本文選題：甘菊　切入點(diǎn)：同源多倍體化　出處：《南京農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：菊花(Chrysanthemum morifoliumTzvel.)是我國十大傳統(tǒng)名花和世界四大切花之一,因其多姿多彩的形態(tài)和豐富的文化內(nèi)涵而深受人們的喜愛。栽培菊花是由菊屬內(nèi)多個(gè)野生種相互雜交形成的異源多倍體,遺傳背景比較復(fù)雜。甘菊(Chrysanthemum lavandulifoium)為菊屬二倍體野生種,遺傳背景簡單,是栽培菊花的供體親本之一,具有耐鹽、抗病蟲害等優(yōu)良特性。多倍體在物種起源及作物生產(chǎn)中起著重要作用,不僅可以提高產(chǎn)量、改善品質(zhì),還可以提高植物對生物和非生物脅迫的抵抗能力等,一直是研究的熱點(diǎn)。多倍體形成過程,其基因組結(jié)構(gòu)和基因的轉(zhuǎn)錄水平會發(fā)生一系列改變,目前相關(guān)的研究多集中在異源多倍體化過程,而有關(guān)同源多倍體的報(bào)道較少。本研究對甘菊同源多倍體化過程的基因組結(jié)構(gòu)及表型變化的機(jī)理進(jìn)行了研究,為菊屬植物進(jìn)化及多倍體的育種利用提供理論依據(jù)。主要內(nèi)容及結(jié)論如下:1.以甘菊試管苗為材料,通過秋水仙素誘導(dǎo)同源四倍體。與二倍體相比,四倍體甘菊株高、葉片、管狀花和舌狀花均表現(xiàn)出巨大化,葉片變大主要是由于細(xì)胞變大和細(xì)胞數(shù)量增加引起。甘菊同源四倍體化過程,基因組結(jié)構(gòu)和基因的轉(zhuǎn)錄均發(fā)生了一定程度的改變。DNA-SRAP ( DNA Sequence-Related Amplified Polymorphism )標(biāo)記共擴(kuò) 增出440條條帶,有1.6%是新條帶,1.1%的條帶發(fā)生了丟失;cDNA-AFLP(cDNA Amplified Fragment Length Polymorphism )分析發(fā)現(xiàn),四倍體中有 8%轉(zhuǎn)錄本(Detectable Transcript-derived Fragments, DTFs)表達(dá)上調(diào),5.9% DTFs 表達(dá)下調(diào);MSAP (Methylation Sensitive Amplification Polymorphism)分析二倍體和四倍體甘菊甲基化變化,共檢測到516個(gè)位點(diǎn),其中有53個(gè)位點(diǎn)在多倍體形成后發(fā)生了變異,以甲基化升高為主。2. NAC轉(zhuǎn)錄因子參與植物生長發(fā)育,利用RACE-PCR技術(shù)克隆得到了甘菊9個(gè)NAC家族成員,其中ClNAC84在二倍體甘菊葉片中的表達(dá)量顯著高于四倍體。分析表明,ClNAC84全長893 bp,開放閱讀框(ORF)687bp,共編碼229個(gè)氨基酸,定位在細(xì)胞核中。同源基因氨基酸序列比對顯示,ClNAC84包括A,B,C,D,E和F六個(gè)亞結(jié)構(gòu)域,其中D亞結(jié)構(gòu)域含有“FVFY”疏水氨基酸。系統(tǒng)進(jìn)化樹分析表明,ClNAC84轉(zhuǎn)錄因子與擬南芥NAC84親緣關(guān)系較近,屬于NAC家族第二類中的TERN亞類,可能與生長發(fā)育相關(guān)。熒光定量PCR分析表明,在二倍體甘菊葉片和花中,ClNAC84相對表達(dá)量較高,根和莖中相對表達(dá)量較低;在四倍體甘菊的花中ClNAC84相對表達(dá)量最高,葉片,根和莖次之,二倍體甘菊葉中ClNA84相對表達(dá)量高于四倍體。3.為了驗(yàn)證ClNAC84基因的功能,構(gòu)建pESPM-ClNAC84酵母表達(dá)載體,轉(zhuǎn)入裂殖酵母菌株SPQ中,超表達(dá)菌株較空載體菌株生長緩慢,且細(xì)胞表現(xiàn)出多核現(xiàn)象,推測ClNAC84基因可能通過減緩細(xì)胞周期而抑制酵母的生長;同時(shí)構(gòu)建了pMDC43-ClNAC84超表達(dá)載體,并建立甘菊遺傳轉(zhuǎn)化體系,通過根癌農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化二倍體甘菊,經(jīng)潮霉素篩選和PCR鑒定后,共獲得6個(gè)超表達(dá)株系,轉(zhuǎn)化率為0.6%。將超表達(dá)株系和二倍體野生型甘菊栽到花盆中,16周后觀察表型,超表達(dá)植株株高、主枝頂部向下的第三片葉面積、葉片長度、寬度、花序直徑、花心直徑、管狀花和舌狀花長度均小于野生型甘菊。利用高效液相色譜儀測定內(nèi)源激素含量,超表達(dá)株系A(chǔ)BA含量顯著上升,IAA含量下降,與超表達(dá)株系形態(tài)分析相吻合。通過石蠟切片法觀察葉片解剖結(jié)構(gòu),發(fā)現(xiàn)柵欄組織上表面細(xì)胞大小未發(fā)生變化,但細(xì)胞數(shù)量卻減少,說明葉片變小是由于細(xì)胞數(shù)量減少引起。4.通過酵母雙雜交系統(tǒng)篩選C1NAC84互作蛋白,共篩選出79個(gè)候選蛋白,其中參與細(xì)胞分裂的相關(guān)蛋白占36.8%,參與抗性脅迫和防御的蛋白占25.0%,參與光合作用的蛋白占11.8%,參與核酸合成的蛋白占6.6%,此外還包括19.7%的其他蛋白,MIP蛋白出現(xiàn)次數(shù)最多(13次)。經(jīng)過酵母雙雜交技術(shù)(H2Y)和雙分子熒光互補(bǔ)(BiFC)驗(yàn)證,C1NAC84與MIP互作,MIP定位在細(xì)胞核內(nèi),進(jìn)一步說明兩蛋白互作,共同調(diào)控甘菊細(xì)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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S682.11

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