The promoter cloning and analyses of bifunctional nuclease gene family from Arabidopsis thaliana

• Main Authors: Yi-Ling Lai, 賴怡伶
• Other Authors: Jei-Fu Shaw
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/22657516182437853512

碩士 === 中興大學 === 生物科技學研究所 === 95 === Nucleases consist of three species: deoxyribonucleases (DNases), ribonucleases (RNases), and bifunctional nucleases (BFNs) which are capable of degrading DNA and RNA. Arabidopsis bifunctional nucleases gene family contains five members, AtBFN1 (At1g11190), AtBFN2 (At1g68290), AtBFN3 (At4g21590), AtBFN4 (At4g21585), and AtBFN5 (At4g21600). In order to study the regulation, tissue-specificity and cellular localization of bifunctional nucleases, the promoter region of AtBFN1 or AtBFN2 was used to drive the expression of the reporter genes fused with or without signal peptide and then cloned into binary vectors pCAMBIA1391 or pBI121, respectively. The four constructs, AtB1P::GUS (AtBFN1 promoter drive β-glucuronidase reporter gene), AtB1SP::GFP (AtBFN1 promoter drive green fluorescent protein fused with AtBFN1 signal peptide), AtB2P::GUS (AtBFN2 promoter drive β-glucuronidase reporter gene), and AtB2SP::GFP (AtBFN2 promoter drive green fluorescent protein fused with AtBFN2 signal peptide), were introduced into the Arabidopsis thaliana (ecotype Columbia) via Agrobacterium-mediated transformation. The transgenic plants were first selected by hygromycin or kanamycin. The Southern blot method is used to identify the insertion copy numbers and the independent lines. Western blot analyses using anti-GFP antibody demonstrated that AtB1SP::GFP protein was mainly expressed in roots, flowers and siliques, whereas AtB2SP::GFP protein was most abundant in roots. The dark-induced senescence of detached leaf assay indicated that AtBFN1 and AtBFN2 were senescence-associated. The results suggested that AtBFN1 participate in etiolation, AtBFN2 was expressed at high levels in the early stage of senescence. We also used GUS staining to identify the tissue specificity and differential expression of AtB1P::GUS and AtB2P::GUS during the development. AtB1P::GUS was expressed in seedling, flower organs and vascular bundles of siliques. AtB2P::GUS was expressed in seedling, flower organs and roots. The cellular localization of AtB1SP::GFP and AtB2SP::GFP fusion proteins were observed by using confocal microscope (Zeiss LSM 510 Meta). AtB1SP::GFP is localized on the root epidermal cell and root hair. AtB2SP::GFP is localized on the root and petal vascular bundle.