SPO11-1调控拟南芥同源多倍体减数分裂的细胞遗传学研究

发布时间：2018-01-07 13:04

本文关键词：SPO11-1调控拟南芥同源多倍体减数分裂的细胞遗传学研究　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：多倍化是植物进化过程中的一种普遍现象,这一现象不仅是推动植物进化的重要力量,也是新物种形成的重要途径。据推测,高达70%的被子植物在进化过程中,都至少经历过一次多倍化。与二倍体物种相比,多倍体在很多方面都展现出明显的优势,如更强的抗逆性、适应性等。基于多倍体在植物界的主导地位,有很多研究都集中于多倍化对植物遗传及表观遗传的影响。但是多倍化对减数分裂相关基因突变后的影响研究较少。减数分裂是一种特殊的细胞分裂方式,在这一过程中,会发生遗传物质的浓缩、配对、联会、重组等一系列受到严格调控的重要事件,任何一个事件出现差错都会导致减数分裂出现缺陷甚至提前终止。多倍体植物含有两套以上的染色体组,与二倍体相比,具有更高的重组频率和更复杂的联会方式。因此,深入研究多倍体植物减数分裂同源重组过程中所涉及到的相关基因具有十分重要的意义。鉴于此,本实验充分利用分子和细胞遗传学手段,研究SPO11-1在同源四倍体拟南芥减数分裂同源重组中的功能,研究结果如下:1、针对拟南芥SPO11-1基因中的特异序列设计干扰片段,经酶切鉴定干扰片段成功定向插入载体,成功构建了RNA干扰载体SPO11-pHellsgate2。2、通过floral-dip法转化拟南芥,经过卡那霉素初步筛选及PCR分子检测,成功获得了二倍体及同源四倍体转基因植株。通过荧光定量PCR检测,二倍体转基因植株SPO11-1基因的表达水平仅为二倍体野生型拟南芥的46%。四倍体转基因植株SPO11-1基因的表达水平仅为野生型同源四倍体拟南芥的40%。3、二倍体、四倍体转基因拟南芥与其所对应的野生型植株在形态学与营养生长的特征上没有显著差别,具体表现为,在叶片面积、叶片形态、生长周期等方面,转基因植株与野生型植株无明显差别。4、二倍体、四倍体转基因植株生殖生长过程出现缺陷,育性显著下降,具体表现为:1)转基因植株的果荚长度明显缩短,且果荚内部可见大量发育不良或败育的胚珠,缺粒现象明显。二倍体转基因植株结实率比其所对应野生型下降51.8%。四倍体转基因植株结实率比对应野生型下降32%。2)花粉亚历山大染色结果显示,二倍体、四倍体转基因植株花药中所含的花粉减少,且出现较多干瘪、皱缩及大小形状不均一的花粉粒,推测这些花粉粒是无活性的花粉粒。二倍体转基因植株异常花粉粒比例比相应野生型上升了49.3%,四倍体转基因植株异常花粉粒比例比相应野生型上升了35%。5、二倍体、四倍体转基因植株的花粉母细胞在减数分裂过程都出现了较为严重的异常现象,表现为粗线期同源染色体不能完全联会,中期Ⅰ及中期Ⅱ出现大量偏离赤道板的染色体以及单价体。后期Ⅰ及后期Ⅱ出现落后染色体,且出现染色体不均等分离的情况,末期Ⅱ分成大小不等的子细胞。根据统计分析,二倍体野生型植株的花粉母细胞在终变期、中期Ⅰ、后期Ⅰ、后期Ⅱ的染色体行为异常比例均为0%,而二倍体转基因植株的花粉母细胞在终变期、中期Ⅰ、后期Ⅰ、后期Ⅱ染色体行为的异常比例分别为66.7%、50%、33.3%、27.3%。四倍体野生型植株的花粉母细胞在终变期、中期Ⅰ、后期Ⅰ、后期Ⅱ的染色体行为异常比例分别为75%、16.7%、0%、0%。四倍体转基因植株的花粉母细胞在终变期、中期Ⅰ、后期Ⅰ、后期Ⅱ的染色体行为异常比例分别为84.2%、33.3%、33.3%、28.6%。这一系列染色体的异常行为即为转基因植株花粉活力降低及育性下降的主要因素。
[Abstract]:Polyploidy is a common phenomenon in plant evolution, this phenomenon is not only an important driving force of plant evolution, but also an important way to the formation of new species. It is speculated that up to 70% of the angiosperms in the evolutionary process, have experienced at least one times. Compared with the diploid species, Polyploid in many ways they show obvious advantages, such as stronger resistance and adaptability. Polyploid dominance in the plant kingdom and based on a lot of studies have focused on the polyploidy of plant genetic and epigenetic effects. But the meiotic polyploidization of less studied gene mutation after meiosis is a special. The method of cell division in the process of concentration, will happen genetic material pairing, synapsis, recombination and a series of tightly regulated events, any errors will lead to reduction of events The number of split defects and even termination. Polyploid plant contains the genome of more than two units, compared with the diploid, with recombination frequency and more complex association. Therefore, in-depth study of plant polyploid meiosis homologous related genes involved in the process of restructuring is of great significance. In view of this. This experiment makes full use of molecular and cytogenetic methods of SPO11-1 in Autotetraploid Arabidopsis thaliana meiotic homologous recombination in the function, the results are as follows: 1, according to the interference design specific sequence in the SPO11-1 gene fragment of Arabidopsis thaliana, identified by enzyme digestion fragment was successfully inserted into the interference vector, RNA interference vector was constructed successfully by SPO11-pHellsgate2.2 transformation of Arabidopsis thaliana Floral-Dip method, detected by kanamycin screening and molecular PCR, successfully obtained four times and homologous transgenic diploid Plants. By fluorescent quantitative PCR assay, the expression level of SPO11-1 gene in diploid transgenic plants is only the expression level of 46%. gene SPO11-1 transgenic tetraploid diploid wild type Arabidopsis only wild type Arabidopsis 40%.3 tetraploid, diploid, tetraploid transgenic Arabidopsis wild type plants corresponding to no significant difference in morphology and vegetative growth the characteristics, specific performance, the leaf area, leaf morphology, growth cycle, transgenic plants and wild type plants had no significant difference between.4 and diploid defects was four times as much as the transgenic plants during reproductive growth, fertility decreased significantly, as follows: 1) transgenic plant pod length was significantly shortened, and the pod is visible inside large dysplasia or abortive ovule, lack of grain was obvious. Diploid transgenic plants than the corresponding wild seed setting rate Type 51.8%. tetraploid transgenic plants decreased seed setting rate than the corresponding wild-type pollen decreased 32%.2) Alexander staining showed that diploid, tetraploid transgenic plants contained in anther and pollen reduced, more dry, shrink the size and shape of pollen grains is not uniform, speculated that the pollen is inactive. Diploid transgenic plants of abnormal pollen grains the proportion of pollen grains was 49.3% higher than the corresponding wild type, transgenic tetraploid abnormal pollen grains increased the proportion of 35%.5 than the corresponding wild-type diploid, tetraploid transgenic plants of pollen mother cells in meiosis appeared abnormal phenomenon is more serious, for pachytene chromosomes cannot synapsis, metaphase I and metaphase II the emergence of a large number of deviating from the board and the unit body. Chromosomes during anaphase I and anaphase II appear lagging chromosomes and chromosome at Unequal separation conditions, ranging from the size of telophase II into sub cells. According to the statistical analysis, the diploid wild type plants pollen mother cells at diakinesis, metaphase, anaphase I, the proportion of abnormal chromosome behavior in anaphase II was 0%, while the diploid transgenic pollen mother cells at diakinesis, metaphase I later, I, the abnormal rate of anaphase II chromosome behavior were 66.7%, 50%, 33.3%, 27.3%. of tetraploid wild type plants pollen mother cells at diakinesis, metaphase, anaphase I, the proportion of abnormal chromosome behavior in anaphase II were 75%, 16.7%, 0%, 0%. transgenic plants of tetraploid pollen mother cells in the end variable period, metaphase, anaphase I, the proportion of abnormal chromosome behavior in anaphase II were 84.2%, 33.3%, 33.3%, 28.6%. this series of chromosome abnormal behavior is the main factors of transgenic pollen viability decreased and the decline of fertility.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943

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