【作者】 常青山；

【导师】 陈发棣；

【作者基本信息】 南京农业大学 ， 观赏园艺， 2011， 博士

【摘要】 菊花(Chrysanthemum×morifolium (Ramat.) Kitamura)原产我国,是我国十大传统名花与世界四大切花之一,具有很高的观赏与应用价值,在花卉生产中占有十分重要的地位。目前,由于菊花以观花为主,研究应用主要集中在花型花色上,叶色研究较少。本研究通过对菊花黄绿叶突变体的形态解剖结构进行对比研究,对类囊体蛋白含量与光谱、叶绿素荧光特性进行分析,及构建黄绿叶突变体叶片中的黄叶组织与绿叶组织抑制差减杂交文库,通过对差减杂交文库分析筛选与黄绿叶突变性状相关的叶色突变基因,并对筛选到的两个基因进行了克隆与表达分析,相关研究对揭示菊花叶色形成机理及遗传改良有积极意义。主要结果如下：1.以菊花黄绿叶突变体’NAU04-1-31’为试验材料,分别测定了菊花黄绿叶突变体叶片中黄叶与绿叶组织的叶绿素含量与类胡萝卜素含量,并比较了两种不同类型叶片组织的显微、超微解剖结构及类囊体膜蛋白的含量。叶绿素含量测定表明：黄叶组织中叶绿素a、叶绿素b和类胡萝卜素含量显著低于绿叶组织,而黄叶组织与绿叶组织叶绿素a/b的比值则没有显著的差异。叶绿体显微与超微结构观察发现：黄叶组织中细胞内叶绿体形状不规则,缺乏正常的叶绿体膜结构,无类裘体,无淀粉粒,嗜锇颗粒较多且积聚成簇状；而绿叶组织细胞内叶绿体较多,形状规则,基粒片层清晰,其内淀粉粒结构清晰且体积较大,嗜锇颗粒较少且分散,说明黄绿叶突变体中黄叶与绿叶组织的颜色差异主要是由于叶绿素含量显著减少所致。类囊体蛋白电泳结果表明：绿叶组织中最少可以分离出15条类囊体膜多肽,而黄叶组织中则仅可分离出4条类囊体多肽,特别是黄叶组织中的光系统Ⅱ的多肽与绿叶相比下降明显。类囊体膜的吸收光谱、荧光发射光谱及激发光谱明显下降,而且峰位发生红移与蓝移,说明突变体类囊体膜的结构可能受到了破坏,黄叶组织对光能的捕获效率下降,且较依赖于Chla捕光并将光能激发传递给PSⅡ反应中心。叶绿素荧光特性表明：菊花突变体黄叶组织与绿叶组织相比,其荧光参数F0较高,而Fv/Fm、Fv’/Fm’、ΦOPSⅡ等参数较低,说明黄叶组织的光合机构可能受到破坏,光化学能力下降；黄叶组织用于光化学反应的光能远小于绿叶组织,而用于热耗散的光能则大大增加。黄叶组织中与光抑制有关的NPQ成分qI增加,说明其更容易受到光抑制的破坏。黄叶组织光系统间的电子传递受阻,放氧复合体受到破坏,单位面积反应中心数目较少,光合性能指数(PIABS)低于绿叶组织。与绿叶组织相比,黄叶组织PS II光化学性能的下降,黄叶组织对光抑制比较敏感,通过提高热耗散能力,来减少过剩光能对黄叶组织光合机构的破坏。2.为了验证黄叶组织与绿叶组织基因表达的差异,将黄绿叶突变体中绿叶组织与黄叶组织通过抑制差减杂交技术构建了抑制差减杂交文库。通过斑点杂交确定为阳性克隆并测序获得339个ESTs序列。其中从黄叶组织中分离到157个ESTs,绿叶组织中分离到182个ESTs,根据测序文件去除低质量序列后,最终获得293个ESTs。利用DNAStar软件对293条ESTs分别进行聚类,共获得150个unigenes,包括93个singletons,57个contigs。所有的ESTs经BlastX比对和功能注释发现在所有的Unigene中,107个unigene与已知基因具有类似功能,占71.3%；27个unigene与已知基因未有显著的同源性或仅具有推定功能,占18%；16个unigene在数据库中未搜索到同源序列,可能为新基因,占10.6%。根据COG分类,可把所有的unigene划分为16类：第一大类为能量生成与代谢,占16%；翻译、核糖体结构与合成为第二大类,占14%;碳转运与代谢及次生物质合成、转运与代谢各占7.3‰细胞骨架、信号转导等其它各类所占比例较少。其中在菊花黄叶中上调表达的镁螯合酶(GUN5)大亚基和ATP依赖的金属蛋白酶(VAR1)有可能参与到与花叶有关的表型形成。3.通过RACE技术,获得了4451bp全长的菊花镁螯合酶大亚基(CmChlH) cDNA序列。序列分析表明：该序列包含4149bp的开放阅读框(ORF),编码一条1383个氨基酸的蛋白。推测的蛋白大小与理论等电点分别为154kDa和5.95。根据ORF序列,在5’端有一段51个氨基酸的叶绿体转运肽。菊花镁螯合酶大亚基与拟南芥、水稻、念珠藻、嗜中温螺旋杆茵分别有85%、82%、67%和43%的同源性,与同为双子叶的拟南芥同源性最高,并具有保守的三个组氨酸残基(H666,H670and H815,)。系统进化树分析表明：菊花与拟南芥、金鱼草及烟草的ChlH有比较近的亲缘关系,属于一类。这些结果表明：菊花ChlH编码镁螯合酶大亚基。通过相同方法获得了2272bp全长的菊花FtsH cDNA序列,命名为CmFtsH。序列分析表明：该序列包含2094bp的开放阅读框(ORF),编码一条698个氨基酸的蛋白质。推测的蛋白大小与理论等电点分别为74.67kDa和5.99,在其5’端有一段长度57个氨基酸的叶绿体转运肽。多序列比对表明：CmFtsH具有保守的两个ATP结合位点(Walker-A, Walker-B),以及推测AAA特征模块——第二同源区(SRH),其C端包括了一个推测的Zn2+结合结构域HEXGH(X代表非保守氨基酸)。菊花CmFtsH与拟南芥12成员中可以引起黄叶突变的最为重要的两个成员AtFtsHl和AtFtsH5亲缘关系最近,分别具有82%与84%的同源性。这些结果表明：菊花FtsH编码FtsH蛋白酶基因。4.组织特异性表达表明,CmChlH与CmFtsH在根、茎、叶和花中均有表达,其中在叶片中表达量最高,在茎中其次,在根与花中表达较低。光抑制实验表明,给予一个较高光强的强光后,其绿叶组织的Fv/Fm减少了0.11,而黄叶组织的Fv/Fm减少了0.45；在其后的微光恢复过程中,绿叶组织可以恢复到接近原来的初始水平,而黄叶组织则无法恢复到原来的水平,甚至降低到一个较低的荧光值。在不同的光强处理下,在绿叶组织中,CmChlH的转录在10μmol/m2/s到450μmol/m2/s时不断上升,在600μmol/m2/s光强下受到抑制,但在黄叶组织中,CmChlH的转录则持续不断上升,在600μmol/m2/s光强下则未受到抑制。当置于黑暗中时,与光下对照相比,CmChlH在两种类型叶片中的表达在早期均下调到一个较低的水平,然后保持在该水平,表明CmChlH受光诱导调节。在绿叶组织中,CmFtsH的mRNA水平从10μmol/m2/s到450μmol/m2/s时稳步上升,在450μmol/m2/s光强下达到顶峰,然后在600μmol/m2/s光强下受到光抑制。在黄叶组织中,CmFtsH的mRNA从10到450μmol/m2/s时也稳步上升,在600μmol/m2/s光强下达到顶点。但在450μmol/m2/s与600μmol/m2/s光强下黄叶组织的转录水平大约是绿叶组织相应光强下的1.4与3.1倍。在黑暗条件下,CmFtsH的转录水平随着时间延长而减少,黑暗处理5h时,两种类型叶片CmFtsH的mRNA水平降至光下对照的一半,在处理12h到48h过程则不断减少。更多还原

【Abstract】 Chrysanthemum（Chrysanthemum x morifolium （Ramat.） Kitamura）, has a long history in China, and is one of the ten most famous flowers in China, which is used as the potted flower, cut flower, and applied as the ground-cover plant. Chrysanthemum is valuable for its ornamental character, and is very important for the flower production. Nowdays the study on chrysanthemum is mostly focused on its colors and types of flowers, but little study on its leaf color. So the study on leaf color will help to increase its ornamental value. In this