摘要
本文以色季拉山的高寒植物垫状雪灵芝为材料,选取分布在海拔4500-4650m范围内的垫状雪灵芝3个居群为研究对象,采用聚丙烯酰胺琼脂电泳技术,对垫状雪灵芝3个居群的48个个体功能叶片的过氧化物酶、细胞色素氧化酶、乙醇脱氢酶、酯酶等同工酶谱带进行测定并编码,依据酶谱带测定结果进行聚类分析;同时结合过氧化物酶、细胞色素氧化酶、乙醇脱氢酶和酯酶等四种同工酶的3个遗传指标进行相关分析。结果表明:
1过氧化物酶同工酶
来自同一居群的个体,不但同工酶谱带相似性高,遗传杂合度小(HeA=0.716;HeB=0.709;HeC=0.705),特别是居群B、C相似性系数大,遗传距离小,这也说明居群内个体遗传分化小;过氧化物酶在不同居群间的相似性系数在四种同工酶中的平均值相对较大,遗传距离相对较小(IAB=0.637;IAC=0.587;IBC=0.646;DAB=0.451;DAC=0.533;DBC=0.437)。这些数据表明垫状雪灵芝过氧化物酶同工酶在不同的居群之间存在一定程度的遗传分化,缺少有效的基因交流。
2细胞色素氧化酶同工酶
垫状雪灵芝的细胞色素氧化酶同工酶在居群内的个体,同工酶谱相似性较高,遗传杂合度比过氧化物酶同工酶较高(HeA=0.752;HeB=0.815;HeC=0.815),在居群A、B、C中基因频率在0.50以上的谱带相对较多,其中A、B居群中各有6条,C居群中有5条,最高值可达到0.94,遗传距离小,这也说明居群内个体遗传分化小;细胞色素氧化酶在不同居群间的相似性系数有最大值和整个种群中四种同工酶的最小值,遗传距离也是如此(IAB=0.418;IAC=0.304;IBC=0.673;DAB=0.873;DAC=1.192;DBC=0.396)。遗传距离最大值是DAC=1.192;这些数据表明垫状雪灵芝细胞色素氧化酶同工酶在不同的居群之间存在较大的遗传分化。
3乙醇脱氢酶同工酶
垫状雪灵芝的乙醇脱氢酶同工酶在居群内的个体,同工酶谱相似性较高,遗传杂合度比细胞色素氧化酶同工酶较小(HeA=0.766;HeB=0.872;HeC=0.736),在居群A、B、C中基因频率在0.50以上的谱带相对较多,其中A居群中有7条,最高值为1.00,B居群中有3条,最高值为1.00,C居群中有7条,最高值为0.88,遗传距离小,这也说明居群内个体遗传分化小,具有高度的遗传同质性;乙醇脱氢酶在不同居群间的相似性系数均较小,而遗传距离相对较大(IAB=0.539;IAC=0.367;IBC=0.513;DAB=0.618;DAC=1.002;DBC=0.667)。遗传距离最大值是DAC=1.002;这些数据表明垫状雪灵芝乙醇脱氢酶同工酶在不同的居群之间存在较大的遗传分化。
4酯酶同工酶
垫状雪灵芝的酯酶同工酶在居群内的个体,同工酶谱相似性极高,遗传杂合度在四种同工酶中是最低的(HeA=0.735;HeB=0.765;HeC=0.726),在居群A、B、C中基因频率在0.50以上的谱带相对较多,其中A居群中各有10条,B、C居群中各有8条,最高值均可达到0.90以上,并且C居群中基因频率为1.00的谱带有3条,居群内个体遗传距离小,这也说明居群内个体遗传分化小;然而,酯酶在不同居群间的相似性系数有不同程度的变化,遗传距离也是如此(IAB=0.392;IAC=0.542;IBC=0.610;DAB=0.937;DAC=0.613;DBC=0.495)。遗传距离最大值是DAB=0.937;这些数据表明垫状雪灵芝酯酶同工酶在不同的居群之间存在较大的遗传分化。
四种同工酶无论是在遗传系数,还是同工酶谱或聚类方面表现出的变化趋势和幅度并不完全一致,这些表明垫状雪灵芝在长期进化的过程中,控制各种同工酶的不同基因并没有协同进化,这种现象在自然界是普遍存在的。青藏高原特殊的气候条件和垫状雪灵芝的地理分布格局所造成的,形成了垫状雪灵芝的同工酶变异特点。
In this paper, the alpine plant Arenaria pulvinata Edgew. of Sejila Mount is used as the material, selecting three populations of Arenaria pulvinata Edgew. distributing the elevation of 4500-4650m for research. The technology of Polyacrylamide agar electrophoresis is used to determine and code the bands of isozyme such as Peroxidase, Cytochrome oxidase, Alcohol dehydrogenase, Esterase and so on , which are from the 48 individual functional leaves of three populations of Arenaria pulvinata Edgew. and then according to the results of the enzymatic bands, we do cluster analysis. At the same time, with three genetic indicators of the four isozymes , Peroxidase, Cytochrome oxidase, Alcohol dehydrogenase, and Esterase, correlation analysis is done. The results indicate:
1.Peroxidase Isozymes
