| Zinc finger proteins(ZFPs) are an important class of transcriptional factors. Zinc finger domain is the important feature of zinc finger protein, which is consist of the different number of cysteine residues (C) and histidine residues (H) combined with zinc ions. ZFPs could combine the cis-acting elements of promoter region of some genes through zinc finger domain and play a role in transcriptional controls. Studies have shown that zinc finger proteins widely involved in plant response to abiotic stresses. At present, a variety of zinc finger protein genes which involved in abiotic stress response have been discovered in rice, most of them belong to C2H2type. Study of C2HC type zinc finger proteins was not yet to be reported in rice, exist in rice. OsLOL2is one of the important members which named LSD1-Like type zinc finger proteins in rice. Studies have shown that OsLOL2played an important role in rice growth and disease resistance. It is not clear that whether OsLOL2involves in oxidative stress and other abiotic stresses or not. In this study, we used RT-PCR technique to clone a known gene (GenBank accession number:AK103785、AJ620677) from cDNA of Nipponbare (Oryza sativa L. spp. japonica). The full length of the former and the latter ORF was1020bp and492bp, which encoded339and163amino acid residuals. By BLAST, we determined that AK103785contained a C2HC zinc finger domain, named OsC2HC-1. AJ620677contained two LSD1-Like zinc finger domain, which had been named OsLOL2. Zinc finger domain of OsLOL2is very conservative. It showed the highest homology to AtLOL2gene of Arabidopsis through the phylogenetic tree. Southern blot analysis indicate that OsLOL2have two copies in rice. Real-time quantitative PCR exhibited that the transcriptional level of OsC2HC-1and OsLOL2increased significantly in rice roots and leaves induced by the stress of80mM NaCl and30mM NaHCO3. The expression of OsLOL2gene in rice leaves has improved significantly under10mM H2O2treatment. OsC2HC-1-GFP and OsLOL2-GFP were transferred to yeast and onion epidermal cells. The yeast cellular localization preliminary showed that OsC2HC-1expressed in the nucleus. Subcellular localization of onion epidermal further indicated that zinc finger proteins OsC2HC-1and OsLOL2were located in the nucleus. Resistance experiment of yeast showed that the growing of OsLOL2transgenic yeast was better than control under NaCl and H2O2stress. Prokaryotic expression conditions of pGEX-6p-3-OsC2HC-1and pQE30-OsLOL2in host strain BL21and M15was optimized by a small amount of induction experiments. The results seemed that the optimal inducing time was4h and5h. GST-OsC2HC-1fusion protein expressed in Escherichia coli should be insoluble and stable form, which was called inclusion bodies. The purified fusion protein was about63kDa, which would be a prerequisite for making antibodies. The analysis of resistant experiments after germination presented that OsC2HC-1-GFP transgenic Arabidopsis significantly enhanced abiotic stress tolerances, including125mM NaCl and1,3mM NaHC03, indicating that OsC2HC-1should be strong responsive to the salt-alkali stress; The germination rate and resistance experiment on/after germination showed that the germination rate of overexpression OsLOL2transgenic Arabidopsis was higher than wild type. The growing of transginc Arabidopsis was better than WT on1/2MS plate which contained100mM NaCl,1.5mM H2O2and3mM NaHCO3. The above results indicated that OsLOL2gene was related to salt-alkalistress as well as oxidative stress. OsLOL2gene was transferred into tobacco(K.326) and rice(Longjing11) by Agrobacterium-mediated method. PCR detection presented that OsLOL2gene had been successfully integrated into the tobacco and rice genome, which prepared materials for further studying the function of OsLOL2gene and laid the foundation for the creation of tobacco and rice with stress resistance. |
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