Long-Fang O. Chen

Publications (3)5.67 Total impact

• Article: Arabidopsis ENDO2: its catalytic role and requirement of N-glycosylation for function.

  Chia-Yun Ko, Yi-Ling Lai, Wen-Yu Liu, Chia-Hui Lin, Yu-Ting Chen, Long-Fang O Chen, Tsai-Yun Lin, Jei-Fu Shaw
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  ABSTRACT: The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 × His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.
  Journal of Agricultural and Food Chemistry 04/2012; 60(20):5169-79. · 2.82 Impact Factor
• Article: Ethylene insensitive and post-harvest yellowing retardation in mutant ethylene response sensor (boers) gene transformed broccoli (Brassica olercea var. italica)

  Long-Fang O. Chen, Jia-Yuan Huang, Yung-Hsiang Wang, Yu-Ting Chen, Jei-Fu Shaw
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  ABSTRACT: A mutant broccoli ers (ethylene-response-sensor, boers) gene was obtained through site directed mutagenesis by replacing the isoleucine with phenylanine at the 62th residue. Two plasmids were constructed with this mutant gene regulated by the CaMV 35S promoter together with the nptII (kanamycin resistance gene) coding sequence and hpt (hygromycin resistance gene), respectively, for the pBI-mERSI62F and pSM1H-mERSI62F plasmids. Genetic transformation of the above two constructs via A. tumefaciens has been conducted to evaluate their effects on floret yellowing of harvested broccoli. Over a hundred transformants have been obtained on the selected cotyledon and hypocotyl explants. PCR and Southern analysis demonstrated integration of the transgenes in the transformants. However, through Southern hybridization, we determined that multi-site integration and DNA rearrangements had occurred in most transformants. Morphological and characteristic alternation such as slower plant growth, shorter plant height, easy branching, late bolting, and relative higher mortality in comparison with other transgenes were noted in some transformants. Transgenic lines showing delayed senescence in leaves and floral heads were obtained. The expression of transgene was confirmed by Northern blot analysis. The transformed progenies also showed ethylene insensitivity in seed germination, detached leaves and harvested florets. Nevertheless, in most lines, the yellowing was only delayed 1–2 days.
  Molecular Breeding 08/2004; 14(3):199-213. · 2.85 Impact Factor
• Article: Senescence-associated genes in harvested broccoli florets

  Yu-Ting Chen, Long-Fang O. Chen, Jei-Fu Shaw
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  ABSTRACT: Broccoli, Brassica oleracea L. var italica, is an important vegetable crop known to be rich in vitamins and sulforaphane. However, rapid postharvest senescence in harvested floral heads reduces its value. Complex factors such as ethylene biosynthesis, temperature, and respiration are involved in this senescence process. Meanwhile, recent developments on postharvest biotechnology, as well as studies on functional genes related to postharvest senescence have provided an opportunity for conquering this problem. In this review, we will discuss the genes involved in broccoli senescence and the physical, chemical, or genetic methods to increase shelf life and reduce quality loss due to postharvest senescence.
  Plant Science.