From the same populations of the individual, not only similarity of isozyme bands, small genetic heterozygosity (HeA=0.716;HeB=0.709;HeC=0.705), particularly the coefficient of similarity is larger and genetic distance is small in populations of B, C,This also shows that the individual’s genetic differentiation in populations smaller enough. The Peroxidase, which coefficient of similarity’s average of four isozyme is relatively bigger in different populations and genetic distance is small(IAB=0.637;IAC=0.587;IBC=0.646;DAB=0.451;DAC=0.533;DBC=0.437),These data indicate that the peroxidase isozyme of Arenaria pulvinata Edgew. being certain degree of genetic differentiation in different populations, and also lack of effective gene flow.
2. Cytochrome oxidase isozymes
The cytochrome oxidase isozymes of Arenaria pulvinata Edgew. in the populations of the individual, which isozyme bands is similar, and the genetic heterozygosity is higher than peroxidase (HeA=0.752;HeB=0.815;HeC=0.815), The bands of gene frequency above 0.50 is relatively more than others in the populations of A,B,C, and there is 6 bands in populations of A, B, each other ,and there is 5 bands in populations of C, its maximum value could reach 0.94, and the genetic distance is small, it also shows that populations of genetic differentiation within the individual is small; The coefficient of similarity of cytochrome oxidase being the maximum in different populations and a factor in the entire population of the four types of isozyme minimum, the same is true of genetic distance (IAB=0.418;IAC=0.304;IBC=0.673;DAB=0.873;DAC=1.192;DBC=0.396). The maximum of genetic distance is DAC = 1.192; These data shows that the cytochrome oxidase Isozymes of Arenaria pulvinata Edgew. being larger genetic differentiation in different populations.
3. Alcohol dehydrogenase isozymes
The alcohol dehydrogenase isozymes of Arenaria pulvinata Edgew. in the populations of the individual, which isozyme bands is similar, and the genetic heterozygosity is lower than cytochrome oxidase isozymes (HeA=0.766;HeB=0.872;HeC=0.736), The bands of gene frequency above 0.50 is relatively more than others in the populations of A,B,C, and there is 7 bands in populations of A, its maximum reach to 1.00, there is 3 bands in populations of B, its maximum reach to 1.00 too, there is 7 bands in populations of C ,its maximum reach to 0.88, but its genetic distance is small, it also shows that populations of genetic differentiation within the individual is small, with a high degree of genetic homogeneity。The coefficient of similarity of Alcohol dehydrogenase are smaller and relatively larger genetic distance in different populations (IAB=0.539;IAC=0.367;IBC=0.513;DAB=0.618;DAC=1.002;DBC=0.667). The maximum of genetic distance is DAC = 1.002; These data show that the alcohol dehydrogenase Isozymes of Arenaria pulvinata Edgew. being larger genetic differentiation in different populations.
