本文選題：茄子　切入點(diǎn)：花柱異型　出處：《華南農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：花柱異型是一種特有的雌雄隔離和多態(tài)性特征,在被子植物中十分常見,包括茄子(Solanum melongena L.)。通常,茄子長(zhǎng)柱花(L-morph)類型坐果能力強(qiáng)、生境適宜性廣,而短柱花(S-morph)類型易落花落果。雖然關(guān)于茄子長(zhǎng)柱花和短柱花坐果能力差異,以及對(duì)光、溫等環(huán)境適應(yīng)性的報(bào)道較多,但茄子花柱異型發(fā)育調(diào)控的分子機(jī)制和品種改良尚未見報(bào)道。我國(guó)華南地區(qū)主栽的紫長(zhǎng)茄屬于兼生長(zhǎng)短柱花類型,坐果數(shù)量少,不宜設(shè)施栽培,生產(chǎn)上亟需對(duì)紫長(zhǎng)茄進(jìn)行改良。本文對(duì)完全長(zhǎng)柱花品系和兼生長(zhǎng)短柱花品系雌蕊發(fā)育、坐果能力、遺傳規(guī)律及分子調(diào)控機(jī)制進(jìn)行研究,主要結(jié)果如下:1.將茄子從現(xiàn)蕾到開花時(shí)期可劃6個(gè)時(shí)期:現(xiàn)蕾期、幼蕾膨大期、露冠期、花冠膨大期、松蕾期和開花期。從花蕾長(zhǎng)度來(lái)判斷,在0~10mm短柱花雌蕊發(fā)育與長(zhǎng)柱花保持一致,到花冠膨大期(約10mm)后短柱花雌蕊基本停止發(fā)育�；òl(fā)育期間,L-morph花花柱長(zhǎng)度與花蕾長(zhǎng)度呈線性關(guān)系�；ㄖL(zhǎng)度與花蕾長(zhǎng)度、子房高度相關(guān)性達(dá)極顯著水平,與花梗粗度達(dá)到顯著水平,而與子房直徑以及花藥長(zhǎng)度相關(guān)性則不顯著。通過(guò)熒光顯微觀察,L-morph花的柱頭和花粉均具有可授性,而S-morph花僅花粉具有活力。光學(xué)顯微觀察完全長(zhǎng)柱花和兼生長(zhǎng)短柱花胚囊中的卵細(xì)胞、中央細(xì)胞、助細(xì)胞和反足細(xì)胞結(jié)構(gòu)完整。2.短柱花自交坐果率僅為5%左右,而長(zhǎng)柱花自交坐果率均在94%以上。雜交試驗(yàn)表明對(duì)S-morph花柱頭進(jìn)行授粉坐果率極顯著低于L-morph花的坐果率。3.完全長(zhǎng)柱花類型與兼生長(zhǎng)短柱花進(jìn)行雜交,F1代植株全部為完全長(zhǎng)柱花類型,B3-3×56和01×119兩組材料的F2代完全長(zhǎng)柱花類型和兼生長(zhǎng)短柱花類型的比值分別為12.14:1和11.85:11,在兩個(gè)組合BC1群體中完全長(zhǎng)柱花類型和兼生長(zhǎng)短柱花類型的分離比例分別為3.64:1和2.56:1。經(jīng)χ2檢測(cè),完全長(zhǎng)柱花類型相對(duì)兼生長(zhǎng)短柱花類型為顯性遺傳,為兩對(duì)基因共同作用產(chǎn)生基因重疊效果;Shapiro-Wilk檢驗(yàn)F2完全長(zhǎng)柱花柱頭長(zhǎng)度也表明花柱長(zhǎng)度由多基因控制的數(shù)量遺傳,存在主效基因作用。4.基于高通量miRNA測(cè)序、微陣列及降解組測(cè)序,我們鑒定了兼生長(zhǎng)短柱花茄子雌蕊發(fā)育過(guò)程中miRNA的表達(dá)及其靶基因。miRNA測(cè)序鑒定了55個(gè)已知的茄子miRNA和35個(gè)與miRBase數(shù)據(jù)庫(kù)中已知miRNA相同序列的miRNA,594個(gè)新的miRNA,而大部分miRNA與其他植物相比有著高度的保守性。在微陣列芯片上的686個(gè)miRNA中,鑒定到10個(gè)差異表達(dá)的miRNA,其中,僅2個(gè)miRNA在L-morph雌蕊發(fā)育中顯著下調(diào)。同時(shí),我們利用qRT-PCR對(duì)差異表達(dá)的miRNA進(jìn)行驗(yàn)證。此外,我們也預(yù)測(cè)并注釋了差異表達(dá)miRNA的靶基因。5.通過(guò)微陣列對(duì)紫長(zhǎng)茄S-morph花和千指茄L-morph花雌蕊miRNA的表達(dá)譜進(jìn)行了鑒定。在微陣列芯片上的686個(gè)miRNA中,我們發(fā)現(xiàn)35個(gè)表達(dá)差異的miRNA,在L-morph花雌蕊中下調(diào)的9個(gè)miRNA,PC-5p-1408812_1和PC-5p-3215765_1下調(diào)最顯著;而上調(diào)的26個(gè)miRNA中,PC-3p-1358_405 and PC-5p-132923_7表達(dá)差異最顯著。利用了qRT-PCR對(duì)通過(guò)微陣列檢測(cè)獲得的表達(dá)特征進(jìn)行了驗(yàn)證,結(jié)果基本與芯片數(shù)據(jù)相一致。此外,我們還進(jìn)行了差異表達(dá)miRNA的靶基因預(yù)測(cè),并對(duì)推測(cè)的靶基因進(jìn)行了GO功能分析。在差異表達(dá)的miRNA的靶基因中,基因編碼轉(zhuǎn)錄因子bHLH、MYB以及AP2等,重要的酶或功能蛋白例如三磷酸水解酶超家族蛋白、蛋白激酶、磷酸酶以及ATP酶等,這些可能對(duì)于花柱異型發(fā)育起重要調(diào)控作用。6.利用iTRAQ技術(shù)對(duì)兼生長(zhǎng)短柱花茄子雌蕊發(fā)育過(guò)程中蛋白質(zhì)組表達(dá)分析,共鑒定了300個(gè)差異表達(dá)的蛋白,其中在花發(fā)育期,S-morph花與L-morph花雌蕊相比有26個(gè)蛋白質(zhì)表達(dá)量下調(diào),23個(gè)蛋白質(zhì)表達(dá)量上調(diào)。在表達(dá)下調(diào)的蛋白中,甲硫氨酸硫氧化物還原酶A3和DNA損傷修復(fù)/耐受性蛋白表達(dá)差異最大。在表達(dá)上調(diào)的蛋白中,甲硫氨酸硫氧化物還原酶和肌氨酸氧化酶的表達(dá)差異最大。這一時(shí)期差異表達(dá)蛋白主要參與能量?jī)?chǔ)備以及對(duì)外界應(yīng)激反應(yīng)、抵御病原體感染等方面。在花成熟期,97個(gè)蛋白表達(dá)上調(diào)和157個(gè)蛋白表達(dá)下調(diào)。在表達(dá)下調(diào)的蛋白中,富亮氨酸重復(fù)蛋白和性器官表達(dá)蛋白的表達(dá)差異最大。在表達(dá)上調(diào)的蛋白中,LAT52和ntp201的表達(dá)差異最大。這一時(shí)期差異表達(dá)蛋白主要參與生物合成過(guò)程、生物分子轉(zhuǎn)運(yùn)和代謝過(guò)程。此外,花成熟期S-morph花雌蕊中乙醇NADP+氧化還原酶、乙醇脫氫酶、半胱氨酸蛋白酶和過(guò)氧化物酶等表達(dá)上調(diào),這些酶參與細(xì)胞死亡過(guò)程,表達(dá)的上調(diào)可能與S-morph花不能坐果有關(guān)。而qRT-PCR結(jié)果并未完全與iTRAQ鑒定的結(jié)果一致,這可能是花形態(tài)特異性轉(zhuǎn)錄后調(diào)控的原因所致。本研究通過(guò)對(duì)茄子花柱異型細(xì)胞形態(tài)學(xué)特征和miRNA、蛋白差異表達(dá)進(jìn)行了研究,這些結(jié)果將會(huì)為揭開被子植物花柱異型發(fā)育模式及其分子機(jī)制的研究提供有用信息,同時(shí)也將對(duì)基礎(chǔ)理論研究以及農(nóng)業(yè)改良、育種等方面提供十分重要的參考價(jià)值。