study, chloroplast microstructure and ultrastructure> SDS-PAGE and spectra anlysis of thylakoid membrane protein^chlorophyll fluorescence characteristics of green leaf tissue and yellow leaf tissue in yellow-green mutant of chrysanthemum were studied. Then suppression subtractive hybridization （SSH） technique was used to identify different genes between the green leaf and yellow leaf tissue of the mutant, the two genes related to the mutation of the leaf color were screened, the expression of the two genes and the physiological difference in the green leaf tissue and yellow leaf tissue were also studied, all these research is signifcant to reveal the mechanism of the formation of leaf color and genetic improvement. The main results are as follows:1. The contents of chlorophyll, carotenoid, anatomic structures of green leaf tissue and yellow leaf tissue of chrysanthemum yellow-green leaf mutant ’NAU04-1-31’ were studied by optical and electron microscopy. The results showed that chlorophyll and carotenoid contents in the yellow leaf tissue were lower than that in green leaf tissue. The ratio of chlorophyll a and chlorophyll b in yellow leaves was equivalent to that in green leaf tissue. In the yellow leaf tissue, there were some abnormal chloroplasts, in which vacuolated structures, a clustered plastoglobuli and no clear starch grains could be observed in the disrupted chloroplasts. In the green leaf tissue, chloroplasts in the green leaf tissue looked normal in their formation of thylakoids and granal stacks, many plump starch grains could be clearly observed, just few disperse plastoglobuli were contained in the green leaf tissue. The analysis of thylakoid membrane protein gel:at least15polypeptides could be isolated from the green leaf tissue, just4polypeptides could be isolated from the yellow leaf tissue. Especially, the polypeptides of PS II in yellow leaf tissue decreased greatly than that in green leaf tissue. The thylakoid membranes of the green and yellow leaf tissue were used to study on their room temperature absorption spectra, chlorophyll emission and excitation fluorescence spectra. Compared to the green leaf tissue, the absorption spectra and fluorescence spectra of the yellow leaf tissue decreased significantly, the peak position red-shifted and blue-shifted, the structure of thylakoid might be destroyed, the efficiency of light harvest decreased, Chla rather than Chlb in the yellow leaf tissue is responsible for harvesting light and being excited by light.Chlorophyll fluorescence characteristics showed:compared to green leaf tissue in the mutant, the yellow leaf tissue had higher Fo, but lower Fv/Fm、Fv’/Fm’、Φ PS Ⅱ、 and so on, which indicated the structure of photosystem might be destroyed; the yellow leaf tissue decreased greatly in photochemical reaction, but greatly increased in heat excitation. The ql component of NPQ in the yellow leaf tissue is higher than in green leaf tissue of the mutant, which indicated the yellow leaf tissue is sensitive to photoinhibition. Chlorophyll fluorescence kinetic parameters indicated PS II electron transport was apparently blocked and oxygen-evolving complex was destroyed, density of reaction centers decreased in yellow leaf tissue, which caused lower performance index on absorption. Compared to the green leaf tissue, the photochemical efficiency of the yellow leaf tissue decreased, the yellow leaf tissue was sensitive to photoinhibition, which increased the heat dissipation to prevent the damage from overmuch energy.2. In order to identify the expression differences between the green leaf