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Isolation And Analysis Of Expressional Regulation Of Phosphate Transporter Genes In PHT1 Family From Three Solanaceous Species

Posted on:2010-10-15Degree:DoctorType:Dissertation
Country:ChinaCandidate:A Q ChenFull Text:PDF
GTID:1220330482471017Subject:Plant Nutrition
Abstract/Summary:PDF Full Text Request
Phosphorus (P) is an essential macronutrient for plant growth and development. It plays key roles in plant metabolic processes, including energy transfer, signal transduction, biosynthesis of macromolecules, photosynthesis and respiration. The primary source for P uptake by plants is inorganic phosphate (Pi) in soils. Due to inorganic fixation and formation of organic complexes, as well as the slow rate of diffusion in soil, P is one of the least available plant nutrients worldwide. Plants have evolved several strategies to improve P acquisition, including alterations in root morphology, modification of soil chemical properties around the roots, formation of symbiotic associations with arbuscular mycorrhizal (AM) fungi and activation of high-affinity Pi transporters.Specific transport systems are essential for the uptake of Pi and for its internal redistribution within plants. Transport of Pi through plant membranes is mediated by a number of families of transporters. The Phtl family including most of the plant phosphate transporters (Pht) identified so far, have been characterized as transporters exhibiting high affinity to Pi. The solanaceae is the third most economically important plant family, exceeded only by gramineae and leguminosae. Although five Phtl genes with orthologous pairs in the two species have been identified so far, the physiological functions and regulatory mechanism of these Phtl genes in the acquisition and translocation of Pi are still unclear. In this study, orthologous genes of five phosphate transporter (Pht) genes, which are members of the Phtl family in tomato and potato, have been cloned from the solanaceous species pepper, eggplant and tobacco. We also isolated the putative promoters of mycorrhiza-regulated Pht1;3, Pht1;4 and Pht1;5 genes from eggplant and tobacco. Detailed investigations, such as RT-PCR analysis, tissue-specific expression and promoter deletion analysis were then carried out in order to elucidate the regulation mechanism of mycorrhiza-inducible/enhancible phosphate transporters. In addition, a putative transcription factor that might interact with Myc-like motif was also isolated from tobacco by yeast one-hybrid system. The main results were as follows:1. Based on the four full-length cDNAs of Phtl;1, Phtl;2, Pht1;3 and Pht1;4 and the partial length of Pht1;5 in tomato and potato, we successfully isolated orthologues of the five Phtl genes from pepper, eggplant and tobacco and obtained all the full-length cDNAs of Phtl; 1-1;5 by the method of rapid amplification of cDNA ends (RACE)-PCR.2. Sequence multiple alignment and phylogenetic tree analysis further demonstrates that all of the new Phtl genes that we cloned from pepper, eggplant and tobacco are the orthologues of five members of the Phtl family in tomato and potato. These Phtl family genes can be roughly divided into three subfamilies, with clade I (Pht1;1 and Pht1;3), clade Ⅱ (Pht1;2), and clade III (Phtl;4 and Pht1;5). Pht1;5 was clearly detected by Southern Blot as a single copy gene in the pepper, eggplant and tobacco genome.3. Overall, expressions of these genes in pepper, eggplant and tobacco showed similar patterns:P-starvation enhancement in both leaves and roots for Phtl;1, P-depletion induction exclusively in roots for Pht1;2, mycorrhizal enhancement for Pht1;3, and mycorrhizal induction for both Pht1;4 and Pht1;5. In the roots of nonmycorrhizal eggplant, SmPht1;3, SmPht1;4 and SmPht1;5 were also expressed under extreme P starvation. Mycorrhizal symbiosis under low-P supply conditions increase the Pi concentration in plants, but with concurrent decrease of the Pht1;1 and Pht1;2 expression. Mycorrhizal symbiosis under high-P supply conditions reduced plant growth, with concurrent enhancement of Pht1;2 expression in the roots of pepper as well as eggplant.4. Based on the known coding region of the Phtl genes, we further isolated the putative promoter regions of Phtl;3, Pht1;4 and Pht1;5 from eggplant and tobacco using inverse PCR. Sequence analysis revealed that the transcription start sites in all the six promoters were very close to the coding region. The TATA-box elements were-25~-30 nucleotides upstream of the transcription start site, whereas there is no CAAT-box located in the-70--80 region in all the promoters.5. In transgenic tobaccos, histochemical analysis of GUS activity show that all the six promoter fragments of Pht1;3-1;5 isolated from eggplant and tobacco were sufficient to drive GUS to be expressed specifically in mycorrhizal roots, and confined in distinct cells containing arbuscule. There was no GUS activity which could be detected in other tissues of the colonized transgenic tobaccos.6. "PLACE" program analysis revealed that several putative cis-elements, such as PIBS (GNATATNC), an element associated with Pi starvation signaling in Arabidopsis and a root motif box (ATATT) are presented in all the six promoter regions. Some of the promoters contain the other two motifs, NODCON2GM(CTCTT) and OSE1ROOTNODULE (AAAGAT) which were the conserved elements that associated with mycorrhiza-and nodule-specific leghemoglobin gene regulation. In addition, we also found a new motif in all the six promoter regions, TTTCTTGTT which is not present in the known promoter regions of non-mycorrhiza-regulated plant phosphate transporters. Since the promoter regions of mycorrhiza-specific phosphate transporters from eudicot tomato, potato, and M. truncatula also contain the new motif, therefore, we name it as Myc-box.7. Both 5’and internal deletion analysis of the putative promoter regions of Phtl;3, Phtl;4 and Pht1;5 revealed that both P1BS and Myc-box elements were necessary to confer the mycorrhiza-specific expression of the genes, deletion one of the two elements could cause strong decrease or even complete abolishment of the GUS activity driven by the promoters.8. A cDNA library of tobacco roots inoculated with a mycorrhizal fungus (G intraradices) was constructed by following the protocol of clontech. A function-unknown protein factor, which might bind to the Myc-box motif, was isolated by yeast one-hybrid system. The corresponding full length cDNA of the isolated protein contains an open reading frame (ORF) of 765bp comprising 255 amino acid residues with the predicted molecular mass of 29 kDa. The deduced amino acid sequence shows 22.3% identity with a tobacco known harpin inducing protein (hin18), therefore, we name it as NtHRL (Harpin-Responsive-Like). Bioinformatic prediction indicates that NtHRL is nuclear localized protein. Since there is no similar model of the protein dimensional structure exist in PDB database, it still needs further data to confirm whether NtHRL is a true transcription factor that regulates the mycorrhiza-specific expression in solanaceous species.
Keywords/Search Tags:Solanaceae, phosphate transporter, gene clone, gene expression, regulation mechanism
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