orf25和atp9基因干扰对烟草雄性不育性的影响

发布时间：2018-07-09 19:10

  本文选题：烟草 + orf25基因　； 参考：《江西农业大学》2017年硕士论文

【摘要】：烟草是我国乃至世界上重要的经济作物,同时也是植物基因工程常用的模式植物。ATP合酶是一种线粒体复合酶,主要由F0和F1两部分组成,异常编码的ATP合酶单个基因或多个基因产物常伴随植物雄性不育的形成。orf25基因是F0部分的亚基基因,其异常表达可能会导致烟草雄性不育的产生。本研究以atp9基因为对照基因,前期已构建orf25和atp9基因的RNA干扰表达载体,通过农杆菌介导法分别转化保持系烟草中烟90(zy90)和K326,并均获得转基因阳性植株。通过分析转RNAi-orf25和转RNAi-atp9烟草在形态学、ATP酶活性、ATP含量、orf25基因相对表达量以及花粉活力上与对照组之间的差异,来探究orf25基因与烟草雄性不育的关系。主要研究结果如下:1、共获得转RNAi-orf25烟草12株,其中1株在苗期死亡,1株未开花;转RNAi-atp9烟草14株,其中有2株未开花。对转基因烟草的形态学特征研究表明:干扰orf25或atp9基因的表达后,植株明显变矮、生长缓慢、分枝增加、叶片数少、叶片变小、易染病。2、生殖生长期非转基因烟草叶、花、茎和根四个器官中,保持系zy90中orf25基因相对表达量显著高于不育系MSzy90,且花中orf25基因相对表达量始终保持最高;保持系K326中atp9基因相对表达量显著高于不育系MSK326,且atp9基因相对表达量在花器官中为最高。RT-PCR分析结果表明,转RNAi-orf25烟草叶、花、茎和根中orf25基因相对表达量明显低于对照组,且花中为最低;同样地,与对照组一致的是,转RNAi-atp9烟草atp9基因相对表达量在花中为最低,并且在四个器官中均明显低于对照组。3、比较生殖生长期非转基因烟草zy90和MSzy90以及MSK326和K326叶、花、茎和根四个器官的ATP酶活性,结果表明均是保持系中的ATP酶活性显著高于不育系。两系皆是花器官中的ATP酶活性最大。两种转基因烟草转RNAi-orf25和转RNAi-atp9中的ATP酶活性和ATP含量均显著低于对照烟草,且花中均明显高于其他三个器官。表明两种烟草RNAi表达载体皆具有干扰效果。4、相比于对照烟草,转基因烟草的花粉活力明显下降。其中有一株转RNAi-atp9烟草育性发生改变,形成雄性不育烟草。5、干扰烟草orf25基因和atp9基因的正常表达均能特异性地减弱目的基因的表达,降低ATP酶活性和ATP含量以及烟草的花粉活力,推测ATP合酶的这两个亚基基因可能具有相似的功能。orf25基因的异常表达虽不能直接引起烟草雄性不育,但是否与其他烟草CMS相关基因共同调控烟草雄性不育性有待于进一步研究。
[Abstract]:Tobacco is an important cash crop in China and even the world. It is also a common model plant. ATP synthase is a mitochondrial complex enzyme, which is mainly composed of F0 and F1. The abnormal encoding of ATP synthase single gene or multiple gene products is often associated with the formation of plant male sterility. Orf25 gene is the F0 subunit gene, and its abnormal expression may lead to the production of tobacco male sterility. In this study, atp9 gene was used as control gene to construct RNAi expression vectors of orf25 and atp9 genes. The vector was transformed into tobacco maintainer line zy90 (zy90) and K326 by Agrobacterium tumefaciens, respectively, and transgenic plants were obtained. In order to explore the relationship between orf25 gene and male sterility of tobacco, the relative expression of orf25 gene and the relative expression of orf25 gene in morphological ATPase activity were analyzed by analyzing the difference between transgenic RNAi-orf25 and transRNAi-atp9 tobacco. The main results were as follows: 1 of 12 tobacco plants transferred to RNAi-orf25, 1 of which died at seedling stage did not bloom, and 14 of them were transferred to RNAi-atp9, 2 of which did not bloom. The morphological characteristics of transgenic tobacco were studied. The results showed that after interfering with the expression of orf25 or atp9 gene, the plants became shorter, grew slowly, increased branches, decreased the number of leaves, reduced the leaves, became susceptible to disease. The relative expression of orf25 gene in the maintainer line zy90 was significantly higher than that in the sterile line MSzy90 in four organs of stem and root, and the relative expression of orf25 gene in flower was always the highest. The relative expression of atp9 gene in the maintainer line K326 was significantly higher than that in the sterile line MSK326, and the relative expression of atp9 gene was the highest in flower organs. RT-PCR analysis showed that the relative expression of orf25 gene in the leaves, flowers, stems and roots of transgenic tobacco was significantly lower than that in the control group. The relative expression of RNAi-atp9 gene was the lowest in the flower, and the lowest in the control group. The activities of zy90 and MSzy90 in non-transgenic tobacco, leaves, flowers, stems and roots of MSK326 and K326 in reproductive period were compared. The results showed that the ATPase activity in both maintainer lines was significantly higher than that in male sterile lines. Both strains had the highest ATPase activity in floral organs. The ATPase activity and ATP content in two transgenic tobacco transRNAi-orf25 and RNAi-atp9 were significantly lower than those in the control, and the flowers were significantly higher than the other three organs. The results showed that both RNAi expression vectors had interference effect. Compared with control tobacco, the pollen viability of transgenic tobacco decreased significantly. One of the transgenic RNAi-atp9 plants changed its fertility to form male sterile tobacco. 5, which interfered with the normal expression of orf25 gene and atp9 gene in tobacco, which could specifically weaken the expression of the target gene, decrease the activity of ATPase, the content of ATP and the pollen vigor of tobacco. It is speculated that these two subunits of ATP synthase may have similar function. The abnormal expression of orf25 gene can not directly cause tobacco male sterility, but whether the two subunits can regulate tobacco male sterility together with other tobacco CMS genes needs further study.
【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S572

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