[Abstract]:Heterostyly is a unique female isolation and polymorphism characteristic, is very common in angiosperms, including eggplant (Solanum melongena L.). Usually, the eggplant flower long column (L-morph) type of fruit setting ability, habitat suitability is wide, and short column flowers (S-morph) type to drop flowers FruitDrop. While on the difference of Eggplant long column and short column flower flowering and fruit setting ability, as well as the light, temperature and other environmental adaptability are reported, but the molecular mechanism and improved varieties of eggplant heterostyly developmental regulation has not been reported in China. The Southern China area planted purple eggplant and belongs to the growth of short column type flowers, fruit number, not greenhouse on the need for improved production of eggplant. The entire cylindrical flowers and growth lines and short column lines flower pistil development, fruit setting ability, to study inheritance and molecular regulation mechanism, the main results are as follows: 1. the eggplant from budding to flowering The period can be divided into 6 periods: bud, bud growth stage, exposed crown, corolla enlargement period, pine flower budding and flowering. Judging from bud length, 0~10mm in short and long column column flower pistil flower consistent to the corolla enlargement period (about 10mm) after a short column flower stop the basic development. During the flower development, flower bud length and the length of the L-morph column. There is a linear relationship between the style length and bud length, ovary height correlation reached extremely significant level, and pedicel degree reached significant level, and the diameter of ovary and anther length correlation is not significant. The fluorescence microscopy, stigma and pollen L-morph flowers have receptivity, and only S-morph flower pollen has vitality. Observe the entire cylindrical flowers and flower growth and short column embryo sac egg cells, micro optical central cell, synergid and antipodal cell structural integrity of.2. short column pollination fruit rate is only about 5%, and Long column pollination fruit rate was above 94%. Hybridization experiments indicated that the S-morph flower stigma pollinated fruit rate was significantly lower than that of L-morph flower fruit rate.3. complete flower type and long column and short column growth flowers hybridization F1 generation plants complete long column flower type, B3-3 * 56 and 01 * 119 two group of materials F2 generation completely long column type and flower growth and short column flower type ratio were 12.14:1 and 11.85:11, the two combinations in the BC1 group completely long column type and flower growth and flower type short column separation ratio were 3.64:1 and 2.56:1. by 2 types of detection, completely and spend a relatively long column students spend the length of column type is dominant, which is the common effect of two gene overlapping effect on gene; Shapiro-Wilk test F2 complete long column flower stigma length also showed that the number of genetic style length controlled by many genes, some major genes based on high-throughput.4. MiRNA sequencing, microarray and Degradome sequencing, we identified the expression of its target gene.MiRNA sequencing and miRNA growth of short column flower pistil development in eggplant eggplant known 55 known miRNA and 35 with miRBase miRNA database in the same sequence of miRNA were identified, 594 new miRNA, and most of the miRNA compared with the other