甜瓜TILLING平台的构建及多壁碳纳米管与过氧化氢酶互作的研究
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摘要
本论文分为两部分，第一部分为甜瓜TILLING平台的构建，第二部分为多壁碳纳米管与过氧化氢酶互作的研究。
甜瓜TILLING平台的构建：
随着测序技术的发展，越来越多的动植物完成了全基因组的测序，科学家们的研究重点逐渐从基因组测序转移到基因功能的阐释上来。传统反向遗传学的研究手段，能够阐释基因的功能，但是它们依赖转基因技术，在转基因体系不成熟的物种中很难应用。定向诱导基因组局部突变（TILLING）技术，是一种不依赖转基因的全新反向遗传学研究手段，结合化学诱变剂和高通量的筛选技术，目前已经成功地应用于动植物基因功能的研究。
甜瓜是世界上一种重要的蔬菜作物，近年来，越来越多甜瓜基因组学的信息被挖掘了出来，尤其是甜瓜全基因组测序项目的完成，使它的研究重点逐渐进入了后基因组学的时代。但是，传统反向遗传学手段限制了甜瓜基因功能的研究，因而在本研究中，以冬甜瓜（Inodorus melon）Piel de Sapo为材料，构建了TILLING的研究平台，希望为甜瓜提供一个有力的反向遗传学工具，本研究的主要内容为以下几个方面：
1. EMS诱变群体的构建及表型变异的调查
本研究以Piel de Sapo DH系的M62-113为野生型亲本材料，选用0.5%，1%和1.5%的甲基磺酸乙酯（EMS）浓度做预备实验，根据M2代种子的发芽率和植株在田间的生命力，最终选用1%的EMS浓度作为大群体的诱变浓度，构建了包含2368个M2家系的EMS群体。经过性状调查发现，甜瓜的EMS群体中包含大量的突变体。约有5%的家系发生了子叶的改变；约有4.3%的家系为侏儒株或者半侏儒株；2.1%的M2家系出现了黄化或者白化现象；2%的M2家系在茎和叶的形态上发生了改变。
2. TILLING体系的建立及突变位点的筛选
在甜瓜TILLING平台的建立试验中，2368个M2家系形成了592个4倍池用于分析。4个与果实成熟相关的基因在群体中筛选得到9个突变位点，其中，Cm-NOR基因没有筛选到突变位点；Cm-ACO1、Cm-DET1和Cm-DHS分别筛选到基因筛选1、5、3个突变位点，产生突变的家系分别为C142；I76、F201、F1876、C487和F1248；F1728、C348和C519。序列分析表明，家系C142、F201、F1876、F1728、C348和C519发生错义突变，I76发生同义突变，C487和F1248的突变发生在非编码区。
3.突变家系的表型分析
对错义突变的6个M2家系做表型分析，结果表明Cm-NOR、Cm-ACO1、Cm-DET1和Cm-DHS的突变体没有发生目标性状的可辨改变。其中，家系C519测序分析没有检测到突变株，这一结果表明家系C519的突变可能为假阳性。因而，在果实成熟的基因中共检测到8个突变位点。除了与果实成熟相关的基因以外，在本研究中，还对基因Cm-PDS的突变家系C384和Cm-eIF4E的突变家系F57和F2036做了表型分析。Cm-PDS基因与类胡萝卜素合成相关，C384的纯合突变株出现白化现象，与拟南芥PDS基因突变体的表型一致，验证了Cm-PDS基因的功能。纯合突变株的表型变化与Cm-PDS基因功能相关，表明TILLING技术是甜瓜反向遗传学研究的有效工具。
4.甜瓜Piel de Sapo EMS突变群体的评价
在甜瓜Piel de Sapo的TILLING平台中，选用7个基因Cm-NOR、Cm-ACO1、Cm-DET1、 Cm-DHS、Cm-eIF4E、Cm-eIFI(iso)4E和Cm-PDS（共12个片段）在EMS诱变群体中筛选到14个突变位点，对甜瓜Pielde Sapo TILLING平台的突变频率进行计算，得出突变频率约为~1/1.5Mb。这个突变密度与目前已经建立TILLING平台的物种比较，处于中间水平。
本研究，首次尝试在冬甜瓜（Inodorus melon）甜瓜Piel de Sapo中构建在TILLING平台，由于经验不足，文献依据不多，最终得到突变群体的效率处于中等水平。但是，本平台的构建，能够用于甜瓜的后基因组学的研究，研究结果能够为构建高效的甜瓜TILLING平台提供依据。多壁碳纳米管与过氧化氢酶互作的研究：
碳纳米管是现代纳米材料中性能独特、应用前景广阔的新型材料。它易于制备，易于生物修饰的优点，使其在生物学领域中得到了广泛的应用。但是，关于碳纳米管的生物学效应缺乏深入系统的研究，碳纳米管与生物大分子的作用机理仍然不够清晰。
