造孢细胞在雄性桑资源染色体鉴定中的应用及FISH分析

发布时间：2019-03-23 15:44

【摘要】：桑树是桑属(Morus L.)中的多年生木本植物。桑树作为一种重要的生态经济林木,在我国有悠久的栽种历史。桑树的细胞学研究始于上世纪初,以染色体数目的研究为重,核型分析较少。染色体作为遗传物质的载体,承担着传递遗传信息的重要作用。对桑树染色体的核型分析,可以得到其细胞学数据,进而对桑树分类与育种工作提供帮助,同时对桑树的起源、系统演化以及亲缘关系研究也具有重要意义。桑树的染色体不但形态较小,而且数目较多,采用传统的压片法制备染色体的效果不理想,这使得桑树细胞学的研究受到极大地限制。去壁低渗法的应用,促进了桑树细胞学的研究。在桑树染色体倍性研究中常用的材料主要有愈伤组织、幼叶、幼芽、根尖和茎尖等。桑树营养器官细胞分裂指数低的特点使得采用上述常用的实验材料得到的中期染色体分裂相较少,阻碍了进一步研究。造孢细胞是由孢原细胞在植物造孢期经多次有丝分裂形成,其数量多并且分裂活力旺盛。造孢细胞经8-羟基喹啉处理后容易得到中期染色体的分裂相,并且分裂相更清晰。造孢细胞因其自身独特的特点,使其成为桑树染色体研究的可选材料之一。本研究采用幼叶、雄花造孢细胞为实验材料制备桑树染色体中期分裂相的样本,对多种桑树种质资源进行了数目及倍性分析。并选取7种四倍体桑树种质资源,以造孢细胞为材料制备染色体中期样本,25S rDNA重复序列为探针进行荧光原位杂交实验,对25S rDNA重复序列位点在这些四倍体桑树种质资源的数目进行了研究,从而推测在桑树中25S rDNA重复序列的位点数目能否作为桑树同源四倍体或者异源四倍体的判断依据。本研究所获结果如下:1、以幼叶为材料的桑树染色体数目及倍性分析取生长最旺盛的幼叶为材料,利用酶解去壁低渗法进行样片的制备。用8-羟基喹啉于室温条件下对幼叶进行避光预处理3h,以体积比为1:1的5%的纤维素酶和5%的果胶酶的混合液进行酶解3h,得到效果最好的样片。以此方法,分析了23种桑树种质资源的染色体数目,发现其体细胞染色体数目有28、35、42、84条四类,且以具有28条染色体的最多。2、以雄花造孢细胞为材料的桑树染色体数目及倍性分析取桑树萌芽期雄花为材料,利用酶解去壁低渗法制备染色体样片。用上述同样的方法进行预处理,但酶解处理时间延长到6h,得到效果最好的样片。通过对分析了11种雄性桑种质资源的染色体数目分析,发现有9种桑树种质资源含有28条染色体,有一种桑树种质资源有42条染色体。另外,首次发现了拥有126条染色体的桑树种质资源。3、25S rDNA重复序列在桑树资源染色体上的FISH分析选择云南野生雄性桑树种质资源(3号、4号、5号、12号、14号)、农桑14、乌皮桑7种四倍体桑树的造孢细胞为材料制备中期染色体样片,以25S r DNA重复序列为探针,利用荧光原位杂交技术对其进行细胞学研究。分析结果发现,这些桑树资源的25S rDNA重复序列位点均为2个,初步推测25S r DNA杂交信号数目可能不能作为判断依据来鉴定桑树四倍体材料的同源或异源性。
[Abstract]:Mulberry is a perennial woody plant in the genus Morus L. The mulberry, as an important ecological economic tree, has a long history of planting in China. The cytological study of mulberry began in the beginning of the last century, and the number of chromosomes was the most important, and the karyotype analysis was less. As the carrier of genetic material, the chromosome bears an important role in the transfer of genetic information. The karyotype analysis of the mulberry chromosome can obtain the cytological data of the mulberry, and further provide the help for the classification and breeding of the mulberry, and also has the important significance for the origin, the system evolution and the relationship of the mulberry. The chromosome of the mulberry is not only small, but also the number is more, the effect of the preparation of the chromosome by the traditional tabletting method is not ideal, and the research of the mulberry cytology is greatly limited. The application of the low-permeability method to the wall promotes the study of the cytology of mulberry. The most common materials used in the study of the ploidy of the mulberry are the callus, the young leaf, the young bud, the root tip and the stem tip. The characteristic of the low cell division index of the vegetative organs of the mulberry leaves the medium-term chromosomal division obtained by the above-mentioned commonly used experimental materials to be less, and the further research is hindered. The spore-forming cells are formed by multiple mitosis in the spore-forming period of the sporogenous cell, and the number of the spore-forming cells is large and the division activity is vigorous. The metaphase of the metaphase can be easily obtained by the 8-hydroxytryptamine treatment, and the division phase is more clear. The spore-forming cell is one of the optional materials for the study of the mulberry chromosome because of its unique characteristics. In this study, a sample of the metaphase-metaphase of mulberry was prepared by using young leaf and male flower-making cells as the experimental material, and the number and ploidy analysis of a variety of mulberry germplasm resources were carried out. in that invention, seven tetraploid mulberry germplasm resource are selected, the medium-term sample of the chromosome is prepare by using a spore-making cell as a material, a 25-S rDNA repeat sequence is used as a probe to carry out fluorescence in-situ hybridization experiment, and the number of the 25 S rDNA repeat sequence sites in the tetraploid mulberry germplasm resources is researched, So that the number of loci of the 25S rDNA repeat sequence in the mulberry leaf can be used as the judgment basis for the autotetraploid of the mulberry or the allotetraploid of the mulberry. The results of this study were as follows:1. The number of the mulberry chromosomes and the ploidy analysis of the young leaves are the most vigorous young leaves as the material, and the preparation of the samples by the low-permeability method of the enzymolysis is carried out. Carrying out light-proof pretreatment on the young leaves under the condition of room temperature by using 8-hydroxybenzene, and carrying out enzymolysis for 3 hours at a volume ratio of 1:1 to 5 percent of the cellulase and the mixed solution of 5 percent of the pectase for 3 hours to obtain the best-effect sample. In this method, the chromosome number of 23 mulberry germplasm resources was analyzed, and the number of somatic chromosomes was found to be 28,35,42 and 84, and the number of chromosomes with 28 chromosomes was the most. The chromosome samples were prepared by means of an enzyme-free-wall low-permeability method. The pretreatment was carried out by the same method as described above, but the processing time of the enzyme was prolonged to 6 h to obtain the best-effect sample. Through the analysis of the chromosome number of 11 male mulberry germplasm resources, it was found that there were 9 mulberry germplasm resources containing 28 chromosomes, and there were 42 chromosomes in the mulberry germplasm resources. In addition, for the first time, a mulberry germplasm resource of 126 chromosomes was first discovered. The FISH analysis of the 25 S rDNA repeat sequence on the chromosome of the mulberry resource selected the germplasm resources (3,4,5,12,14) of the wild male mulberry in Yunnan, and the agricultural mulberry 14. In this paper, the medium-term chromosome samples were prepared by using a 25-S r-DNA repeat sequence as a probe, and the cytological study was carried out by using the fluorescence in situ hybridization technique. The results of the analysis showed that the number of the 25 S rDNA repeat sequences of these mulberry resources was 2, and it was preliminarily estimated that the number of 25S r DNA hybrid signals could not be used as the basis of judgment to identify the homologous or heterogenous of the tetraploid of the mulberry.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S888.2

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