tissue and the yellow leaf tissue, SSH was used to reveal the differentially expressed genes. A total of339（positive） cDNA clones （44.1%） were selected with the dot blot hybridization technology,157clones were isolated from yellow leaf tissue,182clones were isolated from green leaf tissue. After the removal of vector sequences and poor quality sequences,293ESTs of high quality sequences were consequently obtained. Of the293clones,93clones （62%） were singletons and the remaining200ESTs were clustered into57（38%） contigs with occurrence of the common gene ranging from2to23times, and a total of150unigenes were ultimately obtained. Classification of gene function for the150unigenes using a BlastX homology search revealed that107unigenes （71.3%） could be identified or assigned to the putative functions of known genes,27unigenes （18%） were classified to be no significant homology or unknown function, and16unigenes （10.6%） had no matches in public databases. All the unigenes were classified into16primary functional categories, according to the putative function of their homologous genes in the databases. The largest group of genes （16%） was assigned to energy production and conversion. Genes involved in translation, ribosomal structure and biogenesis formed the second （14%）, carbohydrate transport and metabolism and secondary metabolites biosynthesis, transport and catabolism together formed the third （7.3%） largest group, respectively. While other categories were composed of a small number of expressed sequence tags （ESTs）, especially the cytoskeleton category, signal transduction mechanisms category, and coenzyme transport and metabolism were exiguous. The genes of CmChlH and CmFtsH were screened for further study due to its possiblity involved in the formation of the mutation.3. A full-length CmChlH cDNA （chrysanthemum large subunit of Mg-chelatase, AB543917） contains4,451bp with an open reading frame of4,149bp encoding1,383amino acids. The predicted isoelectric point （pI） and molecular weight （MW） of ChlH is5.95and154kDa, respectively. Chrysanthemum CmChlH has a pre-sequence of51N-terminal amino acids and was predicated to be targeted to the chloroplast. The deduced amino acid sequence of CmChlH shared85%,82%,67%and43%of amino acid residues with protein from AtChlH, Arabidopsis thaliana （NP₁96867）; OsChlH, Oryza sativa （ABF95686）, NpChlH, Nostoc punctiforme （YP₀01866414）, HmChlH, Heliobacterium modesticaldum Icel （YP₀01679881）. It is highly homologous to AtChlH （GUN5）, the large subunit of Mg-chelatase. Three conserved histidine residues （H666, H670and H815） are also contained in chrysanthemum putative amino acids of ChlH. These results indicated that CmChlH cDNA represents the large subunit of Mg-chelatase in higher plants.A full-length CmFtsH cDNA （Chrysanthemum, ATP-metalloprotease, AB542716） contains2,272bp with an open reading frame of2,094bp encoding698amino acids. The predicted isoelectric point （pI） and molecular weight（MW） of CmFtsH are5.99and74.67kDa, respectively. CmFtsH has a pre-sequence of57N-terminal amino acids and was predicated to be targeted to the chloroplast. Multiple sequence alignments revealed that consensus regions exist among FtsH homologs from chrysanthemum, Arabidopsis thaliana, Nicotiana tabacum and E.coli. The sequence of CmFtsH Protein contains conserved motifs for a Walker-type ATPase and metalloprotease, such as motifs A and B（Ⅰ, Ⅱ）, the second region of homology （SRH, Ⅲ）, and a zinc-binding domain （Ⅳ）. The deduced amino acid sequence of CmFtsH displayed84%,82%,53%and50%similarity with