本文選題：雄性不育 + 花粉　； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：雜種優(yōu)勢(shì)在農(nóng)業(yè)生產(chǎn)中應(yīng)用廣泛,但是通過(guò)人工雜交產(chǎn)生雜交子代的過(guò)程較繁瑣,通過(guò)創(chuàng)建雄性不育系進(jìn)行雜交育種可以有效降低育種成本。在雄配子發(fā)育成熟的過(guò)程中,絨氈層作為花藥的營(yíng)養(yǎng)細(xì)胞,它的發(fā)育及降解為雄配子提供營(yíng)養(yǎng)物質(zhì)和各種酶,前人的研究表明絨氈層的發(fā)育及PCD異常會(huì)導(dǎo)致花粉敗育。目前已知的調(diào)控絨氈層發(fā)育的轉(zhuǎn)錄因子網(wǎng)絡(luò)在擬南芥和水稻中是保守的,而對(duì)于具體的細(xì)胞學(xué)活動(dòng)的保守性尚不清楚。近期有研究表明,在擬南芥的絨氈層中,NADPH氧化酶RBOHE可以通過(guò)時(shí)空特異性產(chǎn)生活性氧調(diào)控絨氈層的PCD,進(jìn)而影響花粉的發(fā)育。為了進(jìn)一步探究這種由活性氧介導(dǎo)的絨氈層PCD的機(jī)制在其他作物中的保守性,進(jìn)而利用這種調(diào)控機(jī)制創(chuàng)建雄性不育系。我們選擇了兩種重要的經(jīng)濟(jì)作物煙草(Nicotiana Benthamiana)和番茄(Lycopersicon esculentum cv.moneymaker),對(duì)它們的絨氈層展開(kāi)研究。研究結(jié)果及結(jié)論如下:(1)煙草和番茄花藥中活性氧含量是動(dòng)態(tài)變化的通過(guò)NBT和H2DCF-DA染色發(fā)現(xiàn),在煙草和番茄的花藥中活性氧的含量都是伴隨花藥的發(fā)育呈現(xiàn)出先升高后降低的動(dòng)態(tài)波動(dòng),活性氧均是在減數(shù)分裂前期開(kāi)始產(chǎn)生,在減數(shù)分裂時(shí)期和四分體時(shí)期含量逐漸升高,在小孢子前中期達(dá)到最高水平,最后逐漸降低至有絲分裂時(shí)期基本無(wú)活性氧產(chǎn)生。(2)煙草和番茄絨氈層的組織學(xué)及細(xì)胞學(xué)分析為了更有效的研究這兩種作物的絨氈層,我們通過(guò)活體解剖和組織切片的方法探究了這兩種作物對(duì)應(yīng)絨氈層特定發(fā)育時(shí)期的花器官大小。通過(guò)制作半薄切片觀察并描述這兩種作物的絨氈層和小孢子在不同發(fā)育時(shí)期的組織學(xué)特點(diǎn),并通過(guò)透射電鏡觀察到絨氈層PCD進(jìn)程中的細(xì)胞器和膜結(jié)構(gòu)是逐漸變化的。(3)煙草和番茄絨氈層PCD的進(jìn)程分析我們通過(guò)透射電鏡和TUNEL檢測(cè)了這兩種作物中絨氈層PCD的進(jìn)程,煙草絨氈層的PCD主要發(fā)生在11期即有絲分裂時(shí)期,而番茄絨氈層的PCD相對(duì)于煙草發(fā)生較早且緩慢,主要在10期即小孢子后期進(jìn)行PCD,在11期仍可以檢測(cè)到PCD的信號(hào)。(4)煙草花藥中活性氧含量的降低導(dǎo)致絨氈層PCD延遲最終導(dǎo)致花粉敗育通過(guò)活性氧染色和檢測(cè)絨氈層PCD的進(jìn)程,我們發(fā)現(xiàn)煙草和番茄絨氈層的PCD都發(fā)生在活性氧含量升高以后,而這兩者之間是否存在關(guān)聯(lián)尚不清楚。通過(guò)用化學(xué)藥劑DPI抑制煙草花藥中活性氧的產(chǎn)生,發(fā)現(xiàn)在活性氧顯著降低的花藥中絨氈層在11期的降解出現(xiàn)延遲,最終導(dǎo)致產(chǎn)生的花粉部分?jǐn)∮?說(shuō)明在煙草中也存在由活性氧調(diào)控絨氈層PCD的機(jī)制。(5)煙草和番茄中RBOH基因表達(dá)模式和功能分析在擬南芥絨氈層中,活性氧是由NADPH氧化酶RBOHE時(shí)空特異性產(chǎn)生,因此我們推測(cè)在煙草和番茄的絨氈層中也是由RBOH基因產(chǎn)生活性氧調(diào)控絨氈層PCD。為此,我們通過(guò)BLAST找到煙草和番茄中表達(dá)的RBOH基因,并通過(guò)定量PCR對(duì)不同組織中這些基因的表達(dá)量進(jìn)行分析,找到了在花藥中特異性表達(dá)的RBOH,進(jìn)而對(duì)這些基因在花藥中的時(shí)間表達(dá)模式進(jìn)行分析,發(fā)現(xiàn)煙草中的NbRBOHE1、NbRBOHE2和番茄中的LeRBOHE、LeRBOH在花藥中時(shí)間特異性表達(dá),推測(cè)可能是由這幾個(gè)基因在煙草和番茄的絨氈層中發(fā)揮功能。我們進(jìn)一步通過(guò)VIGS對(duì)煙草中的NbRBOHE1進(jìn)行基因沉默,但是并沒(méi)有引起絨氈層和花粉的發(fā)育缺陷,因此我們推測(cè)在煙草花藥中表達(dá)的RBOH基因可能存在功能冗余,下一步可以通過(guò)CRISPR-cas9將更多的RBOH基因敲除,或者通過(guò)在絨氈層中過(guò)表達(dá)RBOH基因,觀察其絨氈層和花粉的發(fā)育。
[Abstract]:Heterosis is widely used in agricultural production, but the process of producing hybrids by artificial hybridization is tedious. Breeding of male sterile lines can effectively reduce the cost of breeding. In the process of mature male gametes, the tapetum is a nutrient cell of anther, and its development and degradation provide nutrition for male gametes. Substances and various enzymes, previous studies have shown that the development of tapetum and PCD abnormalities lead to pollen abortion. The current known transcription factor network regulating tapetum development is conservative in Arabidopsis and rice, while the conservatism of specific cytological activities is not clear. Recent studies have shown that in the tapetum of Arabidopsis, NADPH Oxidase RBOHE can regulate the PCD of tapetum through the time and space specificity, and then affect the development of pollen. In order to further explore the conservation of the mechanism of the tapetum mediated by reactive oxygen (PCD) in other crops, and then use this regulatory mechanism to create male sterile lines. We chose two important economic works. Nicotiana Benthamiana and Lycopersicon esculentum cv.moneymaker were used to study their tapetum. The results and conclusions are as follows: (1) the active oxygen content