在碳纳米管大量应用的同时，它的安全性问题也引起了广泛的关注。目前，“氧化应激学说”是碳纳米管引起生物毒性，被普遍接受的一个原因。在本研究中，选用与氧化应激紧密相关的酶之一过氧化氢酶为靶蛋白，利用透射电镜、红外光谱、圆二色性光谱、紫外-可见吸收光谱及荧光光谱等技术或手段从不同角度研究了碳纳米管与过氧化氢酶的作用机理，一方面能够为碳纳米管与功能蛋白质在分子水平上作用的机理提供一定的数据参考；另一方面也能为阐释碳纳米管引起氧化应激现象，提供新的思路。本论文的主要研究内容为以下几个方面：
1.透射电镜的表征，发现过氧化氢酶有效地结合在碳纳米管的外壁上，吸附饱和值约为40mg过氧化氢酶/100mg碳纳米管；两者的吸附导致了酶活性的降低，抑制率最高达到45%，这有可能碳纳米管导致体内毒性的重要原因之一。
2.通过红外光谱、圆二色光谱的研究，发现与碳纳米管互作后，过氧化氢酶的二级结构发生了变化，α-螺旋比例从26.7%下降到25.1%，β-折叠由20.7%变为23.4%，β-转角由24.6%变为22.2%，这些比例变化表明过氧化氢酶的肽链骨架可能结构变得松散，分子链状结构展开，内部疏水区域的色氨酸、酪氨酸等芳香族氨基酸有可能暴露出来。
3.通过紫外-可见吸收光谱的研究，发现与碳纳米管互作后，过氧化氢酶的肽链骨架结构变得疏松，蛋白质主链酰胺键的微环境由原来的较为疏水变为相对亲水，色氨酸等芳香族氨基酸也因两者的结合而暴露出来。
4.通过荧光光谱的研究，发现与碳纳米管互作后，过氧化氢酶的荧光信号发生了猝灭，根据Stern-Volmer方程曲线和荧光寿命的检测断定这种猝灭为静态猝灭，说明碳纳米管与过氧化氢酶形成了稳定的复合物。同步荧光光谱的结果表明两者的结合为非特异性结合。
5.选用荧光共振能量转移的方法，计算出碳纳米管与过氧化氢酶的荧光基团（色氨酸）之间的平均距离为2.98nm，这个距离说明两者之间具备发生相互作用以及能量转移的条件。
本研究在分子水平上全面分析碳纳米管与过氧化氢酶的相互作用，建立了一种全新研究两者作用机理的方法；以过氧化氢酶为靶蛋白，能够为碳纳米管引起氧化应激反应机理的研究提供了新的思路，丰富了碳纳米材料生物毒理学的研究手段。
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 population
In 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.
甜瓜TILLING平台的构建：
随着测序技术的发展，越来越多的动植物完成了全基因组的测序，科学家们的研究重点逐渐从基因组测序转移到基因功能的阐释上来。传统反向遗传学的研究手段，能够阐释基因的功能，但是它们依赖转基因技术，在转基因体系不成熟的物种中很难应用。定向诱导基因组局部突变（TILLING）技术，是一种不依赖转基因的全新反向遗传学研究手段，结合化学诱变剂和高通量的筛选技术，目前已经成功地应用于动植物基因功能的研究。
甜瓜是世界上一种重要的蔬菜作物，近年来，越来越多甜瓜基因组学的信息被挖掘了出来，尤其是甜瓜全基因组测序项目的完成，使它的研究重点逐渐进入了后基因组学的时代。但是，传统反向遗传学手段限制了甜瓜基因功能的研究，因而在本研究中，以冬甜瓜（Inodorus melon）Piel de Sapo为材料，构建了TILLING的研究平台，希望为甜瓜提供一个有力的反向遗传学工具，本研究的主要内容为以下几个方面：
1. EMS诱变群体的构建及表型变异的调查
本研究以Piel de Sapo DH系的M62-113为野生型亲本材料，选用0.5%，1%和1.5%的甲基磺酸乙酯（EMS）浓度做预备实验，根据M2代种子的发芽率和植株在田间的生命力，最终选用1%的EMS浓度作为大群体的诱变浓度，构建了包含2368个M2家系的EMS群体。经过性状调查发现，甜瓜的EMS群体中包含大量的突变体。约有5%的家系发生了子叶的改变；约有4.3%的家系为侏儒株或者半侏儒株；2.1%的M2家系出现了黄化或者白化现象；2%的M2家系在茎和叶的形态上发生了改变。
2. TILLING体系的建立及突变位点的筛选
在甜瓜TILLING平台的建立试验中，2368个M2家系形成了592个4倍池用于分析。4个与果实成熟相关的基因在群体中筛选得到9个突变位点，其中，Cm-NOR基因没有筛选到突变位点；Cm-ACO1、Cm-DET1和Cm-DHS分别筛选到基因筛选1、5、3个突变位点，产生突变的家系分别为C142；I76、F201、F1876、C487和F1248；F1728、C348和C519。序列分析表明，家系C142、F201、F1876、F1728、C348和C519发生错义突变，I76发生同义突变，C487和F1248的突变发生在非编码区。
