Construction Of Tilling Platform In Melon And Study On Interactions Between Catalase And Muti-walled Carbon Nanotubes

This dissertation is divided into tow parts, one is construction ofTILLING platform in melon; the other is study on interactions betweencatalase and muti-walled carbon nanotubes.Construction of TILLING platform in melon:With the rapid development of sequence technologies, the wholegenome sequence of many species including animals and plants have beenfinished. The focus on genomics has thus been shifted from the collection ofwhole sequenced genomes to the study of functional genomics. Traditionalreverse genetic approaches such as T-DNA, RANi and reverse RNAtechnologies have been used to deduce the gene function in the species ofinterest. However, these technologies rely on the creation of transgenicmaterial, which is not feasible for many species. Targeting Induced LocalLesions IN Genomes (TILLING) is a non-transgenic method, combined withchemical mutagenesis and high-throughout detecting approaches, has beensuccessfully applied on functional genomic in animals and plants.Melon (Cucumis melo L.) is an important vegetable crop. Genetic andgenomic information has been explored significantly due to several nationaland international projects, and the whole genome sequence initiated bySpanish group was already finished. However, the tools employed to conductthe functional validation of various genes in melon were limited. As a result, we constructed a TILLING platform in order to afford one powerful reversegenetic tool for melon. The research contents are as follows:1. The establishment of EMS population and the investigation ofphenotypic variations.The double haploid line, M62-113, belonging to the Piel de Sapocommercial type, was used as the starting cultivar.0.5%,1%and1.5%EMSdosage were selected as the pre-experiment dosage, according to M2seedviability and seedling vigor,1%EMS dosage was chosen as the finalconcentration. Totally,2,368M2families were sampled for DNA and usedfor TILLING purpose. All M2plants were scrutinized for visible variantphenotypes:5%of the M2families showed variation in cotyledons; about4.3%were segregated for "dwarf" or "semidwarf" plants;2.1%showedalbinism and chlorophyll deficiency and over2%of the families showedalterations in leaves and shoot morphology.2. The construction of TILLING system and identifying mutations intarget genes.In the TILLING platform, the M2samples were pooled four-fold to bescreened, a total of592four-fold pools were obtained. Four genes (Cm-NOR，Cm-ACO1，Cm-DET1and Cm-DHS) involved in fruit ripening were selectedto identify mutations, nine mutations were got in eight target amplicons. Nomutation was found in Cm-NOR; one, five and three mutations were found inCm-ACO1, Cm-DET1and Cm-DHS, respectively. The mutant families wereC142, I76, F201, F1876, C487F1248, F1728, C348and C519. The results ofsequence analysis indicated that mutations in C142、F201、F1876、F1728、C348and C519were missense, I76was silent, C487and F1248wereintronic. 3. The phenotype analysis in mutant families.Six missense mutant families were sown and sequenced to detecthomozygous plant, which were performed phenotype analysis. The resultsshowed that no visible changes in fruit ripening of the four genes (Cm-NOR、Cm-ACO1、Cm-DET1and Cm-DHS) were obtained. No heterozygous andhomozygous M2plants were detected in family C519, which indicated themutation in this family may not be true. As a result, there were only8truemutations in this platform. Besides the six mutant families, one mutantfamily in gene Cm-PDS (C384) and the other two in gene Cm-eIF4E (F57,F2036) were also performed phenotype analysis in this study. F57and F2036were resistance to Melon necrotic spot virus; the mutant plants were selfed toproduce more seeds for further analysis. Cm-PDS was involved in carotenoidsynthesis, the mutant line C384showed albino phenotype which wasexpected for PDS disruption. This mutant phenotype indicated TILLING wasa promising approach for advancement in reverse melon genetics.4. The mutation efficiency of Piel de Sapo populationIn Piel de Sapo population, a total of14point mutations were detectedwith7genes (12amplicions). The overall mutation density was calculated bydividing the total base pairs screened, that’s (9,134x2,368)/14=1,544,950,and the mutation density was calculated to be~1/1.5Mb. Compared to otherspecies, the mutation density was moderate.Piel de Sapo TILLING platform was the first one in Inodorus melons, asthe little experience in this species, it was normal that the results were not asgood as expected. However, the information presented here will be useful forcreating new melon populations with a higher mutation rate, which willfacilitate the future genomic and breeding studies in melon. Study on interactions between catalase and muti-walled carbonnanotubes:Carbon nanotubes (CNTs), one of the most excellent nanomaterials, mayspark on abundance of application in biological field because of theirstartling features such as straightforward synthesis and easily modification.However, the biocompatibility of CNTs has not been thoroughly investigated,as a result, illustrating the interaction between the biomacromolecule andCNTs would be of great value.With the wide use of the CNTs, their safety draws a lot of attentionsfrom the government, researchers and public. Now, the toxicology research ofCNTs shows that they will induce oxidative stress in cells. Thus we chosecatalase which is one of the most important oxidation resistance enzymes astarget protein, to study the interactions mechanism between catalase andmuti-walled carbon nanotubes by measns of transmission electronmicroscopy (TEM), fourier transform infrared (FTIR) spectroscopy，circulardichroism (CD) spectroscopy, ultraviolet-visible (UV-vis) absorptionspectroscopy, and fluorescence spectroscopy. The results of these approachesestablished new ways to evaluate the interaction between the CNTs andbiomacromolecules, as well as afford new angles for illustrating the oxidativestress caused by CNTs. The research contents are as follows:1. The image of TEM showed that catalase was adsorbed onto the CNTs,the maximum absorption amount was estimated to be40mg catalase/100mgCNTs, after which the absorption process reached equilibrium. Theenzymatic activity of catalase decrease dramatically after interacting withCNTs, with an inhibition rate of45%for the most, which may be one reasonfor inducing toxic effects in organisms. 2. The results of FTIR and CD showed that the secondary structure ofcatalase changed consequently after adsorbing onto CNTs: α-helix from26.7%to25.1%, β-sheet from20.7%to23.4%, β-turn from24.6%to22.2%,and random coil from28.7%to30.0%. Changes in these ratios showed thatthe backbone structure of catalase became loosen. The chain-structuremolecule became extended, leading to the exposure of Trp and Tyr whichwere originally in hydrophobic regions.3. The UV-VIS absorption manifested that the backbone chain of thecatalase became loosen when interacting with CNTs, and the amido bonds aswell as the Trp residues were exposed to a more hydrophilicmicroenvironment.4. Fluorescence spectra showed that CNTs induced catalase fluorescencequenching, leading to the amino acid residues (especially Trp) which wereoriginally buried in a hydrophobic environment to a hydrophilic one. Thisphenomenon could act as the evidence of protein unfolding. Time-resolvedfluorescence and the Stern-Volmer revealed that the quenching modefollowed a static one, that was, the two substances interacted and formed astable complex. The results of synchronous fluorescence spectrometryindicated that the interaction mode of catalase and CNTs was non-specificbinding.5. By using F rster resonance energy transfer (FRET) calculation, theaverage distance between Trp and carbon nanotubes was estimated to be2.98nm, showing the evidence of formation of stable complexes and highpossibility of energy transfer from Trp to MWCNTs.In this study, we have established an effective and innovativemethodology to evaluate the interaction of CNTs and catalase, which may comprehensively reflect the mechanism of protein-CNT interaction at amolecular level. By using catalase as the target protein, this dissertation mayprovide useful information to the research on CNT-induced oxidative stress incells and organism, which may serve as new research method in the field ofnanotoxicology.