Oligopainting在水稻染色体识别中的应用

发布时间：2018-06-29 22:58

  本文选题：水稻 + 染色体涂染　； 参考：《扬州大学》2017年硕士论文

【摘要】：染色体涂染技术即将整条染色体、某条染色体臂(长臂或短臂)或者染色体某个片段的DNA制备成探针,通过荧光原位杂交(FISH)的方法,将探针杂交到染色体上,从而分析和研究染色体的重组、畸变和同源基因。本研究中,我们以水稻为供试材料,通过生物信息学筛选出了日本晴第9号染色体上25000个的寡核苷酸构建成文库,并将其标记为寡核苷酸探针(oligoprobe)。通过FISH方法,观察了日本晴、Chr11L·9L易位系、2个涉及第9号染色体的非整倍体材料以及基因组为AAC的异源三倍体材料中有丝分裂及减数分裂过程中第九号染色体,揭示了 oligopainting在水稻中的应用价值,主要研究结果如下:1、在本研究中,我们设计了能够覆盖于日本晴第9号染色体0-23Mb的寡核苷酸文库,包含25000个寡核苷酸,每个寡核苷酸包含45个碱基,然后经过乳化PCR扩增、转录、反转录等步骤标记为第9号染色体寡核苷酸探针(oligo-chr9)。2、oligo-chr9能够清晰准确地与日本晴有丝分裂和减数分裂细胞中的第9号染色体杂交,并且能够追踪第9号染色体在分裂过程各个时期的形态变化。结果显示,该寡核苷酸探针可以应用于水稻细胞学的相关研究。3、在本研究中,运用oligo-chr9对一个已知的Chr11L · 9L易位系进行验证,探究基于寡核苷酸探针的涂染技术在染色体结构变异方面的应用价值。结果显示,在该易位系中,oligo-chr9探针能够分别与两对染色体的长臂和短臂进行特异杂交,并且结合CentO探针和5SrDNA探针,说明第9号染色体着丝粒区域发生不均等断裂,11号染色体在5SrDNA区域发生断裂,形成了两对新染色体,Chr11L·9L和Chr9S·11S,验证了之前的研究结果,同时说明该oligo-chr9探针可以应用于籼稻品种。4、利用oligo-chr9探针对本实验室若干染色体数目异常的水稻材料进行鉴定,最终鉴定出有两个与第9号染色体有关的非整倍体材料,Y6077和Y6089。两者均呈第9号染色体增多的现象。其中Y6077为增加1条小染色体,由2个第9号染色体短臂所组成的等臂染色体,但其中一个短臂末端的45SrDNA区域缺失。Y6089增加了 4条染色体,其中两条为第9号染色体短臂形成的等臂染色体,但这2条染色体的着丝粒片段大小不一。5、在基因组为AAC的异源三倍体材料中,我们运用oligo-chr9探针与该材料的根尖细胞染色体做荧光原位杂交,结果显示第9号染色体在A组和C组中具有同源性,是相对保守的。运用oligo-chr9探针追踪该材料在减数分裂过程中第9号染色体的形态变化,发现在异源三倍体AAC的偶线期细胞中,A组的2条第9号染色体和C组的1条第9号染色体有两种配对形式:一是3条染色体都参与配对,形成类似三价体的结构,但是在粗线期、双线期以及终变期细胞中均未观察到这3条染色体形成的三价体;二是3条染色体中,A组的两条第9号染色配对,而C组的该染色体不参与配对,呈单价体。A组的2条9号染色体分离在后期I分离,C组第9号染色体呈单价体。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
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本文编号：2083716