Functional Analysis Of Zinc Finger Protein ZFP157 And AWPM-19-like Protein OsPM19L1 From Rice(Oryza Sativa L.)

Rice is one of the important food crops in the world.There is nearly half of the population cosumes rice.The rice growth and production are largely affected by various environmental stresses.Therefore,the studies on the mechanisms for stress responses in rice are important to ensure the rice growth and development,the high and stable yield.In this research,we aimed to analyze the preliminary functions of an A20/AN1-type zinc finger protein ZFP157 and an AWPM-19-Like protein OsPM19L1 in rice under abiotic stresses.The main research results were described as follows:1.The qRT-PCR and GUS histochemical staining revealed that ZFP157 was constitutively expressed in all the tissues examined.ZFP15 7 was highly expressed in the node,stem,leaf,leaf sheath and primary panicle of rice,while the expression level was low in the root.ZFP157 was induced by 10 ?M MV stress,20%PEG6000,and high temperature.However,ZFP157 was down-regulated by 100 mM NaCl stress.ZFP157 was localized in the cytoplasm and lacked transcriptional activation potential.Using ZFP157 as a bait to screen salt induced cDNA library,two ZFP157 interacting proteins(ZFP157YH1 and ZFP157YH2)were identified.Through BLAST and protein domain analysis,the two proteins were characterized as RING-type domain containing Zinc finger protein and ubiquitin domain containing Rad23 protein.Expression analysis indicated that both genes were down-regulated by salt stress,which was similar with expression of ZFP15 7 under salt stress.Yeast in vitro result showed that ZFP157 could interact with these two proteins.The GST pull-down result showed that ZFP157 could interact with AIP1,the ubiquitin domain containing protein.In vitro ubiquitin assay exhibited that ZFP157 was an E3 ubiquitin ligase.In conclusion,ZFP157 may act as an E3 ligase,participating in the abiotic stresses through the ubiquitin-proteasome pathway.The vectors for 35S:ZFP1157 and CRISPR/Cas9 knock-out were constructed and the T0 generation of 35S:ZFP157 transgenic plants have been gernerated.2.Tissue-specific gene expression analysis revealed that OsPM19L1 was highly expressed in the leaf sheath of rice.Interestingly,expression of OsPM19L1 was highly at early stage of panicle development and decreased thereafter.The qRT-PCR assay indicated that OsPM19L1 was dramatically induced by 20%PEG stress,exogenous abscisic acid,salt and cold stress.Subcellular localization assay suggested that the OsPM19L1-GFP(green fluorescent protein)fusion protein was localized in the membrane system in rice cells.Moreover,it was found that OsPM19L1 expression was enhanced in an ABI5-Likel(ABL1)deficiency rice mutant abll under stress conditions,suggesting that ABL1 probably negatively regulates OsPM19L1 gene expression.Taken together,OsPM19L1 is probably closely associated with the stress tolerance through AB A-dependent pathway in rice.