本文選題：水稻 + 染色體涂染��； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：染色體涂染技術(shù)即將整條染色體、某條染色體臂(長(zhǎng)臂或短臂)或者染色體某個(gè)片段的DNA制備成探針,通過(guò)熒光原位雜交(FISH)的方法,將探針雜交到染色體上,從而分析和研究染色體的重組、畸變和同源基因。本研究中,我們以水稻為供試材料,通過(guò)生物信息學(xué)篩選出了日本晴第9號(hào)染色體上25000個(gè)的寡核苷酸構(gòu)建成文庫(kù),并將其標(biāo)記為寡核苷酸探針(oligoprobe)。通過(guò)FISH方法,觀察了日本晴、Chr11L·9L易位系、2個(gè)涉及第9號(hào)染色體的非整倍體材料以及基因組為AAC的異源三倍體材料中有絲分裂及減數(shù)分裂過(guò)程中第九號(hào)染色體,揭示了 oligopainting在水稻中的應(yīng)用價(jià)值,主要研究結(jié)果如下:1、在本研究中,我們?cè)O(shè)計(jì)了能夠覆蓋于日本晴第9號(hào)染色體0-23Mb的寡核苷酸文庫(kù),包含25000個(gè)寡核苷酸,每個(gè)寡核苷酸包含45個(gè)堿基,然后經(jīng)過(guò)乳化PCR擴(kuò)增、轉(zhuǎn)錄、反轉(zhuǎn)錄等步驟標(biāo)記為第9號(hào)染色體寡核苷酸探針(oligo-chr9)。2、oligo-chr9能夠清晰準(zhǔn)確地與日本晴有絲分裂和減數(shù)分裂細(xì)胞中的第9號(hào)染色體雜交,并且能夠追蹤第9號(hào)染色體在分裂過(guò)程各個(gè)時(shí)期的形態(tài)變化。結(jié)果顯示,該寡核苷酸探針可以應(yīng)用于水稻細(xì)胞學(xué)的相關(guān)研究。3、在本研究中,運(yùn)用oligo-chr9對(duì)一個(gè)已知的Chr11L · 9L易位系進(jìn)行驗(yàn)證,探究基于寡核苷酸探針的涂染技術(shù)在染色體結(jié)構(gòu)變異方面的應(yīng)用價(jià)值。結(jié)果顯示,在該易位系中,oligo-chr9探針能夠分別與兩對(duì)染色體的長(zhǎng)臂和短臂進(jìn)行特異雜交,并且結(jié)合CentO探針和5SrDNA探針,說(shuō)明第9號(hào)染色體著絲粒區(qū)域發(fā)生不均等斷裂,11號(hào)染色體在5SrDNA區(qū)域發(fā)生斷裂,形成了兩對(duì)新染色體,Chr11L·9L和Chr9S·11S,驗(yàn)證了之前的研究結(jié)果,同時(shí)說(shuō)明該oligo-chr9探針可以應(yīng)用于秈稻品種。4、利用oligo-chr9探針對(duì)本實(shí)驗(yàn)室若干染色體數(shù)目異常的水稻材料進(jìn)行鑒定,最終鑒定出有兩個(gè)與第9號(hào)染色體有關(guān)的非整倍體材料,Y6077和Y6089。兩者均呈第9號(hào)染色體增多的現(xiàn)象。其中Y6077為增加1條小染色體,由2個(gè)第9號(hào)染色體短臂所組成的等臂染色體,但其中一個(gè)短臂末端的45SrDNA區(qū)域缺失。Y6089增加了 4條染色體,其中兩條為第9號(hào)染色體短臂形成的等臂染色體,但這2條染色體的著絲粒片段大小不一。5、在基因組為AAC的異源三倍體材料中,我們運(yùn)用oligo-chr9探針與該材料的根尖細(xì)胞染色體做熒光原位雜交,結(jié)果顯示第9號(hào)染色體在A組和C組中具有同源性,是相對(duì)保守的。運(yùn)用oligo-chr9探針追蹤該材料在減數(shù)分裂過(guò)程中第9號(hào)染色體的形態(tài)變化,發(fā)現(xiàn)在異源三倍體AAC的偶線期細(xì)胞中,A組的2條第9號(hào)染色體和C組的1條第9號(hào)染色體有兩種配對(duì)形式:一是3條染色體都參與配對(duì),形成類似三價(jià)體的結(jié)構(gòu),但是在粗線期、雙線期以及終變期細(xì)胞中均未觀察到這3條染色體形成的三價(jià)體;二是3條染色體中,A組的兩條第9號(hào)染色配對(duì),而C組的該染色體不參與配對(duì),呈單價(jià)體。A組的2條9號(hào)染色體分離在后期I分離,C組第9號(hào)染色體呈單價(jià)體。
[Abstract]:The chromosome smear technique makes a probe from the entire chromosome, a chromosome arm (long arm or short arm) or a piece of DNA from a chromosome, and hybridizes the probe onto the chromosome by fluorescence in situ hybridization (fish). Therefore, the recombination, aberration and homology of chromosomes were analyzed and studied. In this study, we selected 25000 oligonucleotides on Japanese clear chromosome 9 by bioinformatics, and labeled them as oligonucleotide probe (oligoprobe). Fish method was used to observe chromosome 9 in the process of mitosis and meiosis in Chr11L9L translocation lines, two aneuploidy materials related to chromosome 9 and aneutriploid materials with AAC genome. The application value of oligopainting in rice was revealed. The main results were as follows: 1. In this study, we designed an oligonucleotide library covering 0-23Mb on Japanese clear chromosome 9, containing 25000 oligonucleotides. Each oligonucleotide contains 45 bases and is then amplified by emulsified PCR and transcribed. Reverse transcription and other steps labeled as chromosome 9 oligodeoxynucleotide probe (oligo-chr9) .2olioligo-chr9 can clearly and accurately hybridize with chromosome 9 in Japanese mitotic and meiotic cells. And it was able to track the morphological changes of chromosome 9 at all stages of the division. The results showed that the oligonucleotide probe could be applied to the study of rice cytology. In this study, oligo-chr9 was used to verify a known Chr11L9L translocation line. To explore the application value of smear technique based on oligonucleotide probe in chromosome structure variation. The results showed that the oligo-chr9 probe could be specifically hybridized with the long and short arms of two pairs of chromosomes, and combined with CentO probe and 5s rDNA probe, respectively. The results indicate that the centromere region of chromosome 9 is not evenly broken, and chromosome 11 breaks in the region of 5s rDNA, forming two pairs of new chromosomes, Chr11L9L and Chr9S11S. the results of previous studies are verified. At the same time, the oligo-chr9 probe can be applied to indica rice variety .4.The oligo-chr9 probe was used to identify some rice materials with abnormal chromosome number in our laboratory. Finally, two aneuploidy materials related to chromosome 9 were identified: Y6077 and Y6089. Both showed an increase in chromosome 9. In order to add one small chromosome, Y6077 consists of two isobaric chromosomes composed of two short arms of chromosome 9, but the deletion of 45s rDNA region at the end of one of the two chromosomes, Y6089, is increased by 4 chromosomes. Two of them were isobaric chromosomes formed by the short arm of chromosome 9, but the centromere fragments of these two chromosomes were of different sizes. Oligo-chr9 probe was used to perform fluorescence in situ hybridization with the root tip cell chromosomes of the material. The results showed that chromosome 9 had homology in group A and group C and was relatively conserved. Oligo-chr9 probe was used to track the morphological changes of chromosome 9 during meiosis. It was found that two chromosome 9 of group A and one chromosome 9 of group C had two types of pairing in the even-line phase of allotriploid AAC: one was that all three chromosomes were involved in the pairing, forming a trivalent-like structure. However, the trivalents formed by these three chromosomes were not observed in the cells of the coarse-line phase, the double-line phase and the terminal stage, and the second was the pair of two chromosome No. 9 in group A of the three chromosomes, but the chromosome in group C was not involved in the pairing. Chromosome 9 of group A was univalently separated from chromosome 9 of group C at anaphase I.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S511
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本文編號(hào)：2083716