刺鳅X染色体DNA文库的构建
THE CONSTRUCTION OF X CHROMOSOME LIBRARY OF SPINY EEL(MASTACEMBELUS ACULEATUS)
-
摘要: 刺鳅(Mastacembelus aculeatus)是具有明显X和Y异形性染色体分化的淡水鱼。本实验室通过显微切割(Mi-crodissection)和兼并引物PCR(DOP-PCR)方法，从雌性刺鳅中期染色体分裂相中分离获得X染色体并扩增其DNA，利用T载体和电转化方法，建立了刺鳅X染色体DNA质粒文库。该文库插入片段的平均长度约为500bp，理论上包含X染色体98％以上的序列。当用荧光原位杂交(FISH)来验证文库的专一性时发现，在无竞争性DNA杂交条件下，整个X和Y染色体上都表现出强烈的杂交信号，并且常染色体上也出现一些随机散布信号;当含有竞争性DNA时，常染色体上的信号消失，仅性染色体上异染色质区域保留有较强信号。就此，本文对刺鳅性染色体上的序列类型进行了探讨。Abstract: The spiny eel (Mastacem belus aculeatus) is a species of the genusMastacembelus (Osteichthyes, Perciformes), whichmainly live in the freshwater of the south ofChina1 The fish isparticularly attractive for cytogenetical study ow ing to possessing well differentiated X and Y sex chromosomes.In the present study, the X chromosomesofM. aculeatus werem icrodissected from themetaphasesof chromosomes of the female. Then, the fragments of X chromosomeswere put into am icro tube and amplified using Degenerated Oligonucleotide-Primed PCR (DOP-PCR). Thereafter, the product of PCR was connected to T vector and the plasm idswere electric transformed into E1 coli1 As a resul,t the library of X chromosome ofM. aculeatus was constructed. The sum of the lengths of the inserted fragments is some 1.08 ?108bp, w ith an average length of 500bp. It is believed that the library coveredmore than 98% sequences of the whole X chromosome theoretically.To check the credibility of the library, fluorescence in situ hybridization (FISH) was applied. The fragmentsof the library were labeledw ith biotin by PCR. Then the probeswere hybridized to the chromosomem etaphases ofboth sex ofM 1 aculeatus in the absence or the presence of competitorDNA1 Strong signalswere detected on the entire sex chromosomes aswell as signals dispersed on all autosomesw ith the condition of FISH in the absence of competitor DNA.W hereas competitorDNA was added, the signals disappeared except those showed on heterochrom atic regions of X and Y chromosomes.H ence, we assorted the repetitive sequences in theX chromosom e ofM. aculeatus into three types. It was interpreted that the signalsof FISH in the presence of competitor DNA showed the distribution of typerepetitive sequences on the sex chromosomes ofM. aculeatus.
-
Keywords:
- M astacembelus aculeatus /
- Sex chromosome /
- Chromosome library /
- FISH
-
-
[1] Devlin R H, Nagahama Y. Sex determination and sex differentiation in fish: an overview of genetic, physiologica,l and env ironmental influences [J]. A quaculture, 2002, 208: 191) 364
[2] Yang D, Yu L N. Sex and sex identification of fish [J]. Acta H ydrobiologica Sinica, 2006, 30 (2): 221) 226 [杨东, 余来宁.鱼类性别与性别决定. 水生生物学报, 2006, 30 (2): 221)226]
[3] Yu X J, Zhou T, LiY C, et al. Chromosomes ofChinese freshwater fishes [M]. Beijing: Science Press. 1989 [余先觉, 周暾, 李渝成, 等. 中国淡水鱼类染色体. 北京: 科学出版社. 1989]
[4] Liu J D, Huang L, YuQ X. The cytogenetic evidences for confirming the ex istence of sex chromosomes in M astacembelus aculeatus (Osteichthyes, Perciformes) [J]. J. Wuhan Univ. (N at. Sc.i Ed.), 1999, 45 (2): 185) 190 [刘江东, 黄琳, 余其兴. 刺鳅性染色体的细胞遗传学确定证据. 武汉大学学报(自然科学版), 1999, 45 (2): 185) 190]
[5] L iu J D, Y i M S, Zhao G. Sex chromosomes in the spiny eel (Mastacem belus aculeatus) revealed by mitotic andmeiotic analysis [J]. Cytogenet Genome Res, 2002, 98: 291) 297
[6] Shibata F, H izume M, Kuroki Y. Chromosome painting of Y chromosomes and isolation of a Y chromosome-specific repetitive sequence in the dioecious plant Rumax axetosa [J]. Chromosoma, 1999, 108: 266) 270
[7] Sambrook J, Fritsch E F, M aniatisT. Molecular cloning, a laboratory manual [M]. Cold Spring HarborLaboratory Press. 1989
[8] Cui JX, R en X H, Yu Q X. NuclearDNA content variation in fishes [J]. Caryologia, 1991, 56: 425) 429
[9] Zhou Y H, Hu Z M, Dang B Y, et al. M icrodissection andm-icrocloning of rye (Secale cereale L. ) chromosome.R [J]. Chromosoma, 1999, 108: 250) 255
[10] Ohno S. Sex chromosomes and sex-linked genes [M]. Berlin-Heidelberg-New York: Springer. 1967
[11] PhillipsR B, ReedK M. Application of fluorescence in situ hybridization (F ISH) techniques to fish genetics: a rev iew [J]. A quaculture, 1996, 140: 197) 216
[12] Phillips R B, Konkol N R, Reed K M. Chromosome painting supports lack of homo logy among sex chromosomes in Oncorhynchus, Salmon and Salvelinus (Salmonidae) [J]. Genetica, 2001, 111: 119) 123
[13] Dong Z J. Preparation of X-andY-chromosome probes ofN ile tilapia (O reochromis niloticus) for in situ hybridization [J]. J. NanjingAgri. Univ, 2004, 27 (3): 136) 138 [董在杰. 尼罗罗非鱼 X和 Y 染色体原位杂交探针的制备. 南京农业大学学报, 2004, 27 (3): 136) 138]
计量
- 文章访问数:
- HTML全文浏览量: 0
- PDF下载量:
下载: