Isolation And Function Identification Of A Novel CCCH-type Zinc Finger Protein Gene GhZFP1 In Cotton

High salinity and fungal pathogen attack are major stresses that are commonly encountered by plants during their developments. To adapt to the changes of environmental stresses, a series of physiological and biochemical reactions occur to eliminate or alleviate the physiological damage induced by improper surroundings. Upon exposure to the stresses, many genes are induced and their products either directly protect plants against stresses or further control the expression of other target genes. To understand the adaptive mechanisms of development of plants and their response to environmental stresses, it is imperative to know the function of crucial genes and their regulation during different stress response in plants。Zinc finger proteins are defined as a superfamily with typical zinc finger domain that involved in many aspects of plant growth and development and have been widely found in many animals and plants. They have been classified into nine types according to the structural and functional diversities: C2H2, C8, C6, C3HC4, C2HC, C2HC5, C4, C4HC3, CCCH (C, H represented Cysteine and Histidine, respectively). Until now, some zinc finger protein related to stress have been isolated and identified, but none of CCCH-type zinc finger proteins has been studied.In this experiment, a NaCl-induced cDNA library from cotton seedlings was constructed, and a new zinc finger protein gene GhZFP1 related to salt stress was identified by differential hybridization screening. Sequence and homology comparison revealed that GhZFP1 belonged to an unusual CCCH-type zinc finger proteins family. In this thesis, our studies are mainly focused on the sequence and expression analysis, function identification of the cotton gene GhZFP1. We used a yeast two-hybrid screen to identify proteins that interact with GhZFP1 expecting that such search might provide new insight into the function and molecular mechanism of GhZFP1 protein. The main results are as follows:1. A NaCl-induced cDNA library of cotton seedlings was constructed with Clontech SmartTM cDNA Library Construction Kit. We screened a salt-induced zinc finger protein gene GhZFP1 by differential hybridization from the cDNA library. The full length cDNA was further isolated by 3'-RACE and RT-PCR. The clone contains 1017 nucleotides and encodes 339 amino acid residues with the predicted molecular mass of 38.4 kDa.2. Sequence and homology comparison revealed that GhZFP1 encoded a putative CCCH-type zinc finger protein and belonged to a poorly studied CCCH-type zinc finger proteins family. Amino acid sequence analysis showed that GhZFP1 contained two typical zinc finger motifs (Cx8Cx5Cx3H, Cx5Cx4Cx3H). Moreover, a potential nuclear localization signal (NLS) and a putative leucine-rich nuclear export sequence (NES) were found in the N-terminal and C-terminal region of the protein, respectively。By the TMpred software, the deduced protein sequence contained one transmembrane (TM) segments at 190-213 amino acid residues, suggesting that it maybe act as an important regulator in responsing to stresses. GhZFP1 is the first CCCH-type zinc finger protein that had been identified and functionally characterized in cotton, and so a series of studies of it will accelerate the study of the whole CCCH-type zinc finger protein family.3. Northern blot results showed that the mRNA accumulation of GhZFP1 was induced by NaCl, PEG and SA treatments and kept increasing in a time-dependent course, but there was no significant GhZFP1 mRNA accumulation under 4?C, CuSO4, CaCl2, abscisic acid (ABA) and ethephon treatments, suggesting that GhZFP1 was not induced by specfic salt stress but induced by different biotic and abiotic stresses. Northern blot results also showed that GhZFP1 expression was up-regulated by NaCl stress in both leaves and stems, but not detected in roots. GhZFP1 mRNA was more abundant in ZMS19 than in ZMS17 under salt stress, indicating that GhZFP1 mRNA transcription levels were not only associated with tissue specificity but also associated with the salt tolerance of field-grown different cotton cultivars. Southern blot showed that it is a single-copy gene in cotton genome.4. The phylogenetic analysis of GhZFP1 indicated that several zinc finger proteins and GhZFP1 were most closely related to each other and significantly clustered together into a single branch in the tree, designated as SRZFP subfamily. Each member of this subfamily contained a conserved 38 amino acid sequence leading to the first putative zinc finger and a putative NES-like sequence at the C-terminal region, outside of these regions and zinc finger domains. Gene structural analysis revealed that each member of this subfamily was intronless gene. Expression profiles of SRZFP members in many microassay databases show that members of SRZFP subfamily might be involved in response to multiple stresses and play important roles in different signal transduction pathways.This is the first report showing that a CCCH-type zinc finger protein of SRZFP subfamily response to stresses in plants, indicating that SRZFP subfamily may be unique for higher plant species.5. GhZFP1-GFP fusion protein was constructed and transiently expressed in onion epidermal cells. The green fluorescence was detected in the nucleus of onion epidermal cells. These results are consistent with our observation that GhZFP1 encodes a nuclear-localized protein.6. GhZFP1 was introduced into tobacco plants. Northern blot analysis shows that GhZFP1 was expressed in plants of each transgenic line. The transgenic tobacco plants overexpressing GhZFP1 grew better than wild type tobacco in 200 mM,250 mM or 300 mM NaCl conditions, and then further verified by measuring changes in chlorophyll fluorescence (Fv/Fm) or endogenous Na+, K+ contents in shoots and roots , thereby validating the increased tolerance to salt stress. The enhanced resistance to Rhizoctonia solani in transgenic tobacco also demonstrated that disease resistance conferred by GhZFP1 was effective against fungal pathogens.7. A hydrophilic 5'fragment of GhZFP1 was selected to construct an E.coli expression vector pET30a-GhZFP1-185, and then it was induced by IPTG to express in E.coli strain BL21(DE3). The fusion proteins were purified and used to immune rabbits to obtain antiserum. The value of antibody reaches 1:1000.8. A yeast two-hybrid library was constructed with BD MatchmakerTM Library Construction & Screening Kits. By using the N-terminal 237 amino acids of GhZFP1 (m1) as bait, we screened and identified 9 positive clones that interacting with it. Northern blot analysis shows that expression of GZIRD21A was very rapidly induced by PEG and SA treatments, and GZIPR5 transcripts were induced by NaCl and SA treatments in cotton. These results are consisted with previous studies and resembled to the expression of GhZFP1, indicating that their interaction help to facilitates formation of higher order complexes and play important roles in transmitting different signal transduction pathway.9. Interaction regions analysis of GhZFP1 showed that it interacted with GZIRD21A and GZIPR5 by different amino acid sequences, To investigate the smallest binding sites of GZIRD21A and GZIPR5 in GhZFP1, a series deletions of GhZFP1 were need to be generated in the future study.