[Screening of specifically expressed genes in amphioxus neurula by construction of a subtractive cDNA library].
OBJECTIVE
To screen specifically expressed genes in the development of nerve, muscle, and body axis of amphioxus, Branchiostoma belcheri tsingtauenese.


METHODS
A subtractive cDNA library was constructed from the 12-hour amphioxus neurula cDNA after subtractively hybridized with the 6-hour amphioxus gastrula cDNA. The total RNA was extracted from the 12-hour neurula and 6-hour gastrula, then reverse transcribed into cDNA. The 12-hour neurula cDNA was designated as the experimental group (the tester) and the 6-hour gastrula cDNA as the control group (the driver). The differentially expressed sequences were exponentially amplified using suppression PCR. Background was subtracted and differentially expressed sequences were further enriched. The PCR products were ligated to the T Vector. After transformation of the recombinant plasmid carrying inserted amphioxus cDNA into E.coli host cells, the cDNA library was constructed successfully.


RESULTS
Two hundred randomly chosen positive clones were sequenced and some of neurula-specifically expressed genes were obtained.


CONCLUSIONS
SSH is an effective method for searching differentially expressed genes. The subtractive cDNA library we generated provides a tool for further study of regulatory mechanisms of amphioxus early embryonic development.
School of Life Sciences, Shandong University, Jinan 250100, China. leizhang@sdu.edu.cn
Lei
L
Zhang
Yong-Jie
YJ
Yang
Yan-Jun
YJ
Zhang
D000818:Animals; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005399:Fishes / Q000196:embryology* / Q000235:genetics*; D015723:Gene Library; D054261:Neurulation / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000235:genetics
D012313:RNA; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases