造孢細(xì)胞在雄性桑資源染色體鑒定中的應(yīng)用及FISH分析
發(fā)布時(shí)間：2019-03-23 15:44
【摘要】：桑樹是桑屬(Morus L.)中的多年生木本植物。桑樹作為一種重要的生態(tài)經(jīng)濟(jì)林木,在我國(guó)有悠久的栽種歷史。桑樹的細(xì)胞學(xué)研究始于上世紀(jì)初,以染色體數(shù)目的研究為重,核型分析較少。染色體作為遺傳物質(zhì)的載體,承擔(dān)著傳遞遺傳信息的重要作用。對(duì)桑樹染色體的核型分析,可以得到其細(xì)胞學(xué)數(shù)據(jù),進(jìn)而對(duì)桑樹分類與育種工作提供幫助,同時(shí)對(duì)桑樹的起源、系統(tǒng)演化以及親緣關(guān)系研究也具有重要意義。桑樹的染色體不但形態(tài)較小,而且數(shù)目較多,采用傳統(tǒng)的壓片法制備染色體的效果不理想,這使得桑樹細(xì)胞學(xué)的研究受到極大地限制。去壁低滲法的應(yīng)用,促進(jìn)了桑樹細(xì)胞學(xué)的研究。在桑樹染色體倍性研究中常用的材料主要有愈傷組織、幼葉、幼芽、根尖和莖尖等。桑樹營(yíng)養(yǎng)器官細(xì)胞分裂指數(shù)低的特點(diǎn)使得采用上述常用的實(shí)驗(yàn)材料得到的中期染色體分裂相較少,阻礙了進(jìn)一步研究。造孢細(xì)胞是由孢原細(xì)胞在植物造孢期經(jīng)多次有絲分裂形成,其數(shù)量多并且分裂活力旺盛。造孢細(xì)胞經(jīng)8-羥基喹啉處理后容易得到中期染色體的分裂相,并且分裂相更清晰。造孢細(xì)胞因其自身獨(dú)特的特點(diǎn),使其成為桑樹染色體研究的可選材料之一。本研究采用幼葉、雄花造孢細(xì)胞為實(shí)驗(yàn)材料制備桑樹染色體中期分裂相的樣本,對(duì)多種桑樹種質(zhì)資源進(jìn)行了數(shù)目及倍性分析。并選取7種四倍體桑樹種質(zhì)資源,以造孢細(xì)胞為材料制備染色體中期樣本,25S rDNA重復(fù)序列為探針進(jìn)行熒光原位雜交實(shí)驗(yàn),對(duì)25S rDNA重復(fù)序列位點(diǎn)在這些四倍體桑樹種質(zhì)資源的數(shù)目進(jìn)行了研究,從而推測(cè)在桑樹中25S rDNA重復(fù)序列的位點(diǎn)數(shù)目能否作為桑樹同源四倍體或者異源四倍體的判斷依據(jù)。本研究所獲結(jié)果如下:1、以幼葉為材料的桑樹染色體數(shù)目及倍性分析取生長(zhǎng)最旺盛的幼葉為材料,利用酶解去壁低滲法進(jìn)行樣片的制備。用8-羥基喹啉于室溫條件下對(duì)幼葉進(jìn)行避光預(yù)處理3h,以體積比為1:1的5%的纖維素酶和5%的果膠酶的混合液進(jìn)行酶解3h,得到效果最好的樣片。以此方法,分析了23種桑樹種質(zhì)資源的染色體數(shù)目,發(fā)現(xiàn)其體細(xì)胞染色體數(shù)目有28、35、42、84條四類,且以具有28條染色體的最多。2、以雄花造孢細(xì)胞為材料的桑樹染色體數(shù)目及倍性分析取桑樹萌芽期雄花為材料,利用酶解去壁低滲法制備染色體樣片。用上述同樣的方法進(jìn)行預(yù)處理,但酶解處理時(shí)間延長(zhǎng)到6h,得到效果最好的樣片。通過(guò)對(duì)分析了11種雄性桑種質(zhì)資源的染色體數(shù)目分析,發(fā)現(xiàn)有9種桑樹種質(zhì)資源含有28條染色體,有一種桑樹種質(zhì)資源有42條染色體。另外,首次發(fā)現(xiàn)了擁有126條染色體的桑樹種質(zhì)資源。3、25S rDNA重復(fù)序列在桑樹資源染色體上的FISH分析選擇云南野生雄性桑樹種質(zhì)資源(3號(hào)、4號(hào)、5號(hào)、12號(hào)、14號(hào))、農(nóng)桑14、烏皮桑7種四倍體桑樹的造孢細(xì)胞為材料制備中期染色體樣片,以25S r DNA重復(fù)序列為探針,利用熒光原位雜交技術(shù)對(duì)其進(jìn)行細(xì)胞學(xué)研究。分析結(jié)果發(fā)現(xiàn),這些桑樹資源的25S rDNA重復(fù)序列位點(diǎn)均為2個(gè),初步推測(cè)25S r DNA雜交信號(hào)數(shù)目可能不能作為判斷依據(jù)來(lái)鑒定桑樹四倍體材料的同源或異源性。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S888.2
本文編號(hào)：2445975
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S888.2
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