4. Esterase Isozymes
The Esterase isozyme of Arenaria pulvinata Edgew. in the populations of the individual, which isozyme spectrum is similar, and the genetic heterozygosity is lowest in these four isozyme. (HeA=0.735;HeB=0.765;HeC=0.726), The bands of gene frequency above 0.50 is relatively more than others in the populations of A,B,C , and there is 10 bands in population of A ,there is 8 bands in population of B , C each other, and the maximum could reach to 0.99, furthermore, there is 3 bands ,which gene frequency is 1.00 and the genetic distance is small, it also shows that populations of genetic differentiation within the individual is small; However, the coefficient of similarity of EST isozyme have different degrees of change in different populations, and the gentic distanceis so (IAB=0.392;IAC=0.542;IBC=0.610;DAB=0.937;DAC=0.613;DBC=0.495). The maximum of genetic distance is DAB = 0.937; These data show that the EST isozyme of Arenaria pulvinata Edgew. being larger genetic differentiation in different populations.
The shown on changes in trends and ranges of the four isozymes is not exactly simility, whether in the genetic factors or the isoenzymatic patterns or the clustering, which shows in the process of long-term evolution of Arenaria pulvinata Edgew.,the different genes which control kinds of isozymes have no co-evolution. This phenomenon is widespread in nature. Because of the special climatic conditions of Qinghai- Tibet Plateau and the geographical distribution pattern of Arenaria pulvinata Edgew., the variant characteristics of isozymes of Arenaria pulvinata Edgew. are formed.
引文
1.胡能书,万国贤.同工酶技术及应用[M].长沙:湖南科学技术出版社,1985
2.靳永年.桑树同工酶研究进展与应用[J].蚕桑通报,1995,26(3):5~9
3.吴易雄.猪血液同工酶遗传多态性的研究应用进展[J].广东畜牧兽医科技,2002,27(1):44~47
4.周光宇.有关同工酶分析的几个问题[J].植物生理学通讯,1983,1(1):1~4
5.金妙华,陈东明,许忠意等.四棱豆不同节位叶片及各器官过氧化物酶同工酶的分析[J].农业与技术,2006,26(1):127~130
6.侯彩霞,沈博礼,刘戈等.镧和铈对小麦幼苗过氧化物酶和淀粉酶活性及其同工酶的影响[J].稀土,1997,18(2):64~66
7.高梅秀,张云贵,李天俊等.茄子种及其品种过氧化物酶同工酶分析[J].西北农林科技大学学报(自然科学版),2002,30(4):65~68
8.曹仪植.植物生理学[M].兰州大学出版社,1998,395~396
9.沙伟,孟凡玲,冯昌军.10 个苜蓿品种过氧化物同工酶(POD)分析[J].北方园艺,2007(1):150~152
10.李娟,宋庭杰,肖忠等.水分胁迫霞白术过氧化物酶同工酶的变化分析[J].中国现代中药,2007,9(4):11~13
