Discussion

The construction of clone library of nuclear or organellar genomes is an important operation in explorating the potentials of genetic manipulation of plants.Only recently a library of the French bean nuclear genome has been established（Sun et al.，1981）.Organellar genes，on the other hand，have been cloned individually（Bedbrook et al.，1977；Coen et al.，1977）prior to the construction of any comprehensive chloroplast genome libraries（Palmer and Thompson，1981）.This is the first clone library of Nicotiana chloroplast genome.Nicotiana chloroplast genome has been the subject of intensive investigations in recent years（Fluhr and Edelman，1981；Jurgenson，1980；Kung et al.，1981；Seyer et al.，1981）.However，there is no report on chloroplast genome library in this genus.In this study，N.otophora，a closely related species of N.tabacum，was selected to construct a clone library of Nicotiana chloroplast genome.A total of 27 out of 28 BamHI fragments of N.otophora chloroplast DNA were ligated into plasmid pBR322 and cloned in E.coli cells.The inability to clone a specific BamHI fragment 8（4.8 kb）after many futile attempts is frustrating.Palmer and Thompson（1981）experienced a similar difficulty in their attempt to clone pea chloroplast genome.They attributed this failure to some unknown feature of its sequence which may interfere with the cloning process or efficiency or even to affect the survival.

This clone library is used as the base for constructing a physical map of N.otophora chloroplast genome.The sequential order of BamHI fragments was aligned by using secondary restriction digestion.Thus，a restriction map of the Smal and BamHI was completed.The genes for rRNA and the LS of RuBPCase have been identified in the clone as well as localized on individual fragments generated by various enzymes.No unusual structural feature was detected in this species.(https://www.daowen.com)

The plasmid-directed protein synthesis system is employed for the in vivo expression experiments.In this maxicell system the p-lactamase is synthesized under the direction of plasmid pBR322 after UV treatment.However the product coded for by the tetracycline resistant gene on this plasmid is apparently not synthesized.A similar result was also reported by others（Erion et al.，1981）.In the case of many cloned ct-DNA fragments tested，only three labeled polypeptides（representing 1%of ct-genome）can be detected.The identity of these polypeptides have not yet been determined.Nevertheless，the in vivo expression of chloroplast genes in E.coli usingthe plasmid-directed protein synthesis system is clearly demonstrated.