發(fā)布時(shí)間：2018-01-07 13:04

  本文關(guān)鍵詞：SPO11-1調(diào)控?cái)M南芥同源多倍體減數(shù)分裂的細(xì)胞遺傳學(xué)研究　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 減數(shù)分裂 SPO11-1 RNA干擾 同源多倍體 擬南芥

【摘要】：多倍化是植物進(jìn)化過程中的一種普遍現(xiàn)象,這一現(xiàn)象不僅是推動(dòng)植物進(jìn)化的重要力量,也是新物種形成的重要途徑。據(jù)推測(cè),高達(dá)70%的被子植物在進(jìn)化過程中,都至少經(jīng)歷過一次多倍化。與二倍體物種相比,多倍體在很多方面都展現(xiàn)出明顯的優(yōu)勢(shì),如更強(qiáng)的抗逆性、適應(yīng)性等。基于多倍體在植物界的主導(dǎo)地位,有很多研究都集中于多倍化對(duì)植物遺傳及表觀遺傳的影響。但是多倍化對(duì)減數(shù)分裂相關(guān)基因突變后的影響研究較少。減數(shù)分裂是一種特殊的細(xì)胞分裂方式,在這一過程中,會(huì)發(fā)生遺傳物質(zhì)的濃縮、配對(duì)、聯(lián)會(huì)、重組等一系列受到嚴(yán)格調(diào)控的重要事件,任何一個(gè)事件出現(xiàn)差錯(cuò)都會(huì)導(dǎo)致減數(shù)分裂出現(xiàn)缺陷甚至提前終止。多倍體植物含有兩套以上的染色體組,與二倍體相比,具有更高的重組頻率和更復(fù)雜的聯(lián)會(huì)方式。因此,深入研究多倍體植物減數(shù)分裂同源重組過程中所涉及到的相關(guān)基因具有十分重要的意義。鑒于此,本實(shí)驗(yàn)充分利用分子和細(xì)胞遺傳學(xué)手段,研究SPO11-1在同源四倍體擬南芥減數(shù)分裂同源重組中的功能,研究結(jié)果如下:1、針對(duì)擬南芥SPO11-1基因中的特異序列設(shè)計(jì)干擾片段,經(jīng)酶切鑒定干擾片段成功定向插入載體,成功構(gòu)建了RNA干擾載體SPO11-pHellsgate2。2、通過floral-dip法轉(zhuǎn)化擬南芥,經(jīng)過卡那霉素初步篩選及PCR分子檢測(cè),成功獲得了二倍體及同源四倍體轉(zhuǎn)基因植株。通過熒光定量PCR檢測(cè),二倍體轉(zhuǎn)基因植株SPO11-1基因的表達(dá)水平僅為二倍體野生型擬南芥的46%。四倍體轉(zhuǎn)基因植株SPO11-1基因的表達(dá)水平僅為野生型同源四倍體擬南芥的40%。3、二倍體、四倍體轉(zhuǎn)基因擬南芥與其所對(duì)應(yīng)的野生型植株在形態(tài)學(xué)與營(yíng)養(yǎng)生長(zhǎng)的特征上沒有顯著差別,具體表現(xiàn)為,在葉片面積、葉片形態(tài)、生長(zhǎng)周期等方面,轉(zhuǎn)基因植株與野生型植株無(wú)明顯差別。4、二倍體、四倍體轉(zhuǎn)基因植株生殖生長(zhǎng)過程出現(xiàn)缺陷,育性顯著下降,具體表現(xiàn)為:1)轉(zhuǎn)基因植株的果莢長(zhǎng)度明顯縮短,且果莢內(nèi)部可見大量發(fā)育不良或敗育的胚珠,缺�，F(xiàn)象明顯。二倍體轉(zhuǎn)基因植株結(jié)實(shí)率比其所對(duì)應(yīng)野生型下降51.8%。四倍體轉(zhuǎn)基因植株結(jié)實(shí)率比對(duì)應(yīng)野生型下降32%。2)花粉亞歷山大染色結(jié)果顯示,二倍體、四倍體轉(zhuǎn)基因植株花藥中所含的花粉減少,且出現(xiàn)較多干癟、皺縮及大小形狀不均一的花粉粒,推測(cè)這些花粉粒是無(wú)活性的花粉粒。二倍體轉(zhuǎn)基因植株異常花粉粒比例比相應(yīng)野生型上升了49.3%,四倍體轉(zhuǎn)基因植株異�；ǚ哿１壤认鄳�(yīng)野生型上升了35%。5、二倍體、四倍體轉(zhuǎn)基因植株的花粉母細(xì)胞在減數(shù)分裂過程都出現(xiàn)了較為嚴(yán)重的異�，F(xiàn)象,表現(xiàn)為粗線期同源染色體不能完全聯(lián)會(huì),中期Ⅰ及中期Ⅱ出現(xiàn)大量偏離赤道板的染色體以及單價(jià)體。后期Ⅰ及后期Ⅱ出現(xiàn)落后染色體,且出現(xiàn)染色體不均等分離的情況,末期Ⅱ分成大小不等的子細(xì)胞。根據(jù)統(tǒng)計(jì)分析,二倍體野生型植株的花粉母細(xì)胞在終變期、中期Ⅰ、后期Ⅰ、后期Ⅱ的染色體行為異常比例均為0%,而二倍體轉(zhuǎn)基因植株的花粉母細(xì)胞在終變期、中期Ⅰ、后期Ⅰ、后期Ⅱ染色體行為的異常比例分別為66.7%、50%、33.3%、27.3%。四倍體野生型植株的花粉母細(xì)胞在終變期、中期Ⅰ、后期Ⅰ、后期Ⅱ的染色體行為異常比例分別為75%、16.7%、0%、0%。四倍體轉(zhuǎn)基因植株的花粉母細(xì)胞在終變期、中期Ⅰ、后期Ⅰ、后期Ⅱ的染色體行為異常比例分別為84.2%、33.3%、33.3%、28.6%。這一系列染色體的異常行為即為轉(zhuǎn)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943

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