in tobacco and tomato anthers is dynamically changed by NBT and H2DCF-DA staining, and the content of active oxygen in the anthers of tobacco and tomato is accompanied by flowers. The development of the drug appeared to rise first and then decrease, and the reactive oxygen species began to occur in the prophase of meiosis. The content of the active oxygen increased gradually in the meiosis and four division period, reached the highest level in the middle of the microspore, and finally gradually decreased to the mitosis period. (2) the group of tobacco and the tapetum of tomato. In order to study the tapetum of these two crops more effectively, we explored the size of the flower organs of the two kinds of tapetum during the specific development of the tapetum through the methods of living dissection and tissue section. The tapetum and the microspore of the two species were observed and described in different developmental stages by making a semi thin section. The cellular organelles and membrane structures in the tapetum PCD process are gradually changed by transmission electron microscopy. (3) the process analysis of PCD in tobacco and tomato tapetum we detected the process of tapetum PCD in these two crops by transmission electron microscopy and TUNEL, and the PCD of the tapetum mainly occurred during the 11 phase of mitosis. The PCD of the tapetum was relatively early and slow relative to tobacco, mainly in the 10 stage, PCD at the later stage of microspore, and the signal of PCD in the 11 phase. (4) the decrease of the active oxygen content in the tobacco anthers led to the PCD delay of the tapetum resulting in the process of pollen abortion through reactive oxygen staining and the detection of the tapetum PCD. PCD in tobacco and tomato tapetum occurred after active oxygen content increased, but it was not clear whether there was an association between the two. By using chemical DPI to inhibit the production of active oxygen in tobacco anthers, it was found that the degradation of tapetum in the 11 phase of the anther which was significantly lower in reactive oxygen species was delayed and eventually resulted in the production of pollen. The mechanism of regulating the tapetum PCD by reactive oxygen species is also found in tobacco. (5) the expression pattern and function analysis of RBOH gene expression in tobacco and tomato, in Arabidopsis thaliana tapetum, the active oxygen is produced by the spatio-temporal specificity of NADPH oxidase RBOHE. Therefore, we speculate that the RBOH gene is also produced in the tapetum of tobacco and tomato. Oxygen regulated tapetum PCD. for this purpose, we found the RBOH gene expressed in tobacco and tomatoes by BLAST, and analyzed the expression of these genes in different tissues by quantitative PCR, and found the RBOH in the anther specific expression, and then analyzed the expression pattern of these genes in the anthers, and found the NbRBOHE in tobacco. 1, LeRBOHE and LeRBOH in NbRBOHE2 and tomatoes are time specific in anthers. It is presumed that these genes may be function in the tapetum of tobacco and tomatoes. We further gene silencing of NbRBOHE1 in tobacco by VIGS, but do not cause the development defects of the tapetum and pollen, so we speculate that smoking is in smoke. The RBOH gene expressed in the herb may have functional redundancy. Next, more RBOH genes can be knocked out by CRISPR-cas9 or through the expression of RBOH gene in the tapetum, and the development of the tapetum and pollen is observed.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q945

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本文編號(hào)：2041329