采用玉米Ubi-1启动子获得低拷贝转基因玉米植株

Transgenic Maize Plants with Low Copy Number of Foreign Genes were Produced with Maize Ubi-1 Promoter

通过基因枪粒子轰击和草丁膦 (PPT)选择获得可育的玉米转基因植株 ,并分析了外源基因在转化体中的拷贝数与启动子之间的关系。用玉米Ubi 1启动子驱动外源基因 ,玉米转化体中外源基因的拷贝数较低 ;可能的原因为Ubi 1启动子通过与其内部同源序列发生重组而被定点整合进玉米基因组 ,共转化的两种质粒DNA在整合至玉米染色体DNA之前已重构成为一个整体。结果显示使用某一植物自身基因的启动子可以降低外源基因在该物种转基因个体中的拷贝数 ,进而避免基因沉默现象的发生。目前已得到第二代转基因玉米种子。

Direct DNA delivery procedures (include biolistics method) often resulted in multiple copies of the transgenes in transformants and certain copies of them were rearranged. Integration of multiple copies of the introduced genes was the main reason of gene silencing which meant inhibition or loss of foreign gene expression in filial generations of transformants. In the present work, we compared the influences of maize Ubi-1 promoter and other promoters on copy number of transgenes in maize transgenic plants. Immature embryos from Zea mays L. plants of sib-pollinated of A188×H99 genotype were used as initial materials. Type-Ⅰembryonic calluses derived from preculture of immature embryos were treated on N6 medium containing 0.6 mol/L sucrose for 3～5 hours and transformed via particle bombardment with PDS1000/He delivery system (Bio-Rad). Bombarded calluses were treated with hyperosmotic N6 medium for 16～20 hours continuously. Then the cultures were transferred onto normal N6 medium and incubated at 26℃ in dark for two weeks and subsequently selected on N6 medium supplemented with 2 or 5 mg/L phosphinothricin (PPT) but without casamino acid for another two weeks. The calluses after selective culture were transferred onto hormone-free MS medium containing 2 or 5 mg/L PPT but without casamino acid, and incubated at 24℃ under 16h illumination for plant regeneration. Regenerated plantlets over 2cm in height were transferred to Magenta box containing 1/2 hormone-free MS medium. Plantlets over 8cm in height were transplanted to soil. After growing for one week in greenhouse, the plants were sprayed with 250mg/L PPT solution. Fertile transgenic maize plants were regenerated and confirmed by Southern blotting and histochemical localization of β-glucuronidase (GUS) activity. Relations between promoter and copy number of transgenes in transformants were analyzed. Maize transgenic plants possessing an intact copy and another incomplete copy of β-glucuronidase gene (gus) were obtained in case gus gene under the control of maize Ubi-1 promoter (pUbi:GUS). Simultaneously the co-transformed phosphinothricin acetyltransferase gene (bar) controlled by CaMV 35S promoter in another plasmid (p35S:BAR) also existed with only one copy. When pDB1 and (pUbi:in2) were cobombarded, the regenerated transgenic maize plant exhibited with only one copy of in2 gene too. It suggested that the copy number of transgenes in maize transformants was low if the transgenes controlled by maize Ubi-1 promoter. The possible reason might be that the foreign genes were integrated site-specifically via homologous recombination between Ubi-1 promoter and its endogenous sequences in maize genome, and two cotransformed plasmids had reconstructed as one intact molecule before integrating into maize chromosome. On the contrary, if p35S:BAR was cobombarded with plasmid pAct:In1 containing rice Act-1 promoter (without maize Ubi-1 promoter), the transgenic maize plants had 4～8 copies of bar gene. These results reflected that utilization of self gene promoter could reduce the copy number of the transgenes in transgenic plants of certain species itself and avoid the occurrence of gene silencing. T2 seeds have been harvested.