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Construction of two-element transposon tagging system and analysis of its genetic behaviour in transformed tobacco plants

Zhang Jingliu, Zhang Wenwei, Zhang Dong, Zhang Dabing, Zhang Lei, An Linsheng, Hong Mengmin

Shanghai Institute of Plant Physiology, The Chinese Academy of Sciences. Shanghai 200032

Abstract

Two plasmids, pKU3(#DELTA#B) and pKU3(#DELTA#H) were reconstructed from plasmid pKU3 by deleting the 5^-terminal region or internal part of maize activator(Ac) transposable element. The Ac element in pKU3 was inserted in the untranslated leader sequence of the neomycin phosphotransferase II(NPT II) gene,when Ac was excised,the destroyed NPT II gene activity was recovered. Thus,the appearance of NPT II activity can be used to detect the excision of Ac element .The pKU3(#DELTA#B) and pKU3(#DELTA#H) alone could not be　transpose from site to site automonously, because two essential sequences,the terminal inverted repeat and coding part of transposase gene, were removed. However, the Ac(#DELTA#H)element may regain the transposibility by complementing the transposase from the Ac(#DELTA#B) element when both elements coexisted within a cell.After the Ac(#DELTA#B) was segregated from Ac(#DELTA#H) element,a stable Ac insertion mutant could be obtained. To test if this two elements system could be used in transposon tagging strategy to clone genes in plants,these two Ac derivatives were introduced into protoplasts from Nicotiana tobacum.After plantlet regeneration,transgenic tobacco plants with kanamycin resistance were obtained. The restoration of kanamycin resistance shows that the Ac(#DELTA#H) element was excised from the untranslated leader sequence of NPT IIgene. Results of Southern blot analysis and polymerase chain reaction assay showed that (i) both the Ac(#DELTA#H)and Ac(#DELTA#B) elements had been integrated into the genome of transformed tobacco plants, and they could be transmitted from F_(0) to F_(1) and F_(2) progenies.(ii)In a few plants the Ac(#DELTA#B) was excised and segregated　from Ac(#DELTA#H). (iii) The copy number of inserted Ac elements　within genome of individual transformed plants was determined as one or more. (iv) Most of the Ac elements were inserted or transposed into structural gene and not into repeated sequences within the genome(data not showed).

Key words: Transposon tagging;Two-elements transposon tagging system;Tobacco;Protoplast;PEG-transformation

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Citing This Article：

Zhang Jingliu, Zhang Wenwei, Zhang Dong, Zhang Dabing, Zhang Lei, An Linsheng, Hong Mengmin. Construction of two-element transposon tagging system and analysis of its genetic behaviour in transformed tobacco plants. Acta Physiol Sin 1997; 49 (1): (in Chinese with English abstract).