烟草和番茄中活性氧介导的绒毡层程序性细胞死亡影响花粉的发育

发布时间：2018-06-19 21:22

本文选题：雄性不育 + 花粉　；参考：《山东农业大学》2017年硕士论文

【摘要】：杂种优势在农业生产中应用广泛,但是通过人工杂交产生杂交子代的过程较繁琐,通过创建雄性不育系进行杂交育种可以有效降低育种成本。在雄配子发育成熟的过程中,绒毡层作为花药的营养细胞,它的发育及降解为雄配子提供营养物质和各种酶,前人的研究表明绒毡层的发育及PCD异常会导致花粉败育。目前已知的调控绒毡层发育的转录因子网络在拟南芥和水稻中是保守的,而对于具体的细胞学活动的保守性尚不清楚。近期有研究表明,在拟南芥的绒毡层中,NADPH氧化酶RBOHE可以通过时空特异性产生活性氧调控绒毡层的PCD,进而影响花粉的发育。为了进一步探究这种由活性氧介导的绒毡层PCD的机制在其他作物中的保守性,进而利用这种调控机制创建雄性不育系。我们选择了两种重要的经济作物烟草(Nicotiana Benthamiana)和番茄(Lycopersicon esculentum cv.moneymaker),对它们的绒毡层展开研究。研究结果及结论如下:(1)烟草和番茄花药中活性氧含量是动态变化的通过NBT和H2DCF-DA染色发现,在烟草和番茄的花药中活性氧的含量都是伴随花药的发育呈现出先升高后降低的动态波动,活性氧均是在减数分裂前期开始产生,在减数分裂时期和四分体时期含量逐渐升高,在小孢子前中期达到最高水平,最后逐渐降低至有丝分裂时期基本无活性氧产生。(2)烟草和番茄绒毡层的组织学及细胞学分析为了更有效的研究这两种作物的绒毡层,我们通过活体解剖和组织切片的方法探究了这两种作物对应绒毡层特定发育时期的花器官大小。通过制作半薄切片观察并描述这两种作物的绒毡层和小孢子在不同发育时期的组织学特点,并通过透射电镜观察到绒毡层PCD进程中的细胞器和膜结构是逐渐变化的。(3)烟草和番茄绒毡层PCD的进程分析我们通过透射电镜和TUNEL检测了这两种作物中绒毡层PCD的进程,烟草绒毡层的PCD主要发生在11期即有丝分裂时期,而番茄绒毡层的PCD相对于烟草发生较早且缓慢,主要在10期即小孢子后期进行PCD,在11期仍可以检测到PCD的信号。(4)烟草花药中活性氧含量的降低导致绒毡层PCD延迟最终导致花粉败育通过活性氧染色和检测绒毡层PCD的进程,我们发现烟草和番茄绒毡层的PCD都发生在活性氧含量升高以后,而这两者之间是否存在关联尚不清楚。通过用化学药剂DPI抑制烟草花药中活性氧的产生,发现在活性氧显著降低的花药中绒毡层在11期的降解出现延迟,最终导致产生的花粉部分败育,说明在烟草中也存在由活性氧调控绒毡层PCD的机制。(5)烟草和番茄中RBOH基因表达模式和功能分析在拟南芥绒毡层中,活性氧是由NADPH氧化酶RBOHE时空特异性产生,因此我们推测在烟草和番茄的绒毡层中也是由RBOH基因产生活性氧调控绒毡层PCD。为此,我们通过BLAST找到烟草和番茄中表达的RBOH基因,并通过定量PCR对不同组织中这些基因的表达量进行分析,找到了在花药中特异性表达的RBOH,进而对这些基因在花药中的时间表达模式进行分析,发现烟草中的NbRBOHE1、NbRBOHE2和番茄中的LeRBOHE、LeRBOH在花药中时间特异性表达,推测可能是由这几个基因在烟草和番茄的绒毡层中发挥功能。我们进一步通过VIGS对烟草中的NbRBOHE1进行基因沉默,但是并没有引起绒毡层和花粉的发育缺陷,因此我们推测在烟草花药中表达的RBOH基因可能存在功能冗余,下一步可以通过CRISPR-cas9将更多的RBOH基因敲除,或者通过在绒毡层中过表达RBOH基因,观察其绒毡层和花粉的发育。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945

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