甘菊同源多倍体化过程遗传变化与NAC84调控细胞分裂的分子机制研究

发布时间：2018-03-12 20:45

本文选题：甘菊　切入点：同源多倍体化　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

【摘要】：菊花(Chrysanthemum morifoliumTzvel.)是我国十大传统名花和世界四大切花之一,因其多姿多彩的形态和丰富的文化内涵而深受人们的喜爱。栽培菊花是由菊属内多个野生种相互杂交形成的异源多倍体,遗传背景比较复杂。甘菊(Chrysanthemum lavandulifoium)为菊属二倍体野生种,遗传背景简单,是栽培菊花的供体亲本之一,具有耐盐、抗病虫害等优良特性。多倍体在物种起源及作物生产中起着重要作用,不仅可以提高产量、改善品质,还可以提高植物对生物和非生物胁迫的抵抗能力等,一直是研究的热点。多倍体形成过程,其基因组结构和基因的转录水平会发生一系列改变,目前相关的研究多集中在异源多倍体化过程,而有关同源多倍体的报道较少。本研究对甘菊同源多倍体化过程的基因组结构及表型变化的机理进行了研究,为菊属植物进化及多倍体的育种利用提供理论依据。主要内容及结论如下:1.以甘菊试管苗为材料,通过秋水仙素诱导同源四倍体。与二倍体相比,四倍体甘菊株高、叶片、管状花和舌状花均表现出巨大化,叶片变大主要是由于细胞变大和细胞数量增加引起。甘菊同源四倍体化过程,基因组结构和基因的转录均发生了一定程度的改变。DNA-SRAP ( DNA Sequence-Related Amplified Polymorphism )标记共扩增出440条条带,有1.6%是新条带,1.1%的条带发生了丢失;cDNA-AFLP(cDNA Amplified Fragment Length Polymorphism )分析发现,四倍体中有 8%转录本(Detectable Transcript-derived Fragments, DTFs)表达上调,5.9% DTFs 表达下调;MSAP (Methylation Sensitive Amplification Polymorphism)分析二倍体和四倍体甘菊甲基化变化,共检测到516个位点,其中有53个位点在多倍体形成后发生了变异,以甲基化升高为主。2. NAC转录因子参与植物生长发育,利用RACE-PCR技术克隆得到了甘菊9个NAC家族成员,其中ClNAC84在二倍体甘菊叶片中的表达量显著高于四倍体。分析表明,ClNAC84全长893 bp,开放阅读框(ORF)687bp,共编码229个氨基酸,定位在细胞核中。同源基因氨基酸序列比对显示,ClNAC84包括A,B,C,D,E和F六个亚结构域,其中D亚结构域含有“FVFY”疏水氨基酸。系统进化树分析表明,ClNAC84转录因子与拟南芥NAC84亲缘关系较近,属于NAC家族第二类中的TERN亚类,可能与生长发育相关。荧光定量PCR分析表明,在二倍体甘菊叶片和花中,ClNAC84相对表达量较高,根和茎中相对表达量较低;在四倍体甘菊的花中ClNAC84相对表达量最高,叶片,根和茎次之,二倍体甘菊叶中ClNA84相对表达量高于四倍体。3.为了验证ClNAC84基因的功能,构建pESPM-ClNAC84酵母表达载体,转入裂殖酵母菌株SPQ中,超表达菌株较空载体菌株生长缓慢,且细胞表现出多核现象,推测ClNAC84基因可能通过减缓细胞周期而抑制酵母的生长;同时构建了pMDC43-ClNAC84超表达载体,并建立甘菊遗传转化体系,通过根癌农杆菌介导法转化二倍体甘菊,经潮霉素筛选和PCR鉴定后,共获得6个超表达株系,转化率为0.6%。将超表达株系和二倍体野生型甘菊栽到花盆中,16周后观察表型,超表达植株株高、主枝顶部向下的第三片叶面积、叶片长度、宽度、花序直径、花心直径、管状花和舌状花长度均小于野生型甘菊。利用高效液相色谱仪测定内源激素含量,超表达株系ABA含量显著上升,IAA含量下降,与超表达株系形态分析相吻合。通过石蜡切片法观察叶片解剖结构,发现栅栏组织上表面细胞大小未发生变化,但细胞数量却减少,说明叶片变小是由于细胞数量减少引起。4.通过酵母双杂交系统筛选C1NAC84互作蛋白,共筛选出79个候选蛋白,其中参与细胞分裂的相关蛋白占36.8%,参与抗性胁迫和防御的蛋白占25.0%,参与光合作用的蛋白占11.8%,参与核酸合成的蛋白占6.6%,此外还包括19.7%的其他蛋白,MIP蛋白出现次数最多(13次)。经过酵母双杂交技术(H2Y)和双分子荧光互补(BiFC)验证,C1NAC84与MIP互作,MIP定位在细胞核内,进一步说明两蛋白互作,共同调控甘菊细胞分裂周期。
[Abstract]:Chrysanthemum (Chrysanthemum morifoliumTzvel.) is one of the four cut flowers ten traditional famous flowers in China and the world, because of its colorful form and rich cultural connotations and loved by the people. The chrysanthemum chrysanthemum is cultivated in many wild species of interbreeding formed allopolyploids, genetic background is complex. Chamomile (Chrysanthemum lavandulifoium) for the genus chrysanthemum and wild diploid species, simple genetic background, is one of the donor parent has cultivated chrysanthemum, salt tolerance, pest resistance and other excellent properties. Polyploidy plays an important role in the origin of species and crop production, not only can improve the yield, improve quality, but also can improve the plant to biotic and abiotic stress resistance etc. that has been the focus of research. The polyploid formation process, the transcription level of the genome structure and gene will develop a series of changes, the current related research focus on Polyploidization process, but there are few reports about autopolyploid. Based on the mechanism of genome structure and phenotypic changes of chamomile autopolyploid process were studied for the genus evolution and breeding of polyploid plants and provide a theoretical basis. The main contents and conclusions are as follows: 1. to chamomile tube seedlings as material, by colchicine induction of Autotetraploid. Compared with diploid, Tetraploid Plant height, leaf, flower and flower showed a huge, larger leaves is mainly due to an increase in the number of cells and the cells became larger. By chamomile autotetraploid process, genome structure and gene transcription are change to a certain extent (.DNA-SRAP DNA Sequence-Related Amplified Polymorphism) markers were amplified 440 bands, 1.6% new bands, 1.1% bands were lost; cDNA-AFLP (cDNA Amplified F Ragment Length Polymorphism) analysis found that there are 8% tetraploid transcripts (Detectable Transcript-derived Fragments, DTFs) 5.9% up-regulated and down regulated DTFs expression; MSAP (Methylation Sensitive Amplification Polymorphism) analysis of diploid and tetraploid chamomile methylation changes, 516 loci were detected, of which 53 loci mutated in polyploid formation with increased.2. methylation, NAC transcription factors involved in plant growth and development, using RACE-PCR technology to clone 9 NAC family members of chamomile is obtained, the expression of ClNAC84 in diploid chamomile in leaves was significantly higher than that of tetraploid. Analysis showed that ClNAC84 was 893 BP, the open reading frame (ORF) of 687bp, encoding 229 amino acids. Located in the cell nucleus. Show the amino acid sequence of ClNAC84 homologous genes, including A, B, C, D, E and F six sub domains, including D sub structure The "FVFY" domain containing hydrophobic amino acids. Phylogenetic analysis showed that ClNAC84 transcription factor is near genetic relationship with Arabidopsis thaliana NAC84, belonging to the TERN subgroup of NAC family in second, may be related to growth and development. The fluorescence quantitative PCR analysis showed that in diploid chamomile leaves and flowers, relatively high expression of ClNAC84, and root the relatively low expression level in the stem; the relative expression was the highest in leaves of tetraploid Chamomile flowers ClNAC84, roots and stems, the amount is higher than that of tetraploid.3. in order to verify the function of the ClNAC84 gene relative expression of ClNA84 in diploid camomile leaves, construct pESPM-ClNAC84 yeast expression vector into the fission yeast strain SPQ, overexpression strains than no-load the strains grow slowly, and the cells exhibited multinucleate phenomenon, suggesting that ClNAC84 gene may inhibit the yeast cell cycle by slowing the growth of; while building the pMDC43-ClNAC84 over expression carrier Body, chamomile and the establishment of genetic transformation system, transformed diploid chamomile by Agrobacterium mediated method. After hygromycin screening and PCR identification, we obtained 6 overexpression lines, the conversion rate of 0.6%. will be super lines and diploid wild type chamomile planted to flower pot expression after 16 weeks of observation over expression of phenotype, plant height, shoot down top third leaf area, leaf length, width, diameter of the inflorescence, flower diameter, flower and flower length were less than the wild type chamomile. By HPLC determination of the content of endogenous hormones, over expression of strain ABA was increased significantly, IAA decreased, consistent with line shape analysis of over expression. By paraffin section method was used to observe the anatomical structure of leaf, found on the surface of palisade cell size did not change, but the number of cells was reduced, indicating smaller leaves is caused due to the reduction of.4. by yeast cells Two hybrid screening of C1NAC84 interacting proteins, we screened 79 candidate proteins, including proteins involved in cell division accounted for 36.8%, in resistance to stress and defense proteins accounted for 25%, accounting for 11.8% of the proteins involved in photosynthesis, protein involved in nucleic acid synthesis accounted for 6.6%, in addition to the other 19.7% protein, MIP protein number most (13). After the yeast two hybrid technique (H2Y) and bimolecular fluorescence complementation (BiFC) test, C1NAC84 and MIP interaction, MIP localized to the nucleus, further illustrate two protein interaction, CO regulation of Gan Juxi cell division cycle.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S682.11

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