Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People's Republic of China.
Plant Cell Rep. 2012 Oct;31(10):1877-87. doi: 10.1007/s00299-012-1301-5. Epub 2012 Jul 10.
The advantages of gene 'stacking' or 'pyramiding' are obvious in genetically modified (GM) crops, and several different multi-transgene-stacking methods are available. Using linker peptides for multiple gene transformation is considered to be a good method to meet a variety of needs. In our experiment, the Bt cry1Ah gene, which encodes the insect-resistance protein, and the mG ( 2 ) -epsps gene, which encodes the glyphosate-tolerance protein, were connected by a 2A or LP4/2A linker. Linker 2A is a peptide from the foot-and-mouth disease virus (FMDV) that has self-cleavage activity. LP4 is a peptide from Raphanus sativus seeds that has a recognition site and is cleaved by a protease. LP4/2A is a hybrid peptide that contains the first 9 amino acids of LP4 and 20 amino acids from 2A. We used the linker peptide to construct four coordinated expression vectors: pHAG, pHLAG, pGAH and pGLAH. Two single gene expression vectors, pSAh and pSmG(2), were used as controls. The six expression vectors and the pCAMBIA2301 vector were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation, and 529 transformants were obtained. Molecular detection and bioassay detection data demonstrated that the transgenic tobaccos possessed good pest resistance and glyphosate tolerance. The two genes in the fusion vector were expressed simultaneously. The plants with the genes linked by the LP4/2A peptide showed better pest resistance and glyphosate tolerance than the plants with the genes linked by 2A. The expression level of the two genes linked by LP4/2A was not significantly different from the single gene vector. Key message The expression level of the two genes linked by LP4/2A was higher than those linked by 2A and was not significantly different from the single gene vector.
基因“叠加”或“金字塔”在转基因（GM）作物中具有明显的优势，并且有几种不同的多基因叠加方法。使用连接肽进行多个基因转化被认为是满足各种需求的好方法。在我们的实验中，Bt cry1Ah 基因，它编码抗虫蛋白，和 mG（2）-epsps 基因，它编码草甘膦耐受蛋白，通过 2A 或 LP4/2A 连接肽连接。连接肽 2A 是来自口蹄疫病毒（FMDV）的具有自我切割活性的肽。LP4 是来自萝卜种子的肽，具有识别位点并被蛋白酶切割。LP4/2A 是一种含有 LP4 的前 9 个氨基酸和 2A 的 20 个氨基酸的混合肽。我们使用连接肽构建了四个协调表达载体：pHAG、pHLAG、pGAH 和 pGLAH。两个单基因表达载体，pSAh 和 pSmG（2），被用作对照。将这六个表达载体和 pCAMBIA2301 载体通过根癌农杆菌介导转化到烟草中，得到了 529 个转化体。分子检测和生物测定检测数据表明，转烟草具有良好的抗虫性和草甘膦耐受性。融合载体中的两个基因同时表达。用 LP4/2A 肽连接的基因的植物表现出比用 2A 肽连接的基因更好的抗虫性和草甘膦耐受性。用 LP4/2A 连接的两个基因的表达水平与单基因载体没有显著差异。关键信息用 LP4/2A 连接的两个基因的表达水平高于用 2A 连接的基因，与单基因载体没有显著差异。