plants are highly conserved. In 686 miRNA of microarray, identified 10 differentially expressed miRNA, among them, only 2 miRNA in L-morph is significantly down regulated in pistil development. At the same time, we use qRT-PCR to verify the differential expression of miRNA. In addition, we also predicted and annotated expression target the.5. miRNA gene by microarray of eggplant S-morph thousand flowers and flower pistil expression refers to eggplant L-morph spectra of miRNA were identified. In 686 miRNA of microarray, we found 35 differentially expressed miR In NA, L-morph spent 9 downregulated miRNA. PC-5p-1408812_1 and PC-5p-3215765_1, most significantly down regulated; while the 26 miRNA increase in the expression of PC-3p-1358_405 and PC-5p-132923_7 the most significant difference. Using the qRT-PCR to validate the expression obtained by microarray detection, results consistent with the microarray data. In addition, we also the differential expression of the predicted target gene of miRNA, and the putative target genes of GO. Functional analysis of target genes in the differential expression of miRNA gene, encoding the transcription factor bHLH, MYB and AP2, enzyme or functional protein important for example three Phosphohydrolase superfamily protein, protein kinase, phosphatase and ATP enzyme so, these may be for heterostyly development plays an important role in the regulation of.6. based on iTRAQ technology and the growth of short column flower pistil development process of eggplant proteome analysis identified 3 00 differentially expressed proteins, which developed in the flowers, flowers and flower S-morph L-morph compared the expression of 26 proteins were down regulated, 23 proteins up-regulated. The downregulated protein, methionine sulfoxide reductase A3 and DNA damage repair / tolerance protein expression differences in the expression. The differentially expressed protein, methionine sulfoxide reductase and sarcosine oxidase. The differentially expressed proteins are mainly involved in energy reserves and external stress response against pathogens and other aspects. In flower maturity, 97 up-regulated and 157 protein expression. The downregulated protein, rich leucine rich repeat protein expression and organ protein. In the differentially expressed proteins in the differential expression of LAT52 and ntp201. The protein expression differences during this period the main parameters With the biosynthesis, biological molecular transport and metabolism. In addition, flower maturity S-morph flower NADP+ oxidation of ethanol in ethanol dehydrogenase reductase, pistil, upregulate the expression of cysteine proteinase and peroxidase, the enzyme involved in the cell death process, the expression of S-morph increased with fruit. Flowers can not be the result of qRT-PCR and iTRAQ is not fully identified results, this may be caused by flower morphology specific post transcriptional regulation. Through the study on Eggplant heterostyly cell morphology and miRNA protein expression differences were studied. These results will provide useful information to uncover the research model and the molecular mechanism of angiosperm heterostyly development, but also on the basis of theoretical research and agricultural improvement, provide very important reference value in breeding and other aspects.

【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S641.1

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本文編號(hào)：1575668