【作者】 倪丽菊；

【导师】 张似青； 王林云； 高诚；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 本研究从染色体、同工酶及蛋白、DNA三个方面研究了来自湖南、宁夏和黑龙江三地的四类东方田鼠的遗传特性和亲缘关系，旨在为进一步明确我国东方田鼠的亚种分类提供遗传学依据。 采用骨髓细胞直接制片法对四类东方田鼠的染色体进行了初步研究。结果显示：湖南东方田鼠的核型为2n=52=12(m+sm)+38(t+st)+XY(m，t)；宁夏东方田鼠的核型为2n=52=10(m+sm)+40(t+st)+XY(m，t)；黑龙江大体型东方田鼠的核型为2n=40=18(m+sm)+20(t+st)+XY(t，t)；黑龙江小体型东方田鼠的核型为2n=40=16(m+sm)+22(t+st)+XY(m，t)。 应用乙酸纤维素膜电泳法分析了四类东方田鼠的10种同工酶及蛋白，并观察了它们在这些生化基因位点上的基因分布特征以研究它们之间的遗传关系。结果表明：四类东方田鼠在所观察的10个生化位点上，大部分都存在着多态性现象；黑龙江小体型东方田鼠与其它三类东方田鼠的电泳结果差异较大，在Es-1、Hbb、Gpi-1、Akp-1、Trf、Ce-2六个位点土都存在其所特有的等位基因，其中Es-1、Hbb和Gpi-1三位点上它的基因型与其它三类鼠完全不同；湖南、宁夏和黑龙江大体型东方田鼠三者间的遗传距离在0.0633～0.2107之间，而黑龙江小体型东方田鼠与其它三类鼠的遗传距离在0.7068～0.8953之间。四类东方田鼠的UPGMA聚类分析图显示：湖南、宁夏和黑龙江大体型东方田鼠聚为一类(湖南鼠与宁夏鼠首先相聚，再与黑龙江大体型东方田鼠相聚)，亲缘关系相对较近；黑龙江小体型东方田鼠单独为一类，与其它三类鼠的亲缘关系均相对较远。 采用垂直板不连续聚丙烯酰胺凝胶电泳法对四类东方田鼠的酯酶(EST)、苹果酸脱氢酶(MDH)和乳酸脱氢酶(LDH)进行了初步研究。结果表明：(1)东方田鼠的肝EST同工酶活性较强，有着丰富的多态性，四类鼠在Ⅰ区较相近，在Ⅲ区和Ⅳ区差异都较大，在Ⅱ区湖南、宁夏和黑龙江大体型东方田鼠较为相似而黑龙江小体型东方田鼠与它们差别较大。(2)四类东方田鼠的肝MDH同工酶变异不大，MDHm都在同一泳动线上且酶活性相似，而MDHs虽然泳动速度都一致但宁夏鼠的酶活性较其它三类鼠弱。(3)四类东方田鼠的肝脏中都只含LDH5；肾脏中，除黑龙江小体型东方田鼠缺乏LDH4和LDH5外，其余三类东方田鼠都含五种LDH同工酶。 应用随机扩增多态DNA(RAPD)技术分析了四类东方田鼠的基因组DNA。RAPD的分析结果表明：四类东方田鼠各类群内个体间的相似性都较高，湖南、宁夏、黑龙江四种群东方田鼠的遗传特性研究大体型东方田鼠以及黑龙江小体型东方田鼠的类群内平均带纹相似系数(ABS)分别为0.9287士0.0436、0.9223士0.0533、0.9515士0.0289、0.8900士0.0328:湖南、宁夏和黑龙江大体型东方田鼠三类鼠之间的遗传相似系数都达0.9以上，遗传距离都小于0.1，黑龙江小体型东方田鼠与其它三类鼠的相似性较低，遗传相似系数在0.6762-0.7203之间，遗传距离在0.3281一0、3913之间;基于遗传距离的UPGMA聚类分析图显示湖南、宁夏和黑龙江大体型东方田鼠聚为一类，黑龙江小体型东方田鼠单独为一类;引物52022可以作为区分黑龙江小体型东方田鼠与其它三类东方田鼠的标记引物，其扩增出的两个特异性DNA片段可以初步作为区分它们的DNA标记。更多还原

【Abstract】 In order to provide genetics basis for the classification of the subspecies of Microtus fortis in china, the chromosome, isozyme, protein, DNA were used to study the genetic characteristics and phylogenetic relationship of four groups of close-bred Microtus fortis from three regions -Hunan, Ningxia as well as Heilongjiang.We observed the chromosomes of the bone marrow cells from four groups of Microtus fortis. The formulas of karyotype were as follows: for voles from Hunan, 2n = 52 = 12( m + sm ) + 38( t + st) + XY ( m, t); for voles from Ningxia, 2n = 52 = 10( m + sm ) + 40( t + st) + XY( m, t); for big voles from Heilongjiang, 2n = 40 = 18( m + sm ) + 20( t + st ) + XY( t, t); for small voles from Heilongjiang, 2n = 40 = 16( m + sm ) + 22( t + st) + XY( m,t).Ten isozymes and proteins were investigated by cellulose acetate membrane electrophoresis, the phylogenetic relationship among four groups of Microtus fortis was analyzed based on the alleles frequency at these biochemical loci. All groups of Microtus fortis showed polymorphism at most of the ten loci investigated. The electrophoretic mobilities of the small voles from Heilongjiang revealed great divergence with the other three groups: the small voles from Heilongjiang had their particular