11.李红燕,杨继华,薛妙男.沙田柚花柱三种同工酶分析[J].广西师范大学学报(自然科学版),1999,17(4):81~84
12.邵继荣,何清海,张述华.水稻多用恢复系 130 的七种同工酶研究[J].西南农业学报,1999,12(1):8~13
13.宋平,曹显祖,曹鹏等.不同染色条件对大麦酯酶同工酶显色的影响[J].江苏农学院学报,1997,18(3):37~39
14.彭婧,陈业志,李贵生.安南龟冬眠前后酯酶同工酶的比较研究[J].四川动物,2008(1):53~55
15.郭凤根,陈娟,王仕玉等.云南紫苏资源的酯酶同工酶分析[J].中国农学通报,2005,21(5):101~103
16.王波,甘炳成,贾定洪等.蘑菇属 17 个菌株的酯酶同工酶分析[J].西南农业学报,2007,20(6):1307~1309
17.兰小中,邓洪平,何平等.濒危植物缙云卫矛细胞色素氧化酶的同工酶变异[J].东北林业大学学报,2000,28(6):40~44
18.兰小中,王景升,郑维列等.巨柏细胞色素氧化酶同工酶变异分析[J].山地农业生物学报,2006,25(4):297~301
19.黄林,陈亚飞,邓洪平.四川大头茶过氧化物酶和细胞色素氧化酶同工酶变异的数量分析[J].西南师范大学学报(自然科学版),2002,27(2):395~400
20.宁黔冀,任虹,段夕全等.重金属离子对扁玉螺细胞色素氧化酶的影响研究[J].烟台大学学报(自然科学版),1999,12(1):42~45
21. 吕跃刚,王际彰.酵母乙醇脱氢酶电极研究[J].微生物通报,1997,24(4):218~219
22. 李继耕.植物同工酶及其在作物遗传研究中应用[J].作物学报,1980,6(4):245~251
23. 王中仁.植物等位酶分析[M].科学出版社,1996,34~36 霍振华,黎中宝,叶兆弘等.新对虾等位酶的遗传控制[J].水产养殖,2001,2:34~36
24.陈劲枫,任刚,余纪柱等.黄瓜属远缘杂种回交自交群体的过氧化物酶同工酶分析[J].武汉植物学研究,2002,20(5):333~337
25.程立生.同工酶电泳技术在动物分类学上的应用[J].生物学杂志,1987,18(2):20~23
26.王梅芳等.3 种江珧同工酶遗传标记[J].2000,湛江海洋大学学报,20(2):1~5
27.王志铮,李太武等.栉孔扇贝六种同工酶的升华遗传分析[J].海洋与湖沼,2002,33(3):232~237
28.王金星,赵小凡,张红卫等.对虾组织蛋白质和同工酶表型及其在虾病中的变化[J].海洋科学,1995(3):46~51
29.蔡玩其.罗氏沼虾莫格球拟酵母病的病理研究[J].水产学报,1996,20(1):13~17
30.赵扬,傅振清,樊新民等.甜瓜成株抗感枯萎病品种过氧化物酶同工酶研究[J].石河子大学学报(自然科学版),1996,(02):41~44
31.王愈.过氧化物酶同工酶酶谱变异与黄瓜抗病的关系[J].本届农学院学报,1994,9(2):61~63
32.陈春林.茶树杂交 F1 代同工酶分析[J].茶叶科学,1996,16(1):31~36
33.杨书婷,桂建芳.两个雌核发运的白莲群体同工酶分析及遗传标记的确定[J].水生生物学报,1999,23(3):264~268
34.杨书婷,桂建芳.雌核发育银鲫和两性生殖彩鲫精子蛋白组份的比较研究[J].动物学报,2001,47(1):79~81
35.刘春光.小麦D2型细胞不育配子发育的细胞形态学特征核同工酶研究[J].遗传学报,1995,22(3):199~205
36.李国风.空间条件对几种粮食作物的同工酶和细胞学特性的影响[J].遗传学报,1996,23(1):48~55
37.卢存福,陈玉珍,简令成.高山植物唐古特红景天粘液细胞及叶肉细胞表面糖蛋白与抗冻性的关系[J].应用与环境生物学报,2003,9(1):16~20
38.卢存福,简令成等.高山植物的抗寒抗冻特性[J].植物学特性,1998,15(3):17~22
39.吴学明.五种高山藏药药用植物茎叶的结构特征研究[J].西北植物学报,1996,16(1):56~60
40.王为义.高山植物结构特异性的研究[J].高原生物学集刊,1985,4:19~32
41.师生波,韩发等.高寒草甸生态系统微气候和植物的生理生态适应[J].高原生物学集刊,1997,13:217~224
42.卢存福,简令成等.低温诱导唐古特红景天细胞分泌抗冻蛋白[J].生物化学与生物物理进展,2000,27(5):555~559
43.杨扬,孙航.高山和极地植物功能生态学研究进展[J].云南植物研究,2006,28(1):43~53
44.冯伟力,王利彦,彭宝珠等.藏药雪灵芝的毒理学研究[J].清海医学院学报,1990,(2):15~17
45.张丽,黎霞,黄叶梅.雪灵芝对大鼠脑缺血再灌注损伤的保护作用[J].四川师范大学学报(自然科学版),2007,30(3):395~397
46.苏甫,王化远.雪灵芝中马替诺皂甙的 2D NMR 研究[J].四川大学学报(自然科学版),2004,41(3):676~679
