CONSTRUCTION OF cDNA LIBRARY OF QINGDAO AMPHIOXUS NEURULAE AND SCREENING OF HEDGEHOG GENE

The mRNA isolated from Qingdao Amphioxus(Branchiostoma belcheri tsingtaunese) neurulae of 18 h period was reverse transcripted into cDNA using NotⅠ-oligo(dT) 18 as a primer.An EcoR Ⅰ adaptor was ligated to the 5′ end of subsequently synthesized double strand cDNA.Then The resulting cDNA was ligated to the vector λExCell which had been double-digested with EcoRⅠ and NotⅠ and had been dephosphorylated with CIP.The mixture of ligation reaction was packed in vitro and the host strain E.coli NM522 was infected by the packing mixture.3.6×10 4 recombinants had been obtained,constituting a cDNA library.In order to examine the efficiency of the cDNA library,we extracted DNA from two clones chosen randomly from these recombinants,and digested the DNA with EcoRⅠ and NotⅠ.The results of 1% agarose gel electrophoresis showed that these two clones had a same 45.5 kb band of vector DNA while their DNA banding patterns of inserts were obviously different.In order to confirm that the inserts were cDNAs of amphioxus,we screened the cDNA library by plaque in situ hybridization using an α- 32P-dCTP labelled amphioxus hedgehog gene fragment as a probe.Sixteen positive clones were obtained through the twice screening.The results showed that the cDNA library of Qingdao Amphioxus neurulae had constructed.