外源基因在烟草绿色组织中的高效表达研究

Research of Exogenous Gene High Level Expression in Green Tissue of Tobacco

研究外源基因在受体绿色组织中的特异表达情况,分别构建由棉花PsbP启动子驱动的GUS基因及CP4 epsps基因的植物表达载体pBI121-P、pBI121-PE。农杆菌介导法转化到烟草中,获得20株转pBI121载体、40株转pBI121-P载体和32株转pBI121-PE载体的烟草阳性植株。组织化学染色分析表明,GhPsbP启动子驱动的GUS基因只在转基因烟草的叶片和茎中表达,在根中不表达;Real-time PCR分析表明,PsbP启动子驱动CP4 epsps基因在叶和茎中的表达量分别是根中的15倍和10倍;草甘膦抗性试验证明,PsbP启动子驱动的CP4 epsps基因在烟草叶及茎的绿色组织中的表达量足以忍受1%浓度草甘膦的毒害。证明GhPsbp启动子可以有效地驱动外源基因在烟草的绿色组织中高效特异的表达。

As a crucial regulatory element, promoter is an important research target of plant genetic engineering. However, most promoters applied to genetically modified crops were usually constitutive promoter. The constitutive promoter can not drive exogenous gene expressing in particular tissue and specific time, so it always results in energy and materials wasting. GhPsbP gene encodes cotton extrinsic protein P of photosystem II oxygen evolution complex, in previous studies, we found the transcription level of GhPsbP is higher in chlorenchyma than other tissues. For studying the GhPsbP promoter expression characteristics and evaluating its application prospect in genetics modified plant,we constructed the fusion expression vectors of GhPsbP promoter with GUS and epsps based on pBIl21 re- spectively, which were named pBI121-P and pBI121-PE. The vectors pBI121, pBI121-P and pBII21-PE were transformed into Nicotiana tabacum var. NC89 using the Agrobacterium-mediated transformation method. By kanamycin screening and PCR molecular identification, we got a total of 20,40 and 32 independent transgenic tobacco lines with pBI121 ,pBI121-P and pBI121-PE,respectively. The result of transgenic tobacco lines with pBI121-P histochemical staining indicated that the GUS only expressed in leaf and stem,which couldn＇ t express in root. The result of transgenic tobacco lines with pBI121-PE real-time analysis showed the CP4 epsps transcription level of leaf and stem were 15 and 10 times higher than root＇ s ,respectively. The glyphosate resistance assay indicated that the expression of CP4 ep- sps in leaf and stem driven by GhPsbP promoter can resistant to the glyphosate effectively. In conclusion, the GhPsbP promoter which can drive exogenous gene expressing in tobacco chlorenchyma effectively, can be applied to some particular genetically modified plants as a chlorenchyma expression regulatory element.