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Functional Analysis Of Two Ubiquitination-Related Zinc Finger Proteins And Zinc Transporter Ozt1from Rice (Oryza Sativa L.)

Posted on:2013-04-20Degree:DoctorType:Dissertation
Country:ChinaCandidate:H X LanFull Text:PDF
GTID:1220330398991325Subject:Application of plant genomics
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Abiotic stresses, such as drought, salt, low temperature, high temperature and heavy metals, are major factors that affect plant growth and crop yield. A20/AN1zinc finger proteins and metal transporters play important roles in plant abiotic stresses responses. In this research, we analyzed the functions of A20/AN1zinc finger proteins ZFP177, ZFP181and zinc transporter protein OZT1in rice abiotic stress. The research results are described as follows:1. ZFP177was constitutively expressed in root, stem, leaf, leaf sheath, panicle of adult rice plant, and markedly induced by high temperature (42℃), low temperature (4℃) and oxidative stress (H2O2). ZFP177was localized in whole cell, including cytoplasm, plasma membrane and nucleus. Using transgenic technology, we obtained over-expression transgenic rice lines of ZFP177. The positive transgenic rice lines were confirmed by PCR, RT-PCR, Southern blot and Western blot. Over-expression lines of ZFP177showed more sensitive to salt and dought stresses. ZFP177protein fused with GAL4BD domain exhibited no transcriptional activity in yeast cell. Using ZFP177as a bait to screen cold induced cDNA and panicle cDNA library of rice, five different ZFP177interacting proteins (AIP1-5) were identified. Through BLAST and protein domain analysis, five ZFP177-interacting proteins were characterized as AIP1(polyubiquitin), AIP2(ubiquitin monomer), AIP3(Ubiquitin extended protein), AIP4(no apical meristem protein) and AIP5(ubiquitin family protein RAD23). Yeast in vivo and pull down in vitro results showed that ZFP177could interact with those proteins through its A20domain. In vitro ubiqutination assay exhibited that ZFP177was an E3ubiquitin ligase, which could be self-ubiqutinated with1-2ubiquitin monomers. The iTRAQ proteomics analysis of ZFP177over-expressing transgenic plants showed that26different proteins were up-regulated and151different proteins down-regulated compared to WT. The proteins involved in ubiquitin-proteasome pathway, indole derivative and amino acid derivative biosynthetic processes were decreased. In brief, ZFP177may act as an E3ubiquitin ligase, participate in the abiotic stresses througth ubiquitin-proteasome pathway.2. ZFP181was constitutively expressed in root, stem, leaf, leaf sheath, panicle of adult rice plants. ZFP181was induced by low temperature (4℃), osmotic stress (20%PEG), salt stress (100mM NaCl) and oxidative stress (H2O2), but suppressed by high temperature (42℃) and have no notable change under ABA (50μM) treatment. ZFP181was mainly localized in cell nucleus. Using transgenic technology, we obtained over-expression, knock down and promoter transgenic rice lines of ZFP181. The transgenic rice lines were checked by PCR, RT-PCR and Southern blot. Histochemical GUS staining of ZFP181promoter transgenic plants showed that ZFP181was constitutively expressed in multiple rice tissues. ZFP181over-expressing plants showed an improved tolerance to drought, salt stress and less sensitive to exogenous ABA. ZFP181over-expressing plants showed dwarf phenotype, with decreased expression of GA biosynthesis pathway related genes and low GA3content. The dwarf phenotype could be rescued partialy by application of exogenous GA3. ZFP181protein fused with GAL4BD domain exhibited no transcriptional activity in yeast. Using ZFP181as a bait to screen cold induced cDNA and panicle cDNA libraries of rice, one interacting protein (EEPD1) was identified. The BLAST and protein domain analysis showed that the protein was an endonuclease/exonuclease/phosphatase family domain containing protein, with conserved Exo_endo_phos domain. In vitro ubiqutination assay exhibited that ZFP181was an E3ubiquitin ligase, which could be self-ubiquitinated, covalently bind one ubiquitin monomer. In brief, ZFP181may act as an E3ubiquitin ligase, participate in the abiotic stresses and growth througth ubiquitin-proteasome and GA signal pathways.3. Subcellular localization analysis in yeast indicated that OZT1was localized to vacuole. Heterologous expression of OZT1in yeast increased tolerance to Zn2+and Cd2+stress but not to Mn2+stress. Together, OZT1is a CDF family vacuolar zinc transporter conferring tolerance to Zn2+and Cd2+stress probably through sequestration those metal into vacuolar.
Keywords/Search Tags:A20/AN1, zinc finger protein, E3ubiquitin ligase, Zinc transporter, abiotic stress, ubiquitin-proteasome pathway
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