造孢細胞在雄性桑資源染色體鑒定中的應用及FISH分析
發(fā)布時間：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)經濟林木,在我國有悠久的栽種歷史。桑樹的細胞學研究始于上世紀初,以染色體數目的研究為重,核型分析較少。染色體作為遺傳物質的載體,承擔著傳遞遺傳信息的重要作用。對桑樹染色體的核型分析,可以得到其細胞學數據,進而對桑樹分類與育種工作提供幫助,同時對桑樹的起源、系統(tǒng)演化以及親緣關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,這使得桑樹細胞學的研究受到極大地限制。去壁低滲法的應用,促進了桑樹細胞學的研究。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。桑樹營養(yǎng)器官細胞分裂指數低的特點使得采用上述常用的實驗材料得到的中期染色體分裂相較少,阻礙了進一步研究。造孢細胞是由孢原細胞在植物造孢期經多次有絲分裂形成,其數量多并且分裂活力旺盛。造孢細胞經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。造孢細胞因其自身獨特的特點,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、雄花造孢細胞為實驗材料制備桑樹染色體中期分裂相的樣本,對多種桑樹種質資源進行了數目及倍性分析。并選取7種四倍體桑樹種質資源,以造孢細胞為材料制備染色體中期樣本,25S rDNA重復序列為探針進行熒光原位雜交實驗,對25S rDNA重復序列位點在這些四倍體桑樹種質資源的數目進行了研究,從而推測在桑樹中25S rDNA重復序列的位點數目能否作為桑樹同源四倍體或者異源四倍體的判斷依據。本研究所獲結果如下:1、以幼葉為材料的桑樹染色體數目及倍性分析取生長最旺盛的幼葉為材料,利用酶解去壁低滲法進行樣片的制備。用8-羥基喹啉于室溫條件下對幼葉進行避光預處理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種桑樹種質資源的染色體數目,發(fā)現其體細胞染色體數目有28、35、42、84條四類,且以具有28條染色體的最多。2、以雄花造孢細胞為材料的桑樹染色體數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行預處理,但酶解處理時間延長到6h,得到效果最好的樣片。通過對分析了11種雄性桑種質資源的染色體數目分析,發(fā)現有9種桑樹種質資源含有28條染色體,有一種桑樹種質資源有42條染色體。另外,首次發(fā)現了擁有126條染色體的桑樹種質資源。3、25S rDNA重復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資源(3號、4號、5號、12號、14號)、農桑14、烏皮桑7種四倍體桑樹的造孢細胞為材料制備中期染色體樣片,以25S r DNA重復序列為探針,利用熒光原位雜交技術對其進行細胞學研究。分析結果發(fā)現,這些桑樹資源的25S rDNA重復序列位點均為2個,初步推測25S r DNA雜交信號數目可能不能作為判斷依據來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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S888.2
本文編號：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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S888.2
【參考文獻】
相關期刊論文 前10條
1 李楊;杜偉;楊光偉;向仲懷;何寧佳;;2份蒙桑和長穗桑野生種質資源的染色體數目觀察[J];蠶業(yè)科學;2014年06期
2 付文炎;劉義飛;黃宏文;;熒光原位雜交技術在植物多倍體起源與進化研究中的應用[J];熱帶亞熱帶植物學報;2014年03期
3 李楊;軒亞輝;王圣;曾其偉;向仲懷;何寧佳;;滇桑(Morus yunnanensis)的染色體核型分析[J];蠶業(yè)科學;2014年02期
4 何寧佳;向仲懷;;桑樹基因組計劃[J];蠶業(yè)科學;2014年01期
5 趙海燕;黃晉玲;;棉花雄性不育材料亞棉A的小孢子敗育研究[J];中國農業(yè)科學;2012年20期
6 何寧佳;趙愛春;秦儉;曾其偉;向仲懷;;桑樹基因組計劃與桑樹產業(yè)[J];蠶業(yè)科學;2012年01期
7 秦儉;何寧佳;黃先智;向仲懷;;桑樹生態(tài)產業(yè)與蠶絲業(yè)的發(fā)展[J];蠶業(yè)科學;2010年06期
8 付琳;徐鳳霞;曾慶文;;觀光木的小孢子發(fā)生及雄配子體發(fā)育的研究[J];熱帶亞熱帶植物學報;2009年05期
9 林天寶;李有貴;呂志強;朱燕;;桑樹資源綜合利用研究進展[J];蠶桑通報;2008年03期
10 孔芳;蔣金金;吳磊;王幼平;;利用原位雜交及CAPS標記分析蕓薹屬A、B和C基因組間的關系[J];作物學報;2008年07期
相關會議論文 前2條
1 唐冰霞;王英;高和瓊;莊南生;;巴西橡膠樹45S rDNA和5S rDNA的熒光原位雜交分析[A];2014年中國作物學會學術年會論文集[C];2014年
2 鄭金雙;侯喜林;張蜀寧;;FISH和GISH應用于二倍體及同源四倍體不結球白菜核型[A];2012年園藝植物染色體倍性操作與遺傳改良學術研討會論文摘要集[C];2012年
相關博士學位論文 前1條
1 云嵐;新麥草多倍體誘導及細胞學研究[D];內蒙古農業(yè)大學;2009年
相關碩士學位論文 前3條
1 李楊;桑屬植物染色體倍性研究及FISH分析[D];西南大學;2015年
2 肖水平;棉屬4個四倍體種的熒光原位雜交研究[D];中國農業(yè)科學院;2010年
3 程曉蕾;石蒜與忽地笑熒光原位雜交研究[D];南京林業(yè)大學;2006年
，本文編號：2445975
本文鏈接：http://sikaile.net/shoufeilunwen/zaizhiyanjiusheng/2445975.html
