油菜自噬相关基因BnATG8过表达对油菜生长发育的影响

发布时间：2018-04-05 06:47

本文选题：甘蓝型油菜　切入点：ATG8　出处：《兰州大学》2017年硕士论文

【摘要】：自噬(Autophagy,AP)是真核生物在正常生长发育过程中或遭受生物和非生物胁迫时细胞质组分降解并再利用的重要方式之一,其对于维持细胞稳态平衡、发育模式重构、自体免疫以及抗氧化胁迫等方面有非常重要作用。关于植物自噬的研究目前主要局限于植物营养生长过程和生物或非生物胁迫条件下细胞的自噬,而对于植物正常发育过程,如有性生殖过程中是否也有自噬活动发生以及功能还不清楚。ATG8(Autophagy related gene 8)是参与自噬活动中最重要的基因之一。此前,本课题组在电镜下发现蜀葵和油菜花粉母细胞中存在类似自噬体或自噬泡的结构,并以甘蓝型油菜为对象,从中克隆出拟南芥ATG8(AtATG8)的同源基因(BnATG8),并将其与绿色荧光蛋白基因(GFP)融合后导入油菜雄性可育(MF)和不育(MS)系,获得相应过表达系(命名为MFAG和MSAG)。初步研究发现,与MF系比较,T1代MFAG(T1-MFAG)分枝数增多,株高和千粒重下降;与MS系比较,T1代MSAG(T1-MSAG)分枝数不变,但株高和千粒重增加。为了解这两种转基因系的遗传稳定性,本研究分别在2015和2016年对T2和T3代遗传性做进一步分析。此外,还借助GFP标签和自噬染色剂MDC对其花粉母细胞及根尖细胞自噬活动做初步分析。结果如下:(1)与MF相比,T2-MFAG和T3-MFAG的抽薹率、株高和千粒重显著降低,相反分枝数增多;与MS相比,T2-MSAG和T3-MSAG的抽薹率、株高和千粒重都显著提高,分枝数却降低。T3与T2之间无显著差异。(2)形态学观察发现,与MF相比,T2-MFAG和T3-MFAG花丝变短、花药变小变瘪且形态多样,药室数变少,花粉母细胞变,花粉数和生活力降低;与MS相比,T2-MSAG和T3-MSAG花丝变长、花药变大变饱满且形态似椭圆有规则,药室都为4个,花粉母细胞增多,花粉粒数和生活力增高。(3)RT-PCR分析发现,油菜自身BnATG8基因在所有品系花中都有表达,而在所有导入了外源BnATG8的转基因系中,载体中携带BnATG8、GFP和HYG基因都有表达,但在不同个体内,表达水平存在差异。在野生型中这些基因均完全不表达。(4)激光共聚焦显微镜观察发现,在MSAG和MFAG转基因系的花粉母细胞、四分体和小孢子中都有GFP荧光,在花粉母细胞和小孢子中,荧光主要集中在细胞质并靠近细胞膜边缘,呈弥散状态。在四分体中,荧光主要集中于细胞核周围且呈明亮斑点状。从花粉母细胞到小孢子阶段荧光强度逐渐增强。(5)MDC染色观察发现,以上转基因系与野生型的花粉母细胞、四分体和小孢子中都有蓝色荧光出现,但荧光都呈弥散状分布。转基因系与野生型之间在荧光强度和分布上无显著差别。(6)抗逆性研究发现,在T2和T3代中,与MS系相比,MSAG转基因系耐盐能力提高;与MF系相比,MFAG转基因系耐盐能力无明显差异。以上结果表明:1)ATG8基因过表达对油菜的生长、育性和抗逆性,特别是育性有着非常重要的影响。2)转基因系的性状是可稳定遗传的,尤其是MSAG系,在生产实践中可能具有一定应用价值。
[Abstract]:Autophagy APs is one of the important ways for eukaryotes to degrade and reuse cytoplasmic components during normal growth and development or under biotic and abiotic stress.Self-immunity and anti-oxidation stress play a very important role.The research on plant autophagy is mainly limited to plant vegetative growth and autophagy under biological or abiotic stress.It is unclear whether autophagy related gene 8 is one of the most important genes involved in autophagy during sexual reproduction.Previously, under electron microscope, we found that there were autophagy or autophagic structures in pollen mother cells of hollyhock and rapeseed, and Brassica napus was taken as the object.The homologous gene BnATG8 of Arabidopsis thaliana ATG8 / AtATG8 was cloned and fused with green fluorescent protein gene (GFP) and introduced into male fertile mf and sterility MS lines of Brassica napus. The corresponding overexpression lines (named MFAG and MSAGN) were obtained.It was found that compared with MF line, the branch number of T1-MFAG increased, and the plant height and 1000-grain weight decreased, but the branch number of T1-MSAG of T1-MSAG of T1-MSAG was unchanged, but the plant height and 1000-grain weight increased.In order to understand the genetic stability of the two transgenic lines, the inheritance of T2 generation and T3 generation were further analyzed in 2015 and 2016, respectively.In addition, the autophagy activity of pollen mother cells and root tip cells was analyzed with GFP label and autophagy MDC.The results were as follows: (1) compared with MF, the bolting rate, plant height and 1000-grain weight of T2-MFAG and T3-MFAG decreased significantly, but the number of branches increased, and the bolting rate, plant height and 1000-grain weight of T2-MSAG and T3-MSAG increased significantly compared with those of MS.The morphological observation showed that compared with MF, the filaments of T2-MFAG and T3-MFAG became shorter, the anthers became smaller and more varied, the number of drug chambers decreased, the pollen mother cells changed, and the pollen number and viability decreased.Compared with MS, the filaments of T2-MSAG and T3-MSAG became longer, the anthers became larger and fuller, and the anthers appeared to be elliptical and regular. The results of RT-PCR analysis showed that the pollen mother cells increased, the pollen grains and viability increased.The BnATG8 gene of rape itself was expressed in flower of all lines, but in all transgenic lines with exogenous BnATG8, BnATG8GFP and HYG genes were expressed in the vector, but the expression levels were different in different bodies.The results of laser confocal microscopy showed that GFP fluorescence was found in the pollen mother cells, tetrad and microspores of MSAG and MFAG transgenic lines, and in the pollen mother cells and microspores in the pollen mother cells and microspores.Fluorescence was mainly concentrated in the cytoplasm and near the edge of cell membrane.In tetrad, the fluorescence was mainly around the nucleus and showed bright spots.From pollen mother cell to microspore stage, the fluorescence intensity increased gradually. MDC staining showed that blue fluorescence appeared in the tetrad and microspore of the above transgenic lines and wild type pollen mother cells, but the fluorescence was distributed in a dispersive form.There was no significant difference in fluorescence intensity and distribution between transgenic lines and wild-type lines. The results showed that in T2 and T3 generation, the salt tolerance of MSAG transgenic lines was higher than that of MS lines, but there was no significant difference between MFAG transgenic lines and MF lines.The results showed that the overexpression of the ATG8 gene had a very important effect on the growth, fertility and stress resistance of rape, especially on fertility. 2) the characters of transgenic lines were stable and hereditary, especially the MSAG lines.In the production practice may have certain application value.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S565.4

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