFP207 could bind to the region containing DST binding site(DBS)in the promoter of OsGA20ox2 in vitro.Furthermore,we found OsZFP207 could bind to the promoter of OsGA20ox2 in vivo by ChIP-qPCR which confirmed that OsZFP207 could depress rice growth by interfering in the gibberellin acid(GA)pathway.4)The expression of OsZFP207 in rice seedlings was found to be induced or impressed when treated with cytokinin(CTK),abscisic acid(ABA),jasmonic acid(JA)and gibberellin acid(GA)by bio-informatics analysis.Some CTK,IAA or cell division related genes were found to have higher expression in OsZFP207 over-expression transgenic lines OE1 and OE13 compared to ZH11 by Affymetrix microarray analysis.Some of the up-regulated genes such as OsCycB2;2,OsCDKB2;1,OsRR5,OsRR6 and WOX11 were confirmed in OsZFP207 over-expression transgenic line OE1 and they were found to be down-regulated in OsZFP207 RNA interference transgenic line R10 by real-time PCR.