Cloning and Gene Library

If study of individual chloroplast genes is desired，it is advantageous to construct a chloroplast genome library，from which a variety of interesting genes can be isolated，purified，amplified and stored for further studies.

When the ct-DNA of N.otophora is completely digested with EcoRI，BomHI and Sma I，at least 40，28 and 15 fragments are generated，respectively.BamHI was used to digest ct-DNA for constructon of a chloroplast genome library，as there is a single BamHI site within the tetracycline resistance gene of pBR322.(https://www.daowen.com)

A“shotgun”procedure was employed for cloning.A mixture of N.otophora ct-DNA and pBR322 was digested with BamHI，ligated，and introduced into E.coli HB1O1.The transformed E.coli cells were first selected on ampicillin plates and then screened for tetracycline sensitivity by duplicate plating on ampicillin and tetracycline plates.A total of 207 recombinants（Ampr，Tcs）were obtained from 2002 colonies（Ampr）.After digestion with BamHI，the chimeric plasmids carrying ct-DNA inserts of various sizes in the recombinants were further analyzed electrophoretically and compared with pBR322 and BamHI-digested N.otophora ct-DNA.Most of the BamHI fragments of ct-DNA（23/28）have been clones in E.coli HB101.pBR322 carries from one to four different BamHI fragments of ct-DNA.The largest inserted ct-DNA fragment（9 kb）is twice the size of pBR322.Fragments larger than this，such as Bam 1，2 and 3，seem to encounter difficulty in being inserted into pBR322.These fragments，from ct-DNA of N.otophora，were separated and recovered from a low-gelling-temperature agarose gel，then ligated to pBR322 and introduced individually into E.coli HB101.Thus，with the exception of Bam fragment 8，a clone library of the entire N.otophora chloroplast genome was constructed［15］.