中文摘要：目前转基因作物的安全性评估需要转基因插入及其表达对寄主植物的生物化学和生理特性所产生影响的详细信息。本文研究了苹果多聚半乳糖醛酸酶抑制蛋白1基因整合的分子特性，其在马铃薯中超表达可增强对黄萎病的抗性。转基因马铃薯AppA6的染色体步行研究表明，在CaMV 35S启动子的控制下包含MdPGIP1转基因的T-DNA插入到基因组，不包含pCAMBIA2300载体的任何non-T-DNA序列。实时荧光定量PCR检测显示，MdPGIP1转基因插入会使转基因马铃薯AppA6的木葡聚糖转移酶（XTH）基因和内源性stpgip1基因的表达增加。这些意想不到的变化是由MdPGIP1转基因或转化相关的体细胞无性系变异组成型表达引起的。研究结果表明，在转基因马铃薯AppA6中，稳定、单一集成的MdPGIP1基因没有破坏任何结构基因从而造成意想不到的效果。
外文摘要：Current safety assessment of genetically modified crops requires detailed information about the insertion of the transgene and the effect of its expression on the biochemistry and physiology of the host plant. Whilst the intended effect of the transformation can be verified through phenotypic screening, molecular approaches are required to observe unintended effects. We investigated the molecular details of the integration of a polygalacturonase inhibiting protein 1 gene from Malus domestica (Mdpgip1), overexpressed in Solanum tuberosum (cv BP1) for enhanced resistance against Verticillium wilt. Genome walking studies of the selected AppA6 transformant revealed that the T-DNA containing the Mdpgip1 transgene under control of the CaMV 35S promoter was inserted into the genome without any non-T-DNA sequences from the pCAMBIA2300 vector. Sequence data indicate that the insertion of the Mdpgip1 transgene was in a gene-rich region of chromosome 1, adjacent to the photosystem Q(B) gene but without disruption of structural genes. Transcriptome-based cDNA-representational difference analysis revealed the distinctive expression of Mdpgip1 in the transgenic AppA6 line, verifying the intended effect. Protein extracts from the transgenic plants inhibited the activities of Verticillium dahliae polygalacturonases in in vitro studies, showing that the transgene is expressed to produce an active PGIP defense protein. cDNA-AFLP fingerprinting revealed genes that were differentially expressed, including genes encoding tryptophan/tyrosine permease, Ef-Tu domain and SKP1-like 1A proteins. qRT-PCR indicated that the Mdpgip1 transgene insertion resulted in increased expression in the AppA6 transgenic of the xyloglucan endotransglycosylase (xth) gene and an endogenous Stpgip1 gene. These unintended changes were either caused by the constitutive expression of the Mdpgip1 transgene or transformation-related somaclonal variation. The results indicate that the stable, single copy integration of the Mdpgip1 gene in the AppA6 transgenic line did not disrupt any structural genes but caused unintended effects that affected gene expression compared to the parental counterpart under the non-stressed experimental conditions investigated.
外文关键词：Agrobacterium-mediated transformation; Differential gene expression; Genetically modified; Genome walking; PGIP; Transgene insertion; Unintended effects
外文关键词：Agrobacterium-mediated transformation; Differential gene expression; Genetically modified; Genome walking; PGIP; Transgene insertion; Unintended effects
作者：Matsaunyane, Lerato B. T.; Oelofse, Dean; Dubery, Ian A.
作者单位：Univ Johannesburg
期刊名称：POTATO RESEARCH
期刊影响因子：1.159
出版年份：2016
出版刊次：6
点击下载：具有苹果多聚半乳糖醛酸酶抑制蛋白1基因的转基因马铃薯AppA6的详细分子生物学特性