大白菜核不育小孢子发育胼胝质沉积相关基因的表达分析

发布时间：2019-01-03 09:15

【摘要】：胼胝质是一种β-1,3-葡聚糖,普遍存在于高等植物中,在植物生长发育过程中起着重要的调节作用,胼胝质异常的沉积和降解都会导致花粉败育,从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为试材,从细胞学、生物化学和分子生物学水平对大白菜小孢子败育过程中的胼胝质沉积进行系统研究,深入探讨胼胝质沉积的相关分子调控机制,以期为揭示大白菜核不育小孢子败育机理研究提供理论参考。研究获得如下结果:1.苯胺蓝染色结合光学显微镜观察小孢子败育过程中胼胝质沉积动态,结果发现小孢子发育至四分体时期不育小孢子的荧光反应与正常可育小孢子基本一致,但四分小孢子形态出现明显的不规则。之后可育花药小孢子荧光反应逐渐减弱,说明四分体周围胼胝质逐渐降解,释放单核小孢子,随后会在花粉外壁检测到荧光,说明胼胝质会随着花粉发育在花粉外壁上沉积;而不育小孢子内荧光反应一直持续,可见未释放的败育小孢子,说明四分体上的胼胝质并未正常降解,无法释放单核小孢子,小孢子未能进一步发育,进而导致败育。2.采用DNS法和苯胺蓝法分别测定小孢子不同发育时期胼胝质沉积相关BG和GSL酶活性,结果表明在小孢子败育过程中,GSL酶活性在不同发育时期总体趋势与可育株一样,随着小孢子发育逐渐下降,但在减数分裂时期显著高于可育株,在单核期极显著高于可育株,而BG酶活性在小孢子败育过程中变化较小,极显著低于可育株,说明在小孢子败育过程中,胼胝质合成增强而降解能力下降,导致胼胝质在不育花蕾中沉积异常。3.采用Real-time PCR对胼胝质沉积相关BG和GSL基因进行表达分析,结果表明,BcGSL1、BcGSL2、BcGSL8和BcGSL10均在花中高表达,但小孢子发育的减数分裂至单核早期,不育花药中这几个基因的表达量均显著低于可育株,说明这几个基因调控的胼胝质合成并未增强;虽然不育花药中BcGSL12基因在四分体时期的表达量极显著高于可育花药,但该时期GSL酶活性依然低于可育株小孢子的GSL酶活性,因此BcGSL12基因在不育花药四分体时期的高表达并未增强胼胝质的总体合成;BcENBG7、BcENBG12基因也在花药中高表达,但四分体时期,不育株花药中这两个基因的表达量均高于可育株小孢子,说明这两个基因调控的胼胝质降解能力并未下降;不育花药中BcA6基因在四分体时期的表达量极显著低于可育花药,导致该时期BG酶活性过低,影响胼胝质的降解。综合分析细胞学、生物化学和分子生物学的研究结果,小孢子胼胝质延迟降解是大白菜核不育小孢子败育的重要原因,四分体时期BG基因BcA6表达量过低,BG酶活性下降可能与胼胝质延迟降解有关。
[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.
【学位授予单位】：云南农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S634.1

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