3.突变家系的表型分析
对错义突变的6个M2家系做表型分析，结果表明Cm-NOR、Cm-ACO1、Cm-DET1和Cm-DHS的突变体没有发生目标性状的可辨改变。其中，家系C519测序分析没有检测到突变株，这一结果表明家系C519的突变可能为假阳性。因而，在果实成熟的基因中共检测到8个突变位点。除了与果实成熟相关的基因以外，在本研究中，还对基因Cm-PDS的突变家系C384和Cm-eIF4E的突变家系F57和F2036做了表型分析。Cm-PDS基因与类胡萝卜素合成相关，C384的纯合突变株出现白化现象，与拟南芥PDS基因突变体的表型一致，验证了Cm-PDS基因的功能。纯合突变株的表型变化与Cm-PDS基因功能相关，表明TILLING技术是甜瓜反向遗传学研究的有效工具。
4.甜瓜Piel de Sapo EMS突变群体的评价
在甜瓜Piel de Sapo的TILLING平台中，选用7个基因Cm-NOR、Cm-ACO1、Cm-DET1、 Cm-DHS、Cm-eIF4E、Cm-eIFI(iso)4E和Cm-PDS（共12个片段）在EMS诱变群体中筛选到14个突变位点，对甜瓜Pielde Sapo TILLING平台的突变频率进行计算，得出突变频率约为~1/1.5Mb。这个突变密度与目前已经建立TILLING平台的物种比较，处于中间水平。
本研究，首次尝试在冬甜瓜（Inodorus melon）甜瓜Piel de Sapo中构建在TILLING平台，由于经验不足，文献依据不多，最终得到突变群体的效率处于中等水平。但是，本平台的构建，能够用于甜瓜的后基因组学的研究，研究结果能够为构建高效的甜瓜TILLING平台提供依据。多壁碳纳米管与过氧化氢酶互作的研究：
碳纳米管是现代纳米材料中性能独特、应用前景广阔的新型材料。它易于制备，易于生物修饰的优点，使其在生物学领域中得到了广泛的应用。但是，关于碳纳米管的生物学效应缺乏深入系统的研究，碳纳米管与生物大分子的作用机理仍然不够清晰。
在碳纳米管大量应用的同时，它的安全性问题也引起了广泛的关注。目前，“氧化应激学说”是碳纳米管引起生物毒性，被普遍接受的一个原因。在本研究中，选用与氧化应激紧密相关的酶之一过氧化氢酶为靶蛋白，利用透射电镜、红外光谱、圆二色性光谱、紫外-可见吸收光谱及荧光光谱等技术或手段从不同角度研究了碳纳米管与过氧化氢酶的作用机理，一方面能够为碳纳米管与功能蛋白质在分子水平上作用的机理提供一定的数据参考；另一方面也能为阐释碳纳米管引起氧化应激现象，提供新的思路。本论文的主要研究内容为以下几个方面：
1.透射电镜的表征，发现过氧化氢酶有效地结合在碳纳米管的外壁上，吸附饱和值约为40mg过氧化氢酶/100mg碳纳米管；两者的吸附导致了酶活性的降低，抑制率最高达到45%，这有可能碳纳米管导致体内毒性的重要原因之一。
2.通过红外光谱、圆二色光谱的研究，发现与碳纳米管互作后，过氧化氢酶的二级结构发生了变化，α-螺旋比例从26.7%下降到25.1%，β-折叠由20.7%变为23.4%，β-转角由24.6%变为22.2%，这些比例变化表明过氧化氢酶的肽链骨架可能结构变得松散，分子链状结构展开，内部疏水区域的色氨酸、酪氨酸等芳香族氨基酸有可能暴露出来。
3.通过紫外-可见吸收光谱的研究，发现与碳纳米管互作后，过氧化氢酶的肽链骨架结构变得疏松，蛋白质主链酰胺键的微环境由原来的较为疏水变为相对亲水，色氨酸等芳香族氨基酸也因两者的结合而暴露出来。
4.通过荧光光谱的研究，发现与碳纳米管互作后，过氧化氢酶的荧光信号发生了猝灭，根据Stern-Volmer方程曲线和荧光寿命的检测断定这种猝灭为静态猝灭，说明碳纳米管与过氧化氢酶形成了稳定的复合物。同步荧光光谱的结果表明两者的结合为非特异性结合。
5.选用荧光共振能量转移的方法，计算出碳纳米管与过氧化氢酶的荧光基团（色氨酸）之间的平均距离为2.98nm，这个距离说明两者之间具备发生相互作用以及能量转移的条件。
本研究在分子水平上全面分析碳纳米管与过氧化氢酶的相互作用，建立了一种全新研究两者作用机理的方法；以过氧化氢酶为靶蛋白，能够为碳纳米管引起氧化应激反应机理的研究提供了新的思路，丰富了碳纳米材料生物毒理学的研究手段。
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 population
In 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.
引文
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