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玉米Ac双因子转座标签系统的构建及其转化烟草后代的遗传分析

张景六, 章文伟, 张栋, 张大兵, 张磊, 安林升, 洪孟民

中科院上海植物生理研究所. 上海 200032

摘要

用使质粒pKU3所携带的玉米Ac因子的5^末端和中间编码转座酶的基因片段分别发生缺失的方法构建成质粒pKU3(#DELTA#B)和pKU3(#DELTA#H)。质粒所携带缺失的Ac因子单独存在时都无自主转座能力,但当Ac(#DELTA#H)和Ac(#DELTA#B)共存于一个细胞时,由于Ac(#DELTA#B)产生转座酶的互补作用促使Ac(#DELTA#H)恢复转座能力,, 而当Ac(#DELTA#B)和Ac(#DELTA#H)因子被分离后即获得Ac(#DELTA#H)因子的稳定插入突变株,它可克服因突变不稳定而给用转座因子标签法分离基因所造成的困难。将双因子系统导入烟草原生质体并获得再生植株，从而选得卡那霉素抗性植株,显示Ac(#DELTA#H)因子已经从原质粒上的NPT II前导顺序中切离。Southern blot和PCR分析表明:Ac(#DELTA#H)和Ac(#DELTA#B)因子已经整合在转化烟草的基因组并能遗传至F_(1)和F_(2)代植株中;有些植株后代中已检测不到Ac(#DELTA#B)因子的存在,说明它们的Ac(#DELTA#B)因子已与Ac(#DELTA#H)因子相分离;各转化植株中Ac(#DELTA#H)因子在基因组中的插入拷贝数从一个到几个不等;初步显示Ac(#DELTA#H)因子多数插入或转座在基因组的结构基因中。

关键词： 转应因子标签; Ac双因子转座系统; 烟草; 原生质体; PEG转化

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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引用本文：

张景六, 章文伟, 张栋, 张大兵, 张磊, 安林升, 洪孟民. 玉米Ac双因子转座标签系统的构建及其转化烟草后代的遗传分析[J]. 生理学报 1997; 49 (1): .

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).