ABSTRACT
[Objective] Subtractive cDNA libraries from the leaves of D. candidum were constructed to supply evidence for the clone of dendrobine-biosynthesis-associated genes. [ Methods ] Suppression subtractive hybridization ( SSH) technology was used. With the cDNA of perennial leaf as the tester and that of annual leaf as the driver, forward and reverse hybridization was performed. The two obtained subtracted cDNA fragments were cloned into PointTMXa-1 plasmid vectors, and then the vectors were transformed into E. Coli JM109. The inserted fragments were amplified by PCR and then identified. [Results] The subtractive cDNA libraries related with dendrobine-biosynthesis-associated genes were successfully constructed. The forward and reverse subtractive libraries included 560 and 220 clones respectively. Detected by PCR, the length of the inserted fragments was 550 bp on average. [Conclusion] The constructed subtractive libraries are suitable for further study on the functional genes associated with dendrobine biosynthesis of D. candidum.
ABSTRACT
AIM: To construct a subtracted cDNA library of differentially expressed genes in human unstable angina lymphocytes. METHODS: Suppression subtractive hybridizations (SSH) were performed between the patients with unstable angina pectoris and stable angina pectoris. Lymphocyte RNA, the obtained forward and reverse cDNA fragments were directly inserted into T/A cloning vector and transformed into E.coli JM 109 to construct a subtractive cDNA library. The inserting fragments were screened by blue and while blot screening and bacterium liqulid PCR. RESULTS: Each subtractive cDNA library contained more than 2000 positive bacteria clones. Most of them distributed between 200-600 bp inserts. CONCLUSION: The library is efficient and lays solid foundation for screening and cloning new and specific expressed genes in unstable angina lymphocyte RNA.