Functional Analysis Of Zinc Finger Protein Gene ZFP185 And Cuticular Wax Synthesis Related Gene OsCUT1 In Rice

Rice(Oryza sativa L.)growth and production are largely affected by abiotic stresses including drought,salt and low temperature.As sessile organisms,plants are frequently exposed to variable environmental stress,causing them to develop efficient mechanisms to sense and adapt to stress conditions.In this research,we analyzed the functions of A20/AN1-type zinc finger protein ZFP185 and a(3-ketoacyl-CoA synthase gene OsCUTl in rice.The main research results are shown below:1.ZFP185 was constitutively expressed in various rice tissues,while highly in leaf and young panicle.Expression of ZFP185 was slightly regulated by abiotic stresses.Histochemical GUS staining of ZFP185Pro:GUS transgenic plants showed strong GUS signals in multiple rice tissues.ZFP185 was localized in the cytoplasm and lacked transcriptional activation potential.Using transgenic technology,we obtained overexpressing and RNAi transgenic rice lines of ZFP185.The transgenic rice lines were confirmed by PCR,RT-PCR and southern blot.Overexpression of ZFP185 in rice resulted in a semi-dwarf phenotype,reduced cell size and the decrease of endogenous GA3 content and expression of gibberellins(GA)biosynthesis-related genes.By contrast,higher GA3 content was observed in RNAi plants.The application of exogenous GA3 could fully rescue the semi-dwarf phenotype of ZFP185 overexpressing plants,suggesting the negative role of ZFP185 in the regulation of GA biosynthesis.Besides GA,it was found that overexpression of ZFP185 decreased abscisic acid(ABA)content and expression of several ABA biosynthesis-related genes.Moreover it was found that ZFP185 increased sensitivity to drought,salt,and cold stresses,implying the negative regulation of ZFP185 in stress responses.Further studies indicated that ZFP185 interacts with an ubiquitin protein ZFP185IP1.Yeast in vivo and GST-pull down in vitro results showed that ZFP185 could interact with ZFP185IP1.In vitro ubiquitination assay exhibited that ZFP185 was an E3 ligase.Our study suggests that ZFP185 may act as an E3 ligase,regulates plant growth by affecting GA biosynthesis and negatively regulates stress responses by modulating ABA biosynthesis in rice.2.OsCUT1 was one of the β-ketoacyl-CoA synthase(KCS)genes involving in cuticular wax synthesis in rice.The BLAST analysis showed there were eight OsCUT1 homologous genes in rice.Expression of OsCUT1 and its homologous genes were down-regulated by dehydration,drought and salt.OsCUT1 transcripts were detected in various rice tissues and its expression was most abundant in leaf and young panicle,but not in root.The expression of OsCUT1 was down-regulated by heat,cold,and ABA treatments,while up-regulated by oxidative stress.Histochemical GUS staining of OsCUT1Pro:GUS transgenic plants showed strong GUS signals in multiple rice tissues except in root.OsCUT1 was localized in the endoplasmic reticulum.Overexpression of OsCUT1 increased the leaf cuticular wax content,decreased the non-stomatal water loss rate of leaves and chlorophyll leaching rate.OsCUT1 overexpressing plants showed an improved tolerance to drought.Under drought stress,OsCUT1 overexpressing plants could improve the photosynthetic rate and water use efficiency.