AtFtsH5（Arabidopsis, NP₅68604）; AtFtsH1（Arabidopsis, NP₅64563）; NtFtsH （Nicotiana tabacum, AAD17230）; EcFtsH （E.coli, P28691）.A phylogenetic analysis shows CmFtsH is highly homologous to AtFtsHl, AtFtsH5in Arabidopsis thaliana. Moreover sequence comparison showed that deduced amino acid sequence of CmFtsH was most homologous to AtFtsH5（VAR1）. Taken together, these results suggested that CmFtsH cDNA identified herein encoded a FtsH protease.4. Tissue specific expression of the two genes indicated:the expression of CmChlH in leaves was the highest, and the next was in stems, only weak expression in roots and flowers. Similarly, we found the expression of CmFtsH was the highest in leaves compared to the green leaf tissue, the expression of CmFtsH in stems was just inferior to that in leaves. Only low abundance mRNA of CmFtsH could be detected in roots and flowers. Following strong light irradiation, Fv/Fm in leaf recovered under dim light was detected. Fv/Fm declined slightly by0.11in the green leaf tissue of the mutant. But the value of Fv/Fm decreased greatly by0.45in the yellow leaf tissue of the mutant, which indicated that the yellow leaf tissue were highly sensitive to photoinhibition in PSII. Recovery of the photoinhibition in the green leaf tissue of the mutant was faster than that in the yellow leaf tissue of the mutant. Fv/Fm in the green leaf tissue of the mutant completely recovered after an overnight adaptation under dim light, While Fv/Fm in the yellow leaf tissue of the mutant did not show a recovery to static levels. Furthermore, their Fv/Fm decreased quickly to a lower value from3to20h. These results clearly indicated that the yellow leaf tissue of the mutant is very sensitive to photoinhibition in PSII, and that the damage caused at the given light intensity is irreversible.The expression of CmChlH to various light intensities was investigated using the green leaf tissue and the yellow leaf tissue in the mutant. In green leaf tissue, the abundance of CmChlH transcript increased steadily from10to150μmol/m2/s light exposure, then increased quickly from150to350μmol/m2/s light exposure, and peaked at450μmol/m2/s light exposure, but suppressed at600μmol/m2/s light exposure. In yellow leaf tissue, the transcript of CmChlH increased steadily from10to450μmol/m2/s, moreover, the transcript of CmChlH kept on increasing from450to600μmol/m2/s. Though there was a higher expression level of CmChlH in the light （0hours）, when placed in the dark, the expression of CmChlH in two types of leaf tissue were both down regulated to a low level, then maintained this level without great change from5to48hours in the dark, whose transcript level were great lower than the control in the light. These results indicated that the expression of CmChlH was induced by light. In the light treatment, we exposed the green leaf tissue and yellow leaf tissue to various light intensities for12h. The results showed that, in green leaf tissue, CmFtsH mRNAs increased slowly from10to450μmol/m2/s, peaked at450μmol/m2/s, then suppressed at600μmol/m2/s. In yellow leaf tissue, CmFtsH mRNAs kept on increasing from10to450μmol/m2/s and peaked at600μmol/m2/s.Similar to the light dependent expression, we found that CmFtsH transcript levels were decreased with the time in the dark. The results indicated that CmFtsH mRNA levels decreased about half of the control after5h in dark in two leaf types, and decreased quickly from12to48hours. Only trace mRNA expression of CmFtsH could be detected after48h in the dark. These results validated that CmFtsH expression is regulated by light condition at the transcript level,too.更多还原