47.江云珠.中国稻种(Oryza,sativa L.)资源同工酶和 SSR 标记遗传多样性研究[D].中国农业科学院研究生院-中国水稻研究所,2002
48.黄晨阳.同工酶和 RAPD 标记在金针菇菌株鉴定和遗传多样性研究中的应用[D].福建农林大学,2002
49.周建云.栓皮栎天然类型划分及其同工酶分析的研究[D].西北农林科技大学,2003
50.余红卫.三疣梭子蟹同工酶的生化遗传分析,中国海洋大学,2005
51.任刚.黄瓜属种间杂交新种(Cucumis×hytivus Chen﹠Kirkbride)的遗传特性、同工酶标记及其分类地位研究[D].南京农业大学,2003
52.王玉波.植物生长物质促控甜菜抽薹过程中相关同工酶的研究[D].东北林业大学,2004
53.王静.西施舌同工酶分析及消化酶活性的研究[D].福建师范大学,2005
54.涂三思.黄姜 POD 和 EST 同工酶及高温胁迫下脯氨酸可溶性糖和丙二醛含量变化研究[D].华中农业大学,2004
55.蒋培东.对生玉米核型、同工酶及其对生性状遗传表现的研究[D].安徽农业大学,2004
56.刘艳.栉孔扇贝亚种间杂交机制的研究-同工酶分析[D].辽宁师范大学,2002
57.熊全沫. 同工酶电泳数据的分析及其在种群遗传上的应用[J]. 遗传,1986,8(1):1~5
58.钟扬,陈家宽,黄德世. 数量分类的方法与程序[M]. 武汉:武汉大学出版社,1990,35~45
59. Market.C.,and Mφller. Multiple forms of enzymes: Tissue, ontogenetic and species specific patterns, Proceeding of the National Academy of Science U.S.A.1959,45:753~763
60. Prakash ,S.et al.1969.A molecular approach to the study of genetic heterozygosity in natural populations. IV . Patterns of genetic variation in cetrol , marginal and isolated population of Drosophila pseudoobscura .Genetics,61:841~ 858
61. CHEN DONG-HWANG,LAO M N-HUNG Preparation and Characterization of YADH-bound Magnetic Nanoparticles[J] Joumal of Molecular Catalysis B;Enzymatic,2002,16:283~291
62. Buth,donold G.And Bobert W. Murphy. The use of Isozymes Characters in Systematic Studies. Biochemical Systematic and Ecololgy.1999,27(2):117~129
63. Practor R R. Marvink,Lansford L,et al.Phosphogluco mutase polymorphis minbrown shrimp Penaeus aztecus. J Fish Res Boad Can,1974,31:1405~1407
64. Quartau J A ,Simoes P C, Crespo A.et al.Taxonomic separation by isozyme electrophoresis of two closely related species of Cicada L.in Portugal.Journal of Natural History.
65. Kemer Die Abhangigkeit der Pflanzengestalt von Kilma und Boden Innsbruck Wager ,1896,29~45
66. Billings W D, Mooney H A. The ecology of arctic and alpine plants Biol Rev,1968,43,481~529
67.Rundel P W,Smith A R,Meinzer F C. Tropical alpine environments plant form and function Cambridge Cambridge University Press,1994
68. Shi SB ,Zhu WY,Li HM,etal Photosynthesis of Saussurea and Gentiana stram inea is not reduced after long-term enhancement of UV-B radiation Environmental and Experimental Botany,2004,51:75~83