启动子对外源基因在转基因植物中表达的影响

Directed Duplication of CaMV35S Promoter Influerues the Expression of the Foreign Genes in Transgenic Plants

将来自ｐＢＩ１２１质粒的ＣａＭＶ３５Ｓ启动子片段插入到ｐＢＩ１２１的ＣａＭＶ３５Ｓ启动子与ＧＵＳ基因之间，构建了串联的ＣａＭＶ３５Ｓ启动子载体ｐＬＢ３８．通过三亲交配，将ｐＢＩ１２１及ｐＬＢ３８分别转移到含ｐＧｖ３８５０的农杆菌中，成为适合本研究的双元载体。以叶圆盘转化法将外源基因转入烟草，获得了２种转基因植株。经ＤＮＡ分子杂交、ＮＰＴⅡ点分析、ＧＵＳ荧光定性及定量分析，证明外源基因已整合进烟草基因组并获得表达。ｐＬＢ３８的ＧＵＳ表达量为ｎＢｌ１２１的３～４倍，这表明启动子数目的不同会直接影响其启动基因的表达水平。

The mediated vector pLB38 was constructed by insertion the 800 bp fragment of CaMV35S promoter from the plasmid pBI 121 into the BamH I site between the CaMV 35S pro- moter and GUS gene of the pBI121 and screening the recombinants containing the directed dupli- cate of CaMV35S promoter with Xba I digest.The plasmid pBI121 and pLB38 were introduced into the Agrobacterium tumefaciens containing Ti plasmid pGV 3850 by triparental-mating tech- nique with the aid of helper plasmid pGJ23. The foreign genes in pLB38 and pBI121 were transferred into the tobcco plants using the leaf -disk co-cultivated method and two kinds transgenic plants were obtained. DNA / DNA dot blot and Southern blot with α-32P labelled probe containing 3kb fragment of CaMV 35S-GUS comfirmed that foreign genes were transferred and integrated into the trans- genic tobacco plant genome,The identification of the product of NPT II gene with NPT II dot as- say,GUS gene with GUS fluorescent assay proved that the foreign NPT II and GUS gene were expressed in transgenic plants.And the GUS activity in pLB38-transformed plants in which the GUS gene was controlled by two directed CaMV35S promoters was three-to four-fold higher than that in the pBI121-transformed plants in which the GUS gene is controlled by single CaMV35S promoter.These results suggested that the promoter number has effect on the expres- sion level of genes controlledy by these promoters.