梅花染色体制片技术优化及基于荧光原位杂交的核型分析
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摘要
植物染色体研究是基因定位、构建物理图谱的重要前提。目前,梅花的核型分析主要通过常规制片法实现,而利用细胞生物学手段进行核型分析还未见报道。一方面由于木本材料的制约,限制了其取材范围。另一方面梅花属小染色体植物,其制片效果还有待改善。本研究对现有梅花染色体制片技术进行改良,并通过制片材料选取,探索梅花的染色体周年性制片技术。首次采用玉米45SrDNA序列作为探针,利用荧光原位杂交(FISH)技术,将探针定位于梅花中期染色上,并基于荧光原位杂交结果进行核型分析。主要研究结果如下：
1、拓展了梅花染色体制片取材类型,除了传统的茎尖取材,还利用梅花未成熟种子、组织培养获得的伸长胚根、一年生实生苗顶端生长点等分生组织,均获得了分散程度高、清晰的染色体制片。
2、经改良的去壁低渗火焰干燥制片法,省略预处理过程,直接进行卡诺低温固定,有效拉伸了染色体长度。这一改良有利于FISH试验结果的显微观察,并提高制片效率。
3、玉米45SrDNA探针在梅花中其染色体中发现有6个信号点,主要分布于第1、3、7号染色体上,不同品种略有差异。信号点主要分布于染色体长臂末端,结合染色体核型参数,推测梅花从进化上看属较原始类型。
荧光原位杂交技术在梅花中的成功探索,为其精确核型分析、基因定位、物理图谱构建等方面的研究,奠定了一定的技术基础,具有较重要的应用价值。
The chromosome study of plants is an important prerequisite of gene location and physical mapping. The karyotype analysis of Prunus mume is commonly achieved by regular method of chromosome sectioning. However, using the cell-biology methods to achieve the karyotype has not been published yet. One reason is that P.mume is a kind of woody plants. The other reason is chromosomes of P.mume are extremely small and short. Thus, the chromosome sectioning method needs to be improved. In the study, the preferable method of chromosome sectioning was picked out. Besides, different kinds of materials were tried in order to make chromosome sectioning of P.m ume all the year around possible. Meanwhile, it was the first time to localize the maize45SrDNA on metaphase chromosomes of P. mume by fluorescence in situ hybridization (FISH) and Karyotype analysis was achieved based on FISH. The main results showed as follows:
1. The limitation of materials in chromosome sectioning of P. mume was broke.There were three other materials, including immature seeds, growing points by root tissue culture, growing points of one-year-old seedlings.They were all proved to be served as materials as well.
2. The pretreatment is eliminated in the improved flame and air-dried method of sectioning.The materials were put in fixed acetic acid-alcohol directly when they were obtained. This process maed the chromosomes stretch to a great extent. Besides, it was convient for FISH observing and improved the efficiency of chromosome sectioning.
3. Three pairs of45SrDNA signals were detected in all metaphases examined, which were mainly distributed on Chormosomel,3and7. The situation may different in different varieties. The results provided some evidence to classification of P.mume.45SrDNA signals were all located at the distal of the long arms of chromosomes. Combining with karyotype parameter, it can be concluded that the evolution of P.mume. was quite low.
The fluorescence in situ hybridization was successfully applied to P. mume. It was meaningful and helpful to make exact karyotype analysis, gene locations and physical mapping.
1、拓展了梅花染色体制片取材类型,除了传统的茎尖取材,还利用梅花未成熟种子、组织培养获得的伸长胚根、一年生实生苗顶端生长点等分生组织,均获得了分散程度高、清晰的染色体制片。
2、经改良的去壁低渗火焰干燥制片法,省略预处理过程,直接进行卡诺低温固定,有效拉伸了染色体长度。这一改良有利于FISH试验结果的显微观察,并提高制片效率。
3、玉米45SrDNA探针在梅花中其染色体中发现有6个信号点,主要分布于第1、3、7号染色体上,不同品种略有差异。信号点主要分布于染色体长臂末端,结合染色体核型参数,推测梅花从进化上看属较原始类型。
荧光原位杂交技术在梅花中的成功探索,为其精确核型分析、基因定位、物理图谱构建等方面的研究,奠定了一定的技术基础,具有较重要的应用价值。
The chromosome study of plants is an important prerequisite of gene location and physical mapping. The karyotype analysis of Prunus mume is commonly achieved by regular method of chromosome sectioning. However, using the cell-biology methods to achieve the karyotype has not been published yet. One reason is that P.mume is a kind of woody plants. The other reason is chromosomes of P.mume are extremely small and short. Thus, the chromosome sectioning method needs to be improved. In the study, the preferable method of chromosome sectioning was picked out. Besides, different kinds of materials were tried in order to make chromosome sectioning of P.m ume all the year around possible. Meanwhile, it was the first time to localize the maize45SrDNA on metaphase chromosomes of P. mume by fluorescence in situ hybridization (FISH) and Karyotype analysis was achieved based on FISH. The main results showed as follows:
1. The limitation of materials in chromosome sectioning of P. mume was broke.There were three other materials, including immature seeds, growing points by root tissue culture, growing points of one-year-old seedlings.They were all proved to be served as materials as well.
2. The pretreatment is eliminated in the improved flame and air-dried method of sectioning.The materials were put in fixed acetic acid-alcohol directly when they were obtained. This process maed the chromosomes stretch to a great extent. Besides, it was convient for FISH observing and improved the efficiency of chromosome sectioning.
3. Three pairs of45SrDNA signals were detected in all metaphases examined, which were mainly distributed on Chormosomel,3and7. The situation may different in different varieties. The results provided some evidence to classification of P.mume.45SrDNA signals were all located at the distal of the long arms of chromosomes. Combining with karyotype parameter, it can be concluded that the evolution of P.mume. was quite low.
The fluorescence in situ hybridization was successfully applied to P. mume. It was meaningful and helpful to make exact karyotype analysis, gene locations and physical mapping.
引文
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