【摘要】：胼胝質(zhì)是一種β-1,3-葡聚糖,普遍存在于高等植物中,在植物生長(zhǎng)發(fā)育過(guò)程中起著重要的調(diào)節(jié)作用,胼胝質(zhì)異常的沉積和降解都會(huì)導(dǎo)致花粉敗育,從而造成植物雄性不育。本研究以大白菜核不育近等基因系10L03為試材,從細(xì)胞學(xué)、生物化學(xué)和分子生物學(xué)水平對(duì)大白菜小孢子敗育過(guò)程中的胼胝質(zhì)沉積進(jìn)行系統(tǒng)研究,深入探討胼胝質(zhì)沉積的相關(guān)分子調(diào)控機(jī)制,以期為揭示大白菜核不育小孢子敗育機(jī)理研究提供理論參考。研究獲得如下結(jié)果:1.苯胺藍(lán)染色結(jié)合光學(xué)顯微鏡觀察小孢子敗育過(guò)程中胼胝質(zhì)沉積動(dòng)態(tài),結(jié)果發(fā)現(xiàn)小孢子發(fā)育至四分體時(shí)期不育小孢子的熒光反應(yīng)與正�？捎℃咦踊疽恢�,但四分小孢子形態(tài)出現(xiàn)明顯的不規(guī)則。之后可育花藥小孢子熒光反應(yīng)逐漸減弱,說(shuō)明四分體周?chē)蓦召|(zhì)逐漸降解,釋放單核小孢子,隨后會(huì)在花粉外壁檢測(cè)到熒光,說(shuō)明胼胝質(zhì)會(huì)隨著花粉發(fā)育在花粉外壁上沉積;而不育小孢子內(nèi)熒光反應(yīng)一直持續(xù),可見(jiàn)未釋放的敗育小孢子,說(shuō)明四分體上的胼胝質(zhì)并未正常降解,無(wú)法釋放單核小孢子,小孢子未能進(jìn)一步發(fā)育,進(jìn)而導(dǎo)致敗育。2.采用DNS法和苯胺藍(lán)法分別測(cè)定小孢子不同發(fā)育時(shí)期胼胝質(zhì)沉積相關(guān)BG和GSL酶活性,結(jié)果表明在小孢子敗育過(guò)程中,GSL酶活性在不同發(fā)育時(shí)期總體趨勢(shì)與可育株一樣,隨著小孢子發(fā)育逐漸下降,但在減數(shù)分裂時(shí)期顯著高于可育株,在單核期極顯著高于可育株,而B(niǎo)G酶活性在小孢子敗育過(guò)程中變化較小,極顯著低于可育株,說(shuō)明在小孢子敗育過(guò)程中,胼胝質(zhì)合成增強(qiáng)而降解能力下降,導(dǎo)致胼胝質(zhì)在不育花蕾中沉積異常。3.采用Real-time PCR對(duì)胼胝質(zhì)沉積相關(guān)BG和GSL基因進(jìn)行表達(dá)分析,結(jié)果表明,BcGSL1、BcGSL2、BcGSL8和BcGSL10均在花中高表達(dá),但小孢子發(fā)育的減數(shù)分裂至單核早期,不育花藥中這幾個(gè)基因的表達(dá)量均顯著低于可育株,說(shuō)明這幾個(gè)基因調(diào)控的胼胝質(zhì)合成并未增強(qiáng);雖然不育花藥中BcGSL12基因在四分體時(shí)期的表達(dá)量極顯著高于可育花藥,但該時(shí)期GSL酶活性依然低于可育株小孢子的GSL酶活性,因此BcGSL12基因在不育花藥四分體時(shí)期的高表達(dá)并未增強(qiáng)胼胝質(zhì)的總體合成;BcENBG7、BcENBG12基因也在花藥中高表達(dá),但四分體時(shí)期,不育株花藥中這兩個(gè)基因的表達(dá)量均高于可育株小孢子,說(shuō)明這兩個(gè)基因調(diào)控的胼胝質(zhì)降解能力并未下降;不育花藥中BcA6基因在四分體時(shí)期的表達(dá)量極顯著低于可育花藥,導(dǎo)致該時(shí)期BG酶活性過(guò)低,影響胼胝質(zhì)的降解。綜合分析細(xì)胞學(xué)、生物化學(xué)和分子生物學(xué)的研究結(jié)果,小孢子胼胝質(zhì)延遲降解是大白菜核不育小孢子敗育的重要原因,四分體時(shí)期BG基因BcA6表達(dá)量過(guò)低,BG酶活性下降可能與胼胝質(zhì)延遲降解有關(guān)。
[Abstract]:Callosum is a kind of 尾-1 and 3-glucan, which is widely found in higher plants and plays an important role in regulating plant growth and development. Abnormal deposition and degradation of corpus callosum will lead to pollen abortion, resulting in male sterility in plants. In this study, the callose deposition during microspore abortion in Chinese cabbage was systematically studied from the cytological, biochemical and molecular biological levels using the gene-sterile near isogenic line 10L03 of Chinese cabbage as experimental material. The molecular regulation mechanism of callose deposition was discussed in order to provide a theoretical reference for the study of the mechanism of microspore abortion in Chinese cabbage. The results are as follows: 1. Aniline blue staining and optical microscope were used to observe the dynamic of callose deposition during microspore abortion. The results showed that the fluorescence reaction of sterile microspore during microspore development to tetrad was basically consistent with that of normal fertile microspore. But the microspore morphology of the quaternion appeared obvious irregularity. The fluorescence reaction of microspore in fertile anthers weakened gradually, indicating that the callose around the tetrad gradually degraded and released mononuclear microspore, then fluorescence was detected in the outer wall of pollen, indicating that callosum would deposit on the outer wall of pollen with pollen development. However, the fluorescence reaction in sterile microspore was persistent, which indicated that the callosum on tetrad did not degrade normally and could not release mononuclear microspore, and the microspore failed to develop further, which resulted in abortion of 2. 2. DNS and aniline blue methods were used to measure the activities of BG and GSL in corpus callosum deposition at different stages of microspore development. The results showed that the general trend of GSL enzyme activity in different developmental stages was the same as that of fertile plants during microspore abortion. The microspore development decreased gradually, but it was significantly higher in meiosis than that in fertile plant, and in mononuclear stage was significantly higher than that in fertile plant. However, the activity of BG enzyme changed slightly during microspore abortion, and was significantly lower than that of fertile plant. The results showed that during microspore abortion, callose synthesis increased and degradation ability decreased, which resulted in abnormal deposition of corpus callosum in sterile flower buds. The expression of BG and GSL genes related to corpus callosum deposition was analyzed by Real-time PCR. The results showed that both BcGSL1,BcGSL2,BcGSL8 and BcGSL10 were highly expressed in flowers, but the meiosis of microspore development was at the early stage of mononuclear development. The expression of these genes in sterile anthers was significantly lower than that in fertile plants, indicating that the callosum synthesis regulated by these genes was not enhanced. Although the expression of BcGSL12 gene in sterile anthers was significantly higher than that in fertile anthers at tetrad stage, the activity of GSL enzyme was still lower than that of microspore in fertile plants. Therefore, the high expression of BcGSL12 gene in the tetrad of sterile anthers did not enhance the overall synthesis of corpus callosum. BcENBG7,BcENBG12 gene was also highly expressed in anther, but at tetrad stage, the expression of these two genes in sterile plant anther was higher than that in fertile plant microspore, which indicated that the ability of callosum degradation regulated by these two genes was not decreased. The expression of BcA6 gene in sterile anthers was significantly lower than that in fertile anthers at tetrad stage, which resulted in the low activity of BG and the degradation of corpus callosum. According to the results of cytology, biochemistry and molecular biology, the delayed degradation of microspore corpus callosum was the main cause of microspore abortion in Chinese cabbage, and the BcA6 expression of BG gene was too low in tetrad. The decrease of BG activity may be related to the delayed degradation of corpus callosum.
【學(xué)位授予單位】：云南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S634.1

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