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（△B）和pKU3（△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（△H）和Ac（△B）共存于一个细胞时，由于Ac（△B）产生转座酶的互补作用促使Ac（△H）恢复转座能力，而当Ac（△B）和Ac（△H）因子被分离后即获得Ac（△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（△H）因子已经从原质粒上的NPTⅡ前导顺序中切离。Sortharnblot和PCR分析表明：Ac（△H）和Ac（△B）因子已经整合在转化烟草的基因组并能遗传至F1和F2代植株中；有些植株后代中已检测不到Ac（△B）因子的存在，说明它们的Ac（△B）因子已与Ac（△H）因子相分离；各转化植株中Ac（△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（△H）因子多数插入或转座在基因组的结构基因中。
Two plasndds, PKU3(△B) and PKU3 (oh) were reconstructed from Plasndd PKU3 by delehng the 5' -terterminal region or internal part of maize AChvator (Ac ) transposable element.The Ac element in PKU3 was inserted in the untranslated leader sequence of the neomycin phosphotransferase Ⅱ (NPT Ⅱ) bine, when Ac was excised,the destroyed NPT Ⅱ gene activity was recovered. Thus, the appearance of NPT Ⅱ achvity can be used to detect the excisionof Ac element(Fig. 1 ). The PKU3(△B) and pain (in) alone could not be transpose from site to site automonously, because tWo essenhal sequences, the terminal inverted repeat and ccing Part of transposase bine,were removed. HOwever, the Ac (△H) element may regain the transposibility by complemenhng the transse from the AC (all) element When bath elements co-existed withen a cell. After the Ac (△B) was segregated from AC (△H) element, a stable Ac inserhon mutant could be obtained. To test if thes two elements system could be used in transal son tabing strategy to clone genes in plants, these two AC derivahves were intreduced into protoplasts from Alter Planilet regrnerahon, transgenic tobacco plants with kanamycin reaistance were obtained.The restorahon of kinamycin resistance shows that the Ac (△H) element was exelsed from the untranslated leader sequence of NPT Ⅱ bine. ResultS of southern blot analysis and polymerase chain reachon assay showed that (i) bath the Ac(△H)and Ac (△B) elements had been integrated intO the grnome of transformed tobacco plants (Fig. 2 ),and they could be transwhtted from F,tO F, and F2 progenies. (ii) In a few plantS the Ac(an) was excised and segreacted from Ac (in) (Fig. 3 ). (iii) aam copy number of inserted Ac elements withen genome of individual transformed Plants was determined as one or more (Fig. 4). (tv) MOst of the Ac elements were inserted or transport intO structUral gene and not into repeated sequences withen the genome (data not showed).
基金
863基金
关键词
玉米
转座因子标签
Ac双因子
转座系统
烟草
transposon tagging, two-elements transposon tagging system, tobacco. protoplast, PEG-transformation