alleles at six loci (Es-1, Hbb, Gpi, Akp-1, Trf-1, Ce-2) and the phenotypes of them were completely different at three (Es-1, Hbb, Gpi-1) of these loci. The genetic distances among the voles from Hunan, Ningxia and the big voles from Heilongjiang were ranged from 0.0633 to 0.2107 while that between the small voles from Heilongjiang and the other three groups were ranged from 0.7068 to 0.8953. The phylogenetic tree constructed by UPGMA showed that: the voles from Hunan, Ningxia and the big voles from Heilongjiang could be gathered in one group (the voles from Hunan and Ningxia gathered firstly, then gathered to the big voles from Heilongjiang ), but the small voles from Heilongjiang were in the other group alone. These results indicated that the phylogenetic relationship among the voles from Hunan, Ningxia and the big voles from Heilongjiang is closer than that between the small voles from Heilongjiang and them.We present a preliminary study on the zymograms of esterase (EST), lactatedehydrogenase (LDH), malate dehydrogenase (MDH) from four groups of Microtus fortis by vertical discontinous polyacrylamide gel electrophoresis method. The number, mobility and dye intensity of the enzyme band were analyzed. The results revealed that: (1) The zymogram of EST isozyme in the livers from all Microtus fortis existed widespread polymorphism. They were similar to each other at the I area while quite different at the III and IV area. At the II area, the voles from Hunan, Ningxia and the big voles from Heilongjiang were close but the difference between the small voles from Heilongjiang and three other groups was obviously. (2) The liver MDH isozymes of Microtus fortis were rather conservation in evolution. The MDHm zones of all voles were located to the same migratory line with the similar activity. The migratory rates of the MDHs zones from all voles were identical but the activity of MDHs of the voles from Ningxia was weaker than that of the other voles. (3) There was only LDH5 in the livers of all groups of Microtus fortis. The LDH4 and LDH5 were absent in the kidneys of the small voles from Heilongjiang, but five kinds of LDH isozymes were all found in the kidneys of the other three groups.By means of the random amplified polymorphic DNA (RAPD) technique we analyzed the genome DNA from four groups of Microtus fortis. The RAPD results indicated as the following: The average band similarity index ( ABS) within each group of Microtus fortis is high: for voles from Hunan, 0.9287+0.0436; for voles from Ningxia, 0.9223+0.0533; for big voles from Heilongjiang, 0.9515 + 0.0289; for small voles from Heilongjiang, 0.8900 + 0.0328. The genetic similarity coefficients (I) among the voles from Hunan, Ningxia and the big voles from Heilongjiang